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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

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AnAnecdotal

History

ofNematology

J. M. Webster, K. B. Eriksson & D. G. McNamaraEditors

An A

necdotal History of N

ematology

Nematology 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 ournematological world’s most fascinating discoveries and about their discoverers.

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 Historyof

Nematology

AnAnecdotal

History

ofNematology

J. M. Webster, K. B. Eriksson & D. G. McNamaraEditors

Sofia–Moscow2008

AN ANECDOTAL HISTORY OF NEMATOLOGYJ. M. Webster, K. B. Eriksson & D. G. McNamara (Editors)

First published 2008ISBN 978-954-642-324-5 (paperback)ISBN 978-954-642-426-6 (e-book)

Book and cover design: Zheko Aleksiev

© PENSOFT PublishersAll rights reserved. No part of this publication may be reproduced, stored in a retrievalsystem or transmitted in any form by any means, electronic, mechanical, photocopying,recording or otherwise, without the prior written permission of the copyright owner.

Pensoft PublishersGeo Milev Str. 13a, Sofia 1111, BulgariaFax: [email protected]

Printed in Bulgaria, 2008

TABLE OF CONTENTS

– Contributors 7

– Preface 11

– Prologue 14

1. Our early stars 17Bengt Eriksson

2. Nematological nebulae in Europe and the USA 33John M. Webster and Seymour D. Van Gundy

3. First catch your nematode! – the development of methods for recovering nematodes from soil 59David McNamara

4. Through nematode diversity to living soil processes – holistic studies aid progress 67Gregor Yeates

5. Nematode physiology: Significant developments in the understanding of the biology of simple eukaryotic animals 80Howard Ferris and Haddish Melakeberhan

6. Molecular taxonomy of nematodes 98Pierre Abad and Philippe Castagnone-Sereno

7. A history of potato cyst nematode research 107Ken Evans and David L. Trudgill

8. Cereal cyst nematode complex 129Roger Rivoal

9. The science and art of soybean cyst nematode research 137Terry L. Niblack and Don P. Schmitt

10. Nematodes/Viruses/Plants: “A 32-year love affair” 152Derek J.F. Brown

11. Horticultural hazards: In and out of hot-water baths and other transient technologies 168Simon R. Gowen and Philip A. Roberts

12. The spread of nematology to developing countries: A case study 187Michel Luc

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

14. Quarantine nematodes 219David McNamara

15. The pinewood nematode: a personal view 231Helen Braasch and Manuel M. Mota

16. History of the development of nematodes as biocontrol agents 246Parwinder S. Grewal and Harry K. Kaya

17. Dynamics of nematological infrastructure 258Roland N. Perry and James L. Starr

18. Nematology: Dreams and visions of the future 272(i) A nematology dream: Miscalculations, and false prophecies? 272

Ernest C. Bernard

(ii) Vision of nematology in Canada in the next 50 years 276Guy Bélair

(iii) The future of nematode systematics and phylogeny over the next 50 years 279Virginia R. Ferris

(iv) A nematologist’s dream 283Florian Grundler

(v) My nematology dream 286Paulo Vieira

(vi) The devil’s advocate 288Derek J.F. Brown

(vii) My dream, not a nightmare 292Forest Robinson

(viii) My dream of the future of nematology and chemical communication research – 50 years from now 295Ekaterina Riga

(ix) C. elegans as a model system for space travel 297Robert Johnsen and David Baillie

CONTRIBUTORS

PIERRE ABAD, UMR Interactions Plantes Microorganismes et SantéVégétale, INRA/CNRS/Université de Nice Sophia Antipolis,BP 167, 06903 Sophia Antipolis Cedex, [email protected]

DAVID L. BAILLIE, Molecular Biology and Biochemistry, Simon FraserUniversity, Burnaby, British Columbia, V5A 1S6, [email protected]

GUY BÉLAIR, Agriculture and Agri-Food Canada/Agriculture etAgroalimentaire Canada, St. Jean-sur-Richelieu, Quebec, J3B 3E6, [email protected]

ERNEST C. BERNARD, Entomology and Plant Pathology, TheUniversity of Tennessee, Knoxville, TN 37996-4560, [email protected]

HELEN BRAASCH, Kantstrasse 5, D-14471 Potsdam, [email protected]

JANETE A. BRITO, FDACS, DPI, Nematology Section, PO Box 147100, Gainesville, FL 32614-7100, [email protected]

DEREK J.F. BROWN, RD Consultants, “Penbro”, 3, Neofit Rilski Street,2778 Banya, Razlog Municipality, [email protected]

PHILIPPE CASTAGNONE-SERENO, UMR Interactions PlantesMicroorganisms et Santé Végétale, INRA/CNRS/Université deNice Sophia Antipolis, BP 167, 06903 Sophia Antipolis Cedex,[email protected]

BENGT ERIKSSON, Department of Forest Mycology and Pathology,Swedish University of Agricultural Sciences, Box 7026, SE-750 07, Uppsala, [email protected]

CONTRIBUTORS 7

KENNETH EVANS, Nematode Interactions Unit, RothamstedResearch, Harpenden, Hertfordshire, AL5 2JQ, [email protected]

HOWARD FERRIS, Department of Nematology, University ofCalifornia, Davis, CA 95616, [email protected]

VIRGINIA FERRIS, Department of Entomology, Purdue University,West Lafayette, IN 47907-2089, [email protected]

JAVIER FRANCO, Fundación PROINPA, Casilla Postal 4285, El Paso,Cochabamba, [email protected]

ROBIN M. GIBLIN-DAVIS, Fort Lauderdale Research and EducationCenter, University of Florida/IFAS, Fort Lauderdale, FL 33314, [email protected]

SIMON R. GOWEN, School of Agriculture, Policy and Development,The University of Reading, Reading, RG6 6AR, [email protected]

PARWINDER GREWAL, Department of Entomology, The Ohio StateUniversity, Wooster, OH 44691, [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, [email protected]

RENATO N. INSERRA, FDACS, DPI, Nematology Section, PO Box 147100, Gainesville, FL 32614-7100, [email protected]

ROBERT JOHNSEN, Molecular Biology and Biochemistry, Simon FraserUniversity, Burnaby, British Columbia, V5A 1S6, [email protected]

8 CONTRIBUTORS

HARRY K. KAYA, Department of Nematology, One Shields Avenue,University of California, Davis, CA 95616, [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 PlantProtection Organization, Paris, [email protected]

ROSA H. MANZANILLA-LÓPEZ, Plant Nematode Interactions Unit,Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, [email protected]

HADDISH MELAKEBERHAN, Agricultural Nematology Laboratory,College of Agriculture and Natural Resources, Michigan StateUniversity, East Lansing, MI 48824, [email protected]

MANUEL M. MOTA, NemaLab-ICAM, Departmento Biologia,Universidade de Évora, 7002-554 Évora, [email protected]

TERRY L. NIBLACK, Department of Crop Sciences, University ofIllinois, Urbana, IL 61801, [email protected]

ROLAND N. PERRY, Plant Pathogen Interactions Division, RothamstedResearch, Harpenden, Hertfordshire, AL5 2JQ, [email protected]

PATRICK QUÉNÉHERVÉ, Laboratoire de Nématologie Tropicale,PRAM, IRD, BP 8006, 97259 Fort-de-France, Martinique,[email protected]

EKATERINA RIGA, Nematology, Washington State University, Prosser,WA 99250, [email protected]

CONTRIBUTORS 9

ROGER RIVOAL, UMR INRA/ENSAR, Biologie des Organismes etdes Populations Appliquée à la Protection des Plantes (BiO3P),BP 35327, 35653 Le Rheu Cedex, France. [email protected]

PHILIP A. ROBERTS, Department of Nematology, University ofCalifornia, Riverside, CA 92521, [email protected]

FOREST ROBINSON, USDA-ARS, 2765 F&B Road, College Station, TX 77845, [email protected]

JESSÉ ROMÁN, Crop Protection Department, AgriculturalExperiment Station, PO Box 21360, Rio Piedras, Puerto Rico 00928. [email protected]

DONALD P. SCHMITT, 14844 Highway 5, Marceline, MO 64658, [email protected]

JAMES L. STARR, Department of Plant Pathology and Microbiology,Texas A&M University, College Station, TX 77843-2132, [email protected]

DAVID L. TRUDGILL, Scottish Crop Research Institute, Invergowrie,Dundee, DD2 5DA Scotland, [email protected]

SEYMOUR D. VAN GUNDY, Department of Nematology, Universityof California, Riverside, CA 92521, [email protected]

PAULO CEZANNE VIEIRA, NemaLab/ICAM, Universidade de Évora,Évora, [email protected]

JOHN M. WEBSTER, Department of Biological Sciences, Simon FraserUniversity, Burnaby, British Columbia, V5A 1S6, [email protected]

GREGOR W. YEATES, Landcare Research, Private Bag 11052,Palmerston North 4442, New [email protected]

10 CONTRIBUTORS

PREFACE

Derek Brown first had the idea of writing this anecdotal history ofnematology and, when he invited us three to undertake the editorialwork, we were convinced by his reasons as to why now was theideal 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 ofentomology and plant pathology have much longer histories. Lessthan 150 years have passed since the true pioneers of nematologywere in action. Those of us of a certain age knew the previous gen-eration of nematologists, who themselves knew some of the pio-neers. So we realized that it would be a good idea to get the storiesabout the “old days” down on paper before it was too late. One ofthe advantages of being old (and there are a few!) is that youbecome rather good at history – because you were part of it or, atleast, you think that you remember that you were! A new genera-tion of nematologists is now holding centre stage, and, although theyknow well how their technology was developed and they under-stand perfectly the significance of their results in the context of thenematological study, they, perhaps, do not always fully appreciatethe broader biological relationships or how and by whom the exist-ing body of knowledge was obtained.

Nematology has also been, up to now, a “small” science with alimited number of adherents. Even though it has often been subdi-vided into different subject areas, everyone who called him/herselfa nematologist knew nearly every other nematologist, no matter inwhich country they lived and worked. However, as the subject areasthemselves have become more specialized, they have tended to driftapart, so that there is an increasing trend for nematologists involvedin particular fields of the science to know only minimal amountsabout areas of nematology other than their own. We believe thatthis trend is not good for nematology as a whole and we feel that itis a distinct advantage for those in different areas to be aware ofeach other’s work and to share ideas. We are all working on nema-todes, after all! By presenting a history of discovery in nematology inthe form of different chapters relating to different subject areas, wehope that we can, in some small measure, help to bridge theexpanding gap. To appreciate fully the significance of the advances

PREFACE 11

in nematology, it is necessary not only to understand something ofthe knowledge that has been gained but also to recognize theachievements of the men and women who have been responsiblefor the advances. By producing this book, we hope to promote anappreciation of the subject matter of nematology and of the peopleinvolved in its discovery, within our own tiny sector of the broadfront of scientific progress. We hope that this does not sound toograndiose and we hope that it does not dissuade you from readingand enjoying this book as you trip through time, technique and title!

We began preparation of the book by, firstly, trying to decidewhat was to be covered by the name “nematology”. We agreed thatwe should only include the study of nematodes relevant to plantprotection, as this has traditionally formed a quite self-containedarea of study, and, of course, has been the particular area of expert-ise of the three editors throughout their careers. This definitionincludes all soil and plant-inhabiting species, and those soil speciesthat are not plant parasites (whose relationships with each other andwith other soil organisms contribute to the general health of thesoil). Also included are the entomopathogenic species, which playan important role in controlling insect pests of plants. We have notincluded in our particular definition of nematology the study ofmarine and fresh-water nematodes which have long had their ownparticular specialists; nor have we included nematodes that are para-sitic in humans and other mammals, which are generally covered inmedical and veterinary research. The field of study surrounding thegenetic elucidation of Caenorhabditis elegans, though contributingenormously to our understanding of the Nematoda and of living sys-tems, is not included in detail as the mass of information emanatingfrom that field would have required another volume.

We then divided up the science of nematology into some of itsmajor subdivisions, and invited experts in these areas to write a chapteron their history. We asked them to highlight the principal milestonesalong the road to the present, to tell us about the leading personalitieswho achieved these milestones (and about others who were just inter-esting personalities), and we asked them to make the history personaland anecdotal. And much to our surprise, very few of those experts thatwe approached declined to participate, despite the fact that everyone isalways very busy these days (just as nematologists have always been),and despite the fact that “anecdotal” is a strange word, possibly difficultfor the non-English speakers among our authors (and there are a few!).

12 PREFACE

Just as we have defined „nematology“ for our own particularpurposes, so have we adopted our own interpretation of “anecdotal”.Originally, an anecdote was simply something that has not beenpublished. The word now generally refers to the telling of somebiographical incident that may or may not be true; in fact, ofteninformation that could be considered to be untrustworthy. Ourintent, however, was to include, within the historical narrative, ref-erences to the personalities engaged in the progress of nematologicalresearch, to try to add stories (but true stories!) about these peoplethat might bring the history to life. We left it to the individualauthors to decide how, and how much, they should incorporate ofthe anecdotal elements.

We also invited a number of nematologists to stick their necksout and predict how their own particular subject area will developin future years. Predictions of any kind usually provide immenseentertainment to readers in the future, who can smugly laugh andcompare the way things really turned out with the totally erroneousvision of the prophets. Of course, such prophets are not usuallyexperts in their chosen field of prediction. Our prophets, on theother hand, are true experts and visionaries and we are quite confi-dent that the future will arrive just as they predict!!!

With such a wide range of individuals contributing their ownviews, it is inevitable that the style and format of different chapterswill vary. Nevertheless, we hope also that our readers will beenlightened and amused. We recognize that, despite our own enthu-siasm, very few people outside of the field have even heard ofnematodes. So, to those readers who are not already nematologists,this book might convince you of what we have always known to betrue: that nematodes are delightful little creatures and that nematol-ogy is an endlessly fascinating, multifaceted subject.

Finally and wholeheartedly, we thank all our contributors, andthe many other nematologists who contributed in diverse ways, andhope that everyone involved will feel that this has been a worth-while project.

JOHN M. WEBSTER

BENGT ERIKSSON

DAVID MCNAMARA

August, 2007

PREFACE 13

PROLOGUE

According to the famous Russian nematologist and philosopher A.Paramonov, nematodes are an ancient group of organisms exhibitingtrue biological progress, manifested by a high level of taxonomicdiversification, intensive speciation, diversity of life cycles and lifestrategies, trophic specialization and the occupation of various habi-tats. 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 excellentidea, now realized through the efforts of many outstanding nematol-ogists who have shared their personal memories, feelings, thoughtsand a lot of information accumulated through many years committedto nematology. The main driving forces, both objective and subjective,the circumstances, ideas and concepts marking the development ofour science and the progress of nematological research all over theworld are traced. The photographs present a rich gallery of pioneernematologists and teams – insights into early epochs and into therichness and excitement of more recent times.

B. Eriksson leads us back to the beginning of the science ofnematology and its “early stars”. The first nematological centres wereestablished in Europe and North America, inspired and pushed for-ward by the discovery of the microscope, elucidation of the patho-genic role of plant parasitic nematodes, the social needs, and….thecuriosity of the scientists (J.M. Webster & S.D. Van Gundy’s chapter).The centres had, and still have, a significant influence on the estab-lishment of nematology on the world scene and on its continuedadvance. It is fascinating to follow the genealogy of the differentschools and research branches with their inputs into uncovering thesecrets of the nematodes, showing their economic importance andlearning how to manage their control. Special attention is given tothe events of nematology in Latin America where a range of remar-kable scientists have studied the diversity of nematode pests under avariety of agricultural and horticultural circumstances. Developmentof nematology in this part of the world has often occurred in collab-oration with colleagues from centres in North America and Europe.

Progress in science is highly dependent on the development ofresearch methodology. D. McNamara outlines the evolution andapplication 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 pro-voked 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. Castagnone-Serrano review the modern molecular approaches used in nematodetaxonomy and phylogenetic reconstructions, briefly showing themain problems and shortcomings, and prospecting the future of thisquickly-evolving branch of nematology.

I was touched by the stories told by G. Yeates and M. Luc – theyfully confirm the concept that the history of a science is (nothingbut) the summary of the personal experiences of the scientists themselves.

In a treatise of this kind we must not omit the prime nematodessuch as the cyst nematodes of potato, soybean and cereal, the virusvectors, the burrowing nematode, the stem and bulb nematodes, thepinewood nematode and Caenorhabditis elegans – all have their ownsagas marking the different developmental phases of nematology as awhole. Most of these nematodes are quarantine objects and, nowa-days, quarantine services are becoming very important for preventingthe spread not only of well-known pest organisms but also of poten-tially invasive species, as pointed out by D. McNamara.

The flourishing of nematology is intimately connected with theestablishment and diversification of nematology infrastructure – jour-nals, newsletters, conferences and societies – as distinctly shown byR. Perry and J. Starr. All these structural units help substantially tostimulate the exchange of knowledge and ideas, facilitate contactbetween scientists and promote international collaboration.

Of special interest is the final chapter on dreams and visions ofthe future of nematology. Comfortingly, the majority of predictionsare positive, foreseeing new horizons, new findings, and an increasein 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 tothe 50th Anniversary of the European Society of Nematologists(ESN) which was celebrated at the 28th Symposium of the ESN inBulgaria, in 2006. A memorable congress, and one which I was hon-oured to be allowed to organize. We have to say a big THANK YOUto our colleagues who have contributed the various chapters and

PROLOGUE 15

have shown us the many-sided face of our science in a vivid andinspired manner. We are very much obliged also to the editors J. M.Webster, K. B. Eriksson and D. G. McNamara and to D. Brown whocame up with the idea for this book, and finally to PENSOFT pub-lishers. This work has particular value for the young and future gen-erations; it shows the long road that has been traveled from the firstdiscoveries and inventions to present-day nematology.

I leave you, dear Reader, with a wonderful history of nematologypresented with a sense of humour and true love for those little butvery powerful creatures – the nematodes.

VLADA PENEVA,Past-President,European Society of Nematologists.January, 2008

16 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 itsrulers or sovereigns. So, who are the excellencies, the “stars”, in thehistory of nematology? Before we identify some of the more obvi-ous figures and pioneers, let us describe in historical terms thedevelopment of the study of these ‘beautiful little beasts’, common-ly called nematodes or roundworms, and so poetically described byB.G. Chitwood, as cited:

“The grace of movement of some lowly soil-inhabiting forms finds littleequal among other living organisms, being comparable to the gliding ofsnakes, which Solomon noted as one of the four mysteries of life. The complex patterns of their body markings and of the head and other partsmight 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 his-tory back to parallel the history of any field of zoology. If not men-tioned specifically as nematodes, or even roundworms, the pheno-mena associated with them were well-known from the ancients inscriptural times. Often cited are the biblical passages in Numbers 21,where Moses “fiery serpent” made from brass is supposed, rightly orwrongly, to be the Guinea worm (Dracunculus medinensis) causingcutaneous lesions on the extremities of man. To remove the parasitethe adult female is rolled out of the ulcer and onto a forked stick,the so called ‘Indian barber’ technique (Maggenti). The way it is sofiguratively described in being extracted from the body tissues has a

OUR EARLY STARS 17

striking resemblance to the rod of Asclepius, the symbol of medi-cine. Human-parasitic nematodes (e.g., Ascaris, Dracunculus), or atleast the diseases they cause, seem to have been known since beforethe times of Moses, as indicated in an Egyptian papyrus dating fromthe 1550s B.C., and published in 1889 by the German Egyptologist,G. Ebers, as “Der Papyrus Ebers”. Probably the oldest reference toparasitic nematodes is found in the Yellow Emperor’s Classic ofInternal Medicine from China, some 2700 B.C., where a disease isaccurately described that thousands of years later is known as anAscaris infection.

A giant of these ancient times is, of course, the Greek philoso-pher and natural scientist, Aristotle (384–322 B.C.), also known asthe Father of Zoology. In his “Historia Animalium” he refers tonematodes, 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 genera-tion”, a theory that was not refuted until the 19th century by LouisPasteur, and that we were again confronted with in nematology inthe 1700s.

In his historical parallelisms Chitwood considered that after theancient times there followed the many hundred years of the “darkages” 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 onhuman medicine. In his classic “Canon Medicinae” Avicenna refersto parasitic worms, notably ascarids and dracunculids. The “dark”and superstitious medieval period was followed in the eighteenthcentury by the Age of Enlightenment in science, literature, cultureetc. In that enlightened period are some prominant figures that we,perhaps somewhat generously may enrol, in the cadre of nematolo-gist, most notably Linnaeus and Needham.

…to the age of enlightenment

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

18 OUR EARLY STARS

van Leeuwenhoek, who later in his career as a naturalist (died 1723at 91 years old), started to build microscopes. Science began toflourish, and nematologists now had the means to study also thelesser creatures in soil and plants. Probably the first person ever tostudy nematodes under a microscope was Borellus, who in 1656published the first information on a free-living nematode, viz. the‘vinegar eelworm’ (Turbatrix aceti). I personally, remember how thelocal vinegar producer helped me each year by providing thesenematodes 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 para-sitic nematode, the wheat gall nematode, Anguina tritici, with theline, “Sowed cockle, reap’d no corn”. However, those of us nematolo-gists who are more or less lenient with the possible nematologicalconnection in this statement, may surrender to the fact that thesecockles might have contained the “longitudinal fibres” that an Englishclergyman discovered some 150 years later. Hence, it was the poetwho unwittingly was the first to point at a plant parasitic nematode,later named Anguina tritici, causing the wheat “cockles”. It was as aclergyman that Turbevill Needham in 1743 described, before a proba-bly startled audience of the Royal Society of London, what he sawwhen he examined shrunken and blackened wheat kernels under themicroscope: “I dropped water upon it…when to my great surprisethese imaginary fibres…took life, moved irregularly…”. It is not sur-prising, therefore, that this report contributed to the prevailing theo-ries on spontaneous generation (generatio spontanea).

An “all category star” whose 300th anniversary we celebrated in2007 and who ought to be mentioned in this context is the Swedishpolymath, botanist, physician and natural scientist, Carl Linnaeus(1707–1778), who was raised to the nobility with the name Carl vonLinné. In his ‘Systema Naturae’ (1758) he pioneered a practical classi-fication of organisms by his binomial system with a genus and adescriptive species attribute – “Deus creavit, Linnaeus disposuit”(God created, Linnaeus ordered), as he so proudly declared.Linnaeus named several animal and human parasitic nematodespecies, including Ascaris lumbricoides and Dracunculus medinensis,which he recorded under the group Vermes (“worms”). He wasfirst to give a scientific name to a free-living nematode, the vinegareelworm, which he named Chaos redivivus (“the resurrectedchaos”), with the comment: “…reviviscit ex aqua per annos exsicca-

OUR EARLY STARS 19

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, alsoknew that the wheat seed gall nematode was, at that time, knownas Vibrio tritici.

Modernists in Europe

In his attempts to identify historical epochs Chitwood chose 1870 asthe beginning of recent nematology, “because following this timethere was a marked uptrend in the amount and average quality ofthe 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 nema-tology through his famous “100-new-species-of-free-living-nema-todes” paper, and Thorne (1961) appears to consider these publica-tions as the beginning of the science of nematology. Raski (1959)defines the period of “early modern nematology” as the years1845–1907, beginning with the great work of F. Dujardin, “one ofthe major taxonomic works in early nematology”. With theupswing in nematology after the Second World War the writerdares to suggest that the period of “contemporary nematology” per-haps began with the introduction, in 1943/44, of the soil fumigantsDD 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. Theywere primarily concerned with nematode taxonomy and the differ-entiation of forms living in fresh-water, soil and marine habitats.Plant-parasitic nematodes came suddenly and dramatically into thepublic eye when H. Schacht (1859) identified the beet cyst nema-tode (Heterodera schachtii) as a threat to the beet sugar industry inEurope. Investigations into the control of this nematode dominatedthe literature of much of European nematology during this period,with researchers such as Schmidt, Strubell, Liebscher and Kühn. In1829, root galling, caused by Subanguina radicicola, was reportedfrom Norway, where the entomologist W.M. Schoeyen (1885) illus-trated and described it as Tylenchus hordei, without knowing that ithad already been described by Greeff in Germany. Another signifi-cant event during these years was the discovery of the root-knot

20 OUR EARLY STARS

nematode, described as “Vibrios” by M.J. Berkeley in 1855, andnamed, Meloidogyne, by E.A. Goeldi in 1892. The increased interestin plant parasitic nematodes was excellently summarized by KatiMarcinowski (1909) in her book “Parasitisch und semiparasitisch anPflanzen lebende Nematoden” (Parasitic and semiparasitic nema-todes living on plants).

Let us linger on three particular figures in Europe that stick outas “stars” during this time.

Henry Charlton Bastian (1837–1915) has a reputation as an out-standing taxonomist (see above) and was prominent in the classifica-tion of the Nematoda. He contrasted the animal parasitic nematodeswith the free-living, and subdivided the latter into continental (soiland fresh water nematodes) and marine forms. Johann Adam OttoBütschli (1848–1920) was not only a nematologist, in the modernsense of the word, but also a pioneering histologist and professor inHeidelberg, Germany. His embedding methods for thin tissue sec-tions paved the way for all biological research, and proved thatnematodes could be useful study objects in embryology and genetics.With his excellent, detailed drawings Bütschli set the standard fornematode illustration. Many morphological details that he discov-ered are still used in nematode taxonomy. Johannes Govertus deMan (1850–1930), the “Altmeister der Nematodenkunde” (The oldmaster of nematode science) (Micoletzky, 1925) and “one of the mostinteresting personalities ever engaged in nematology” (Thorne), com-pletes this remarkable triumvirate of founders of the science ofnematology. De Man’s first nematological paper (1876), dealing withsoil nematodes, was the beginning of nematology in The Netherlandsand Belgium (Coomans). Financially independent, and an all-roundzoologist, he felt free to pursue any problem and did much of hislater research in private life. His devotion to nematodes resulted innearly 50 papers among a total of about 170 scientific publications.Andrassy (1976) grades him as “the first modern nematologist” andrecommends his beautifully illustrated monograph (de Man, 1884) onnematodes in soil and fresh water as “the bible of nematologists”.

The versatile German plant pathologist Julius Kühn was a lead-ing figure in the campaign against the beet cyst nematode. Kühn(1871) probably was first to use soil fumigation against nematodeswhen he applied carbon disulfide to nematode infested beet fields.He also tried trap crops, although with limited success, and croprotation, which became the principle control method in the sugar

OUR EARLY STARS 21

beet industry. Kühn has his “star” status also for his discovery ofDitylenchus dipsaci, found in teasel (Dipsacus fullonum), and thecausal agent of “Stockkrankheit” in rye and several other crop plants.

Another celebrity is described as follows: “…somewhat old-fashioned in appearance with his round-lensed glasses…full of vitali-ty…for a scientist he was unusually smartly dressed (!) …travellingto and from work on his high stepping bicycle with its large frontbasket…and upon his arrival sustained himself with a piece ofcheese!”(Hooper, 1994, Ann. Rev. Phytopathol.). “He” is the “Fatherof Nematology” in Britain, Tom Goodey (1885–1953). In his earlyscientific career Tom Goodey interested himself in soil protozoa. Inhis forties he started as a parasitologist in St. Albans, and from 1926he specialized in free-living and plant-parasitic nematodes. In 1947,he was transferred to Rothamsted as head of the new NematologyDepartment where he stayed until a year before his death. His majorresearch contribution was on the biology of the stem nematode, buthe published also on nematodes that cause galls on plants. TomGoodey was a real ambassador for nematology and instigated train-ing courses in nematology, the second one of which, in Harpendenin 1951, was followed by an international symposium. This markedthe beginning of regular, international nematology symposia inEurope, eventually under the auspices of the European Society ofNematologists. His famous textbook, “Plant Parasitic Nematodesand the Diseases they Cause” (1933) was a landmark in the develop-ment of the science, and a couple of years before his sudden deathhe completed his well-known book, “Soil and FreshwaterNematodes” (1951), which was revised and enlarged in 1963 by hisson, J. Basil Goodey. Hooper points at Tom Goodey as a personwith many interests, most notably as a singer and actor. His theatreand concert hall performances were so professional and successfulthat he adopted a stage name, Roger Clayson, so as not to conflictwith his profession as a scientist! Imagine our symposia banquetswith Tom Goodey performing!

A nematological troika

In the Russian nematological starry sky I see three fixed stars thatrepresent very different personages with a profound influence onmodern Russian nematology and with worldwide impact. Ivan

22 OUR EARLY STARS

Nikolaevich Filipjev created a new scientific school in Russia –nematology, and is looked upon as the founder of Russian nematol-ogy. He was born in 1889 in St. Petersburg and died in 1940. Hecame from a wealthy family and had a good education. As a student,he was advised to study free-living, marine nematodes, and had theopportunity to make a short visit to Naples, Italy. At the age of 21he published his first nematological paper, which was about thenervous system of nematodes. During the years 1923–1933 Filipjevhad a position in Petrograd (St. Petersburg) where he lectured onentomology and evolutionary theory. He even wrote on evolution-ary aspects of modern genetics, the first of its kind in Russia.

Filipjev’s entomological interests took him to the USA where, in1928, he presented a paper on the general classification of nematodeswhich was of fundamental importance for nematode taxonomicalresearch. Just as important was his book on systematics, “Nematodesthat are of importance for agriculture”, published 1934 in Russian.A year later he asked the Dutch parasitologist, S.J.H. SchuurmansStekhoven Jr., to cooperate in editing a second edition of his book,in English. An intimate cooperation between the two followed, andresulted in the co-authored classic, “A Manual of AgriculturalHelminthology”, published in 1941 (after Filipjev’s death). The book– with the motto “Concordia parvae res crescent” (“Collaborationfosters small undertakings”) – was “dedicated to the memory of thepioneers in the field of agricultural helminthology, Cobb, Filipjev,de Man, Micoletsky and Ritzema Bos”. Filipjev’s epoch-making con-tribution to systematics is his classification of the class Nematoda, inwhich he grouped free-living and parasitic families into a total ofeleven orders.

At the same time as his international reputation increased, beingelected a member of scientific societies in various countries, Filipjevexperienced increasing hardships in his own country. In 1931 hewas arrested and charged with counter-revolutionary, subversiveactivity. The charges, however, were dropped, but in 1933 he wasagain arrested and exiled to Alma-Ata, Kazakhstan, from where hewas prohibited to leave. He became persona non grata, but wasallowed to continue his research. It was during these years that hewrote all his major works on the Nematoda and prepared his funda-mental revision of nematode taxonomy. In 1937, Filipjev was arrest-ed a third time and charged as an “enemy of the Soviet people”. Hiscorrespondence with colleagues abroad continued until the end of

OUR EARLY STARS 23

1937 when “the sendings suddenly stopped” (SchuurmansStekhoven). He was taken to court and sentenced in March, 1938.Though the exact particulars remain obscure, Filipjev’s death is offi-cially reported as being on the 22nd October, 1940. According to hisbiographers, S.Ya. Tsalolikhin et al. (Russian J. Nematol. 8:173–9), afellow-prisoner was an eyewitness to Filipjev’s execution, but offi-cial documentation is lacking. His good name and scientific recogni-tion were officially restored in 1956. His two sons also perished inthe 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 pur-poseful 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 againsthim. He appears as being of an independent nature, kind to hisfriends and colleagues but uncompromising and even sarcasticwith scientific opponents. Deeply committed to his science hesometimes appeared lost in his thoughts and was absentminded –found to buy tickets twice for the same train and tried to get hiswatch repaired when he had forgotten to wind it. (How many ofyou, fellow nematologists, have not been frenetically searching foryour specs until your colleague tells you that there is somethingon your nose…!)

While I.N. Filipjev was held in high esteem internationally as ataxonomist, Alexander Alexandrovich Paramonov (1892–1970)became renowned for his evolutionary concepts and hypotheses onnemic relationships and nematode evolution. He had wide biologi-cal interests, which ranged from studies of fur-bearing animals tomammalian skulls and entomology to general biology. From 1925onwards, his research involved also free-living, soil and phytopara-sitic nematodes. His main interest was evolutionary theory and themajor principles of phylogeny. After 1945, he concentrated his stud-ies on plant nematodes in the broad sense, and also on saprozoicforms. He drew up an ecological classification of soil and plantnematodes, considering their (biocoenotic) interrelationships withfungi, bacteria and other organisms. His particular interest was theevolution of parasitic adaptations of plant nematodes. Paramonov,who was known for his almost encyclopaedic knowledge, held vari-ous positions at museums and academies in Moscow and Leningrad,where he lectured on zoology, entomology, general biology and

24 OUR EARLY STARS

Darwinism. Among his 130 publications on various biological sub-jects, about 50 deal with nematodes. Paramonov summarized hisresearch 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 chargedwith active participation in the revolutionary student movement.He was expelled from the university but continued his studies inHeidelberg in Germany, where he was taught nematology byProfessor Otto Bütschli. The frosty connections between Russia andGermany forced Paramonov to return to Russia where he in spite ofpersonal restrictions, managed to finish his undergraduate studies in1922. During the following years he held academic positions inMoscow. In 1948, he was dismissedfrom the Academy because of his criti-cism of the biological concepts ofLysenko. He was restored to favour in1952, and devoted his time until hisdeath to develop phytohelminthology asan independent scientific discipline inRussia.

Although Paramonov deprecatedpolitical and scientific trends of opinionthat appeared iniquitous or totallywrong he treated those that he consid-ered to be offenders with indulgence,and was an attentive listener in discus-sions. Eino Krall, Estonia (Fig. 1), whomet Paramonov and even visited hishome in Moscow and experienced theofficialdom of the 1950s, found him to be a very nice and humani-tarian person, who treated his students and colleagues with respectand amiability. “A brilliant lecturer and teacher he was always sur-rounded by young people to whom he was a sincere friend”, to citehis biographer.

If Filipjev and Paramonov were the prominent founders ofRussian 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 Sovietnematology. Dr. Kirjanova was born in Alma Ata and graduated in

OUR EARLY STARS 25

Fig. 1. Eino Krall (right), Estonia, fraternizing with Vicente Campos,Brasil.

1928 at the Middle AsianUniversity in Tashkent. Shewas awarded a post-graduatestudentship and moved, in1930, to Leningrad and theZoological Institute of theUSSR Academy of Science,where she worked until herdeath. There she worked withI.N. Filipjev for a couple ofyears. She even cooperatedwith Filipjev and SchuurmansStekhoven in their co-authoredbook.

Ekaterina Sergeyena (Fig. 2), as she was commonlycalled, was concerned mainlywith the systematics and ecol-ogy of free-living and plantparasitic nematodes but inter-ested herself also in insect parasitic nematodes and hair-worms. A life-time achieve-

ment of hers was to put together, as completely as possible, a nema-tode collection for the USSR. Nematologists visiting her wereexpectantly 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 nematodesamples, together with 3,000 hairworm samples. Throughout, sheworked untiringly on the identification to species level of her sam-ples, with detailed recording of various aspects pertaining to theirtaxonomy and to the damage they caused. A colleague commentedon her sedulous work that “not even a bomb-hit could remove herfrom 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 inTajikistan….!

26 OUR EARLY STARS

Fig. 2. Ekaterina Sergeevna Kirjanova chatting upBen Chitwood at the Warsaw ESN Symposiumin 1967.

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 inhistorical overviews to characterize one of Nematology’s most dis-tinguished individuals, Nathan Augustus Cobb (1859–1932) – “TheFather of Nematology”, not only in the United States but world-wide. He introduced the term “nema”, though “nematode” hasbeen the commonly used word , and also the name “nematology”(first in 1914) as a new branch of study in science and distinct fromhelminthology.

Cobb’s name is connected with various methods (Cobb decanti-ng and sieving) and techniques (Cobb aluminium slide). For manyof us he is also well-known for his magnum opus, “Contributions toa Science of Nematology”, a monumental compilation of his greatestworks in nematology through the years 1914–1935. In this contextwe must not forget the name W.E. Chambers, who illustrated thepapers with unsurpassed, beautifully detailed drawings, “under theauthor’s personal supervision”(!), as Cobb self-confidently(?) pointedout in his paper “The Mononchs”.

Was he adventurous – or maybe, rather restlessly always on themove? At least that is the impression one gets when reading aboutCobb travelling all over the world, Europe (to complete his doctor-ate in Jena, Germany), America, Asia, Australasia, Oceania, finally in1915 to settle down in North America, where he became recognizedas the founder of nematology in the United States. His family, withsix children, followed him, probably suffering some hardships, find-ing dad at times flogging soap and watches to survive.

Certainly, he was a versatile and talented man! Preceding hislast 15 or so years as a full-time nematologist he held positions as aplant pathologist and studied fungal and bacterial diseases of plants.He even developed an interest in the breeding and cultivation ofwheat, selection of wheat varieties and the handling of grain in com-merce, the cultivation of sugarcane and won approval for his stan-dardization of cotton grading! He was influential, as exemplified bythe legacy he left, the Division of Nematology (in the United StatesDepartment of Agriculture), which was officially established in1929, and ensured the continuation of his work. In the group ofdedicated workers who belonged to the staff or were influenced byCobb we find many of the names of contemporary nematologists,

OUR EARLY STARS 27

such as G. Steiner, B.G. Chitwood, G.Thorne, J.R. Christie, and A.L. Taylor, all ofwhom rose to stardom.

If N.A. Cobb, as the first full-timenematologist in the United States, madethe most of his talents, Benjamin G.Chitwood (1907–1972), supervised by Cobbin the late 1920s, is alternatively referred toas “the greatest systematist of Nematoda”,“likely the most outstanding nematologistof all time” and “one of the early architectsof the Science of Nematology”, whosecareer had a profound influence on nema-tology. As a professional, after he receivedhis Ph.D. degree at the age of 24, B.G.Chitwood held positions as nematologistand zoologist in the U.S. Department ofAgriculture, and later held short term posi-tions as university professor, chief nematol-ogist in Florida, consultant in the Kaiser

Foundation Research Institute and various short term assignmentsuntil he retired, in 1964. Difficult to get on with and apparently apoor mixer, occasionally involved in controversy, his personal lifewas marred by adversity. In his later years he abandoned nematol-ogy and took up other interests. We know him best for his numer-ous contributions to zoology, parasitology and nematology, and forhis tremendous classic “An Introduction to Nematology”, co-authoredwith his wife, M. B. Chitwood.

Apparently, students and collaborators found Ben Chitwood notonly a brilliant scientist, but also exigent as a teacher and often acomplex character – would that be a correct definition of a genius?But, “Ben was also willing to put himself out for his students andgive unlimited amounts of his time to them”, as Father R.W. Timmobserved in a personally kept obituary. Father Timm has given ussome vivid, on-the-spot accounts of what it was like to be withthis remarkable man, who excelled not only at theorizing but wasalso eminently practical. Students were taught how to collect“spaghetti worms” in the slaughter-house and from bear faeces inthe 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 OUR EARLY STARS

Fig. 3. Herman Nilsson-Ehle,Swedish geneticist, renownedfor his pioneer breeding workon cyst nematode resistance inthe early 20th century.

parasitologist” (it reminds me of the smell from a dissected seal in asmall lab in St. Albans during my training years). Students beingexamined were urged to gather and classify all nematodes to befound in a snake from a road kill, from cow manure or retrievedfrom plant tissues. If you have available the SON NematologyNewsletter of March, 1973 I recommend that you read FatherTimm’s most amusing anecdotes! I met or rather saw Dr.Chitwoodonce at a symposium in the late 1960s – and, sadly, that was a per-son, as I remember him, sitting alone on a sofa, smoking cigarettes,going downhill….

Cobb, as the prominent “Father of Nematology”, surroundedhimself with several prospective nematologists who, as time wentby, became the founders and architects of the science in its modernshape. One of them was Gerald, “Jerry”, Thorne (1890–1975), whoseimpact on nematology developed after the Second World War.Influenced by Cobb and making acquaintance with the sugar beetcyst nematode in 1917, he joined the Nematology Section of theBureau of Plant Industry of the U.S. Department of Agriculture.During his 38 years in this organization his official headquarters wasthe Regional USDA Laboratory at Salt Lake City, Utah, his nativestate. After his mandatory retirement, in 1956, he was an indefatiga-ble professor and lecturer at several universities, and a consultantand expert, advising widely in North America and worldwide.What an enviable “otium cum dignitate” (leisure with dignity), tocite Cicero, or perhaps more appropriate in this case, to put it“otium est pulvinus diaboli” (“leisure is the devil’s cushion”)! Withunimpaired energy and dedication, Thorne continued working untila few weeks before he died. Again, the name of a nematologist isinevitably associated with a book, “Principles of Nematology”(1961), a classic that had a profound influence on nematology andnematologists in the latter half of the 20th century.

I met professor Thorne once, during an ESN symposium, andwas very surprised (flattered) when this world famous, nematologi-cal giant approached me, an embryo nematologist in the street, andasked 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 inUtah, in the 1950s.

Another “star”, old enough considering its origins long before allChinese and Egyptian notations, but probably not as brilliant as the

OUR EARLY STARS 29

nematological heroes we have remembered thus far, would be ourco-operative friend, Caenorhabditis elegans. “Rundmask fårNobelpris” (“Roundworm receives Nobel Prize”) was the announce-ment in empathic letters in Swedish newspapers in the autumn of2002. Handsomely, our gracile study object gave its collaborators,Sydney Brenner, Robert Horvitz and John E. Sulston, quite a bit ofthe cash flow we experience each year when the Prize is bestowedon those who (hopefully) deserve it. With its 959 cells and some-what 20,000 genes, C. elegans – oh yes, kindly assisted by the threelaureates(!) – has helped us to sniff at the secrets behind plagueslike cancer, AIDS and Alzheimer’s. Caenorhabditis, as benefactor ofmankind, is no doubt a unique star in the nematological sky. By1998, C. elegans had already placed itself at our disposal and hadbecome “a landmark in biology: determination of the essentiallycomplete 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 myquestion from the introductory lines. Who was most important: the“collector” who surveyed and described the manifoldness of nema-tode species and tried to open our eyes to their biology, the ingen-ious one who invented and constructed apparatus, techniques andequipment for laboratory work, or the brave and imaginative enthu-siast who entered the untrodden paths, launching and defendingnew ideas and theories (Fig. 3). I think it was Einstein who main-tained that “fantasy is more important than knowledge” – but isn’tknowledge, acquired by the assiduous microscopist and observer,the necessary sound and solid platform on which to gain the fruitfulresults of our imaginations! Most important, though, are the vitaldiscussions where concepts and ideas are brought face to face, with-out which there would be no progress in science. Some of us havevivid memories from symposia sessions of such “fights” betweenstars and giants of all ages and extraction.

Among all the giants we must also remember the patient labo-ratory assistants and technicians who with skill and care extractedthe soil samples, prepared the solutions and mounted the nematodesto be studied by the star. Remember Chambers who helped Cobb

30 OUR EARLY STARS

with the incredible,artistic drawings thathave stimulated teachersand students for almosta century to try to gaina closer aquaintancewith the “nems”. Howmany of us have notstared, at the full, enface view of the grislyMononch head “aboutto seize its victim”,sending thrills of fasci-nation along the back-bone of students?

A tribute also tothose, not mentionedabove, who welcomed“nematologists inembryo” to their insti-tutes and laboratoriesto be trained and to gainfirst hand experiencefrom the master. Thewriter remembers vividlythe friendly and helpful atmosphere at Rothamsted in the early1960s, with Fred Jones and all his colleagues chatting at the morningcoffee-breaks in the cosy little lab prep room, with Basil Goodeywho devoted much of his time to teach us beginners nematode tax-onomy based on his newly issued book, with David Hooper sokindly teaching us the “Seinhorst two-flask method”, and makinglife-long bonds of friendship between staff and more or less occa-sional visitors and researchers. Some of the giants from ages ago liveon with their names tied to techniques and methods. Wim Seinhorst(Fig.4) is one of them. We know too about de Man indices meas-ures, the Demanian system named by Cobb in tribute to de Man,Cobb’s slide and Peter’s 1 ml nematode counting slide, the Fenwickcan, Seinhorst’s and Oostenbrink’s elutriators, just to mention a few.And during a two months’ stay and training in Wageningen, TheNetherlands, I found my humble self accommodated in a room on

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Fig. 4. J.W. Seinhorst receives the insignia (hat, ring & diploma) as Doctor Honoris Causa at theSwedish University of Agricultural Sciences, 1983.

Ritzema Bos weg… – I found myself as at home with myDitylenchus and Aphelenchoides pets. Many others, besides myself,have similar precious memories and experiences from the labs atRothamsted, Wageningen, Gent and elsewhere.

It has been my privilege to select and pause at some of the “earlystars”, and I think you will agree, dear reader, that the choices I madewere not too controversial. I would not be surprised, however, if onsimilar reflection, you miss one or two or several names that certainlyreached the status of grandeur and have left behind a lot of anecdotes(Fig. 5). Therefore, stop a minute in your hustle and bustle, andremember what they meant to you; I know they deserve it!

In preparing this article I acknowledge the help I had from his-torical reviews as indicated in the text, obituaries etc, as well asfrom stimulating talks with Eino Krall.

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Fig. 5. Professor Sigurd Andersen (in shirt-sleeves in the middle) demonstrating his cerealcyst 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.

2.NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

JOHN M. WEBSTER

Department of Biological SciencesSimon Fraser University, Burnaby, Vancouver, Canada

&SEYMOUR D. VAN GUNDY

Department of Nematology, University of California, Riverside, USA

Research into plant parasitic nematodes evolved steadily throughoutthe first part of the twentieth century and then entered a rapid phaseof expansion in mid-century as clusters of nematologists with signifi-cant technical and financial support became established in researchcenters in many parts of the world. The collaboration and productivityof researchers in these emerging and expanding nematological researchcenters tended to overshadow the excellent research done by scattered,lone research nematologists and their teams. This may have beeninevitable as the thrust of the collective expansion focused onincreased food production rather than on curiosity driven research. Thetrigger for this expansion was, undoubtedly, a combination of the dis-covery, in the 1940s, of chemical nematicides and the increased aware-ness by both agricultural and government agencies of significant nema-tode-induced loss of crop yield during a period of projected, world-wide, food shortage and major replant problems in perennial crops.

This period of expansion of nematological research led, concur-rently, to a wider recognition in plant pathology and agriculture ofthe economic importance of plant parasitic nematodes. Nematology,as it became commonly known, expanded to include the study ofinsect parasitic and free-living, soil nematodes because of their closeassociation with agriculture. Inevitably, through association andextensive networking and exchange of ideas, nematology came toinclude also free-living nematodes of freshwater and marine habitatsas well as nematode pests of forest trees.

NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA 33

The “enhanced food production” mandate worldwide in the1950s and 1960s triggered different approaches to solving nematodeproblems in different countries due to socio-political and culturaldifferences, variation in climatic and soil conditions in the agricul-tural areas and to the contemporary level of nematological expertisein different jurisdictions. Europe and the USA already had a historyof nematological research, although its organization and manage-ment had developed along different lines.

Europe, due to its longer history in zoological research, had itsnematological foundations scattered in the laboratories of eminent,lone, research zoologists in established institutions. There was rarelymore than one or two per country, they were usually located in uni-versities and their research was eclectic and individualistic. Thechange in momentum and the expansion in nematology to one witha distinct agricultural focus resulted in the establishment in severalEuropean countries of large centers of nematological research eachwith a distinct utilitarian focus.

In the USA, nematologists had for many years been located inthe laboratories of the US Department of Agriculture (USDA) in itsnetwork of regional centers and through the Land Grant Universitysystem of Agricultural Experimental Stations. It was within theUSDA research framework that, early in the twentieth century, N.A. Cobb not only established a strong and influential nematologi-cal research reputation, but trained what was to become the nextgeneration of nematological researchers and teachers. These well-trained researchers either remained in the USDA or relocatedto some of the major universities across the country, where theywere well-positioned to lead the development of major nematologi-cal research centers during the rapid growth phase of the 1950–1960s.This lends itself to a genealogical approach to the history of nema-tology in the U.S.A.

It is upon this array of different, evolving centers of nematologi-cal research in Europe and the United States that this chapter focus-es. It will help to identify the role and contributions of theseresearch centers, and of the associated nematological personalities, inthose formative years of change.

34 NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

Early research centers in Europe

As the ravages of the Second World War faded low agricultural pro-ductivity was manifest, in part due to several years of monocroppedland and to widespread crop diseases. The potato cyst nematode andbeet cyst nematode, together with some local, severe outbreaks ofstem eelworm and lesion nematode, were the major nematodepests. Unfortunately, and sadly, there were virtually no young,trained nematologists. There were, however, a few older, seniornematologists in countries across Europe who recognized that thecontrol of nematode pests of potatoes, sugar beet and fodder cropscould significantly help to improve Europe’s struggling agriculturalrenewal. They had the foresight to realize the necessity of movingaway from the traditional approach of “lone star” nematologists,working diligently but undervalued and in relative isolation, andmoving towards the concept of larger, well-funded, collaborativeresearch teams with international links. Significant initiatives weretaken to establish research units in nematology at some of the majorresearch centers in crop production and protection.

Within the space limitations of this chapter the emergence anddevelopment of some of these nematological units during this criti-cal period of change are addressed. Unfortunately, the particularfocus in time and structure of the chapter results in omitting refer-ence to many of Europe’s nematologists of the day, in particularthose that worked alone or in smaller centers.

In England, a new Department of Nematology was establishedin 1947 at one of Europe’s largest centers of agricultural research,Rothamsted Experimental Station. The already distinguished nema-tode taxonomist, Tom Goodey, was brought in as Head from anearby research outpost of the University of London, namely theInstitute of Agricultural Parasitology at St. Albans. He and his initialstaff of eight, including his son J.Basil Goodey, D.W. Fenwick, M.T.Franklin and B.G. Peters provided substantial taxonomic expertiseplus ecological and control interests. This department was to growinto one of the world’s most famous nematological research centreswith particular emphasis on cyst nematodes, their taxonomy, biolo-gy, population genetics and control.

The department flourished, and it attracted and spawned manydistinguished 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 35

founding members of the Department these early years includedC.C. Doncaster (nematode behaviour), K. Evans (potato cyst nema-tode), 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 (cystnematode hatching; ultrastructure ), D.L. Trudgill (Heteroderidae),H.R. Wallace (nematode ecology and locomotion), J.M. Webster(nematode host-parasite relationships), T.D. Williams (cereal nema-tode management), A.G. Whitehead (nematode control) and R.D.Winslow (cyst nematode populations).

This remarkable department prospered (reaching a complementof about 45 nematologists) (Fig.1) at a time when agricultural researchfocused on maximizing crop yields, on the search for new nemati-cides, on the development of resistant cultivars, and when new pestproblems ensured the allocation of increased resources for research.The increasing concerns worldwide, in the 1970s, over the use ofnematicides stimulated the search for alternative methods of nema-tode pest management and increased support of research into basicnematode biology and ecology. However, the early 1980s markedthe beginning of a marked decline in resources for nematologicalresearch. This was mainly the result in some countries, including theUK, of decreasing direct government support for agriculturalresearch in favour of the concept that agriculture should be treatedlike any other industry by financing its own research. This trend was

36 NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

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

compounded by the overproduction of European agriculture andthe 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 wasunsustainable, and some smaller research groups were disbanded.Rothamsted Research (as it became known), like several otherresearch centers, moved away from discipline-based structures andthe Nematology Department was closed in 1987. Currently, a smallerbut active group of nematologists (about 21 including visitingresearchers from around the world), led by B.R. Kerry, conduct theirnematological research within a program on rhizosphere biology,and so encompass nematode-host recognition processes, nematodeinteractions with the rhizosphere microbial community and nema-tode management. This multifaceted approach to plant nematodecontrol involves extensive collaboration with researchers in thepublic and private sectors in the UK (e.g., CABI Bioscences and theUniversity 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 MallingResearch Station, were focused on interactions between nematodesand other pathogens, in particular, the virus vectors Xiphinema andTrichodorus. Concurrent with these activities were those of C. Ellenby(The University, Newcastle-on-Tyne) on potato resistance to cyst ne-matodes, R. Cook (Welsh Plant Breeding Station) on resistance to stemeelworm 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 (CommonwealthInstitute 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 expandingresearch needs a graduate diploma programme was established in1958 at Imperial College, University of London. B.G. Peters headedthe programme, the first of its kind in Europe. Through fundingfrom Shell Research, the marketers of D-D, one of the most suc-cessful nematicides, a customized research and training facility wasbuilt to house the twelve-month diploma programme and theincreasing number of researchers. A prominent research group assist-ed Peters and N.G. M. Hague (cyst nematode control) to developresearch excellence in nematicides, especially soil fumigants, (F. Call,

NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA 37

O.F. Lubatti and A.B. Page). Later, with the evolving researchemphasis, N. Croll (behaviour), A.A.F. Evans (ecology and survival),W. Hominick (insect nematodes), and D.J. Wright (physiology andcontrol) joined the center.

Within the Scottish Horticultural Research Institute, subse-quently renamed the Scottish Crops Research Institute (SCRI), amajor nematological research group was established by C. E. Taylorin the Zoology Department. Its focus was on the nematode vectorsof viruses causing disease in raspberry and strawberry crops. He wassoon joined by P. Thomas (trichodorids) and W. Robertson (virusretention in longidorid and trichodorid vectors). At that time chemi-cal control of virus vectors was an important research endeavour.

In the 1970s, Taylor was appointed Director of the Institute, andD. Trudgill became Head of the Department, and was joined by T.Alphey, B. Boag and D. Brown. The mandate of the Departmentexpanded to include research on potato cyst nematodes (PCN)because Globodera pallida was increasingly recognized as a seriousthreat to UK potato production, replacing G. rostochiensis which bythen was beginning to be controlled by resistant potato cultivars. Inthe mid 1970s, the Scottish Plant Breeding Station (SPBS) inEdinburgh was closed and relocated at the SCRI, bringing J. Forrestand M. Philips to further develop PCN research while Brown ledresearch on nematode vectored potato viruses. Later, V. Blok wasappointed to help expand the application of molecular techniques,and J. Jones, B. Griffiths and R. Neilson to expand research on nem-atode-plant community interactions (Fig. 2). Subsequent internalreorganization and restructuring at SCRI, in the 1990s, resulted inclosure of the Zoology Department and the nematologists retired ormoved into several newly created departments.

In The Netherlands, the long tradition of Wageningen as a cen-tre for plant pathology and agricultural research provided the foun-dation for one of the world’s great centres of nematology. M.Oostenbrink and J.W. Seinhorst, “nematological giants” of the 1950sand 60s, began to build the nematological reputation of theAgricultural University of Wageningen and of the independentInstituut voor Plantenziektenkundig Onderzoek (I.P.O.), respective-ly. The research excellence of these two centers evolved over subse-quent 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 Nema-

38 NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

tological Section in 1956 but it did not become a Department ofNematology within the University until 1972. Within it P.A.A. Loofbecame a world authority in nematode taxonomy and T. Bongerssupervises a large, international collection of nematodes. The other“giant”, Seinhorst, focused his attention, initially with H. den Ouden,developing mathematical models of nematode populations toexpress their growth in relation to plant host growth. He retiredfrom Head of Department in 1983.

The intensity and emphasis of agricultural cropping in The

Netherlands resulted in large population increases of potato cystnematode and, on flowers and onions, of stem eelworm. This led tothe first Dutch, commercial advisory service with a focus on nema-todes in soil and on crops (P. Kleijburg). The research focus inWageningen on plant parasitic nematode populations and their con-trol continued until about 1980. Thereafter, changing methods ofnematode control and increasing environmental pressures led tochanging research emphases as can be seen in the major contribu-tions of J.J. s’Jacob, J. Kort, K. Kuiper, in the early days and, morerecently, of T. Bongers, H. Hoestra, P.W.Th. Maas, A. Mulder, J. vanBezooijen, L. den Nijs, T. Been, C. Schomaker, and F. Gommers atthe University. The application of molecular genetics for the identi-

NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA 39

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

fication of nematode species and of species within populationsbecame 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 attractedworldwide attention also through its international nematology train-ing programme, led by J. van Berkum. It operated very successfullyover several years within the Agricultural University of Wageningentogether with adjunct programmes in India and Venezuela.

Another major European center of nematology emerged at theUniversity of Gent, Belgium, under the leadership of L.A.P. de Coninck.This center developed into one of the world’s largest and most success-ful groups of nematode taxonomists. The laboratory had the good for-tune in its early years to have a series of astute leaders in L.A.P. deConinck, A. Coomans (dorylaimids, fresh water nematodes), and E.Geraert (plant parasitic tylenchids) who successfully built the nematodetaxonomy reputation and maintained it into the twenty-first century.The Department’s research activities, and those in collaboration withcolleagues in Brussels, extended beyond that of light-microscope studiesof the morphology and systematics of free-living and plant-parasitic

40 NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

Fig. 3. Research staff at the Wageningen laboratories with a visiting group. Numberingfrom 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.

nematodes and into the biochemistry, phylogeny, ecology and (recently)embryology (G. Borgonie) of nematodes. W. Decraemer (desmoscole-cids, trichodorids, general morphology), N. Smol (marine nematodes), P. De Ley (rhabditids, phylogeny), G. Gheysen (biotechnology), J. vanFleteren (biochemistry, molecular phylogenetic research) and M. Vinxand A. Vanreusel (marine nematodes, ecology) played leading roles inthe success of this department. They developed also a graduate degreeprogramme in nematology, and it continues to this day as the onlyremaining programme of its type in Europe. They were greatly helpedby cooperation with A. De Grisse (criconematids, electron microscopy)and M. Moens (applied nematology, biochemical identification), in theFaculty of Agriculture, and with J. Coosemans, D. de Waele and A.Elsen (applied pest management) at Leuven. Nematologists from otherEuropean countries have contributed to the success of this programmewhich has graduated almost 150 Masters (mostly from African andAsian countries) and almost 40 Ph.D.’s in nematology.

Although nematology in the Nordic countries dates back to theearliest record of a plant parasitic nematode, Subanguina radicicola inNorway in 1849 (studied by W.M. Schoeyen), the occurrence of cystnematodes became the economic driver of nematology in Denmarkand Sweden. In the 1930s, I. Wåhlstedt mapped the occurrence ofcereal cyst nematode (Heterodera avenae s.l.) in Sweden, and P.Bovien led research in plant and insect-associated nematodes inDenmark. The 1950’s expansion of nematology in the Nordic coun-tries 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) inDenmark, M. Stoen in Norway and O. Roivainen in Finland, all ofthem more or less specializing in plant breeding and plant protectionresearch. In 1962, they formed the Nordic Working Party which wasinstrumental in developing nematology in Scandinavia within themodern sense of the science. One of the activities emerging from thisgroup was the 1962 handbook “Nematoder på Växter” (Nematodeson Plants) which has been widely used in teaching and advising.

The prime mover in nematological research was Bingefors. Fromthe late 1950s, he also laid the basis for teaching nematology at theSwedish University of Agricultural Sciences, Uppsala. He was sup-ported by B. Eriksson and C. Magnusson in Uppsala (Ultuna), and S.Andersson and A. Banck in Alnarp (near Lund) who subsequentlyestablished specialized laboratories (Fig. 4). The Alnarp department

NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA 41

that originated in the former Plant Protection Institute became thelaboratory for routine testing of soil and plant material for nema-todes. Seinhorst claimed that Andersson had made this laboratory“one of the best equipped and fitted for its purpose in Europe”. Atthe Swedish Museum of Natural History, Stockholm, B. Sohleniusand S. Boström developed international recognition in nematodeecology and taxonomy, respectively, while at Lund University B. Nordbring-Hertz and H.B. Jansson focused on nematophagousfungi. In Norway, nematology was nurtured initially by M. Stoen, andlater by C. Magnusson who moved from Ultuna to the NorwegianInstitute 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 recentyears have restricted nematological teaching and research through-out 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 nema-tode; entomopathogenic nematodes) in Norway.

In southern Europe, M. Ritter established, within INRA, a major

42 NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

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 Marja-Leena Magnusson, Sven Bingefors, Anita Banck, Sven Boström, Sigrid Bingefors, VioletaInsunza, Stig Andersson, Riita Hyvönen, Lucyna Wasilewska (visiting), Björn Sohlenius.

nematological research center at Antibes to help solve agronomic prob-lems due to nematodes in France. At that time, there was a wide arrayof research interests, from plant parasitic nematodes to entomopatho-genic nematodes and from chemical nematicides to biocontrol strate-gies maintained by J.B. Berger, G. de Guiran, C. Laumond, K. Netscherand C. Scotto La Massese. During the 70s, A. Dalmasso introduced bio-chemical approaches to nematode taxonomy and by the end of the1980s the new molecular techniques were incorporated into the nema-tode research programme. As well, the research focus changed fromidentification of root-knot nematode subspecific groups to nematodeinteractions, and this group now has a staff of 20 including eight scien-tists. In 2003, the INRA center was moved from the Cap d’ Antibesarea 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 ofresearchers with strong molecular approaches to nematologicalresearch. It included P. Castagnone-Sereno, M.-N. Rosso, B. Favery,C. Djian-Caporalino and D. Esmenjaud (Fig. 5). Their research focuswas on understanding the molecular dialogue between root-knotnematode and the plant, and on the key steps leading to the devel-

NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA 43

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.

opment of the pathogen (compatible interaction) or its rejection bythe plant (incompatible interaction). Another focus was on thegenetic variability of root-knot nematodes and on the identificationof natural resistance genes in plants in order to develop durableresistance management of this nematode pest. The INRA researchgroup was the first to initiate a complete genome sequencing of aplant 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 asleading the ORSTOM nematology research centers in Senegal andCote d’Ivoire. He and G. Merny also were responsible for startingthe Revue de Nématologie for the publication of international articles on nematodes.

Since the early 1950s, much of the nematological research in theformer West Germany has been centered at the Institut fürHackfruchtbau, Biologische Bundesanstalt für Land- undForstwirtschaft (BBA), the present-day Institut für Nematologie undWirbeltierkunde, Münster. Initially it was under the directorship of H. Goffart, and successively since then under the leadership ofW. Steudel, B. Weischer and J. Müller. It has maintained a perma-nent 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 stillis) responsible for basic and applied nematology related to Germanagriculture, horticulture and forestry, with the main focus being oncyst, stem, foliar and virus-transmitting nematodes. Being the mainnematological center in West Germany, numerous diagnostic cours-es and more than 40 nematological workshops have been organizedthrough this federal German research institute since its foundation.

Two significant centers of nematology emerged later at theInstitut für Phytopathologie, Christian Albrechts Universität, Kieland at the Institut für Pflanzenkrankheiten, Universität Bonn whereU. Wyss and R. Sikora, respectively lead small, vibrant teams thattrained the next generation of nematologists.

In East Germany, important nematological research related toagriculture was centered at the Institut für Phytopathologie undPflanzenschutz, 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 fororganizing annual nematological meetings. At the Ernst-Moritz-

44 NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

Arndt-Universität in Greifswald, L. Kämpfe researched nematodephysiology and behaviour.

Germany, like other European countries, also has been fortunatein having many internationally recognized nematologists and theirresearch teams working at institutions throughout the country.Through the latter part of the twentieth century, nematologicalresearch followed a pattern somewhat similar to that elsewhere inEurope, namely a period of rapid growth followed by a period ofrationalization and contraction. At the turn of the century, a rela-tively wide, stable situation seemed to prevail in which there werefewer research nematologists, and those that remained were inte-grated into collaborating research groups rather than in identifiablenematological research centers.

Interestingly, some of the first nematological activities in Italy wereby N.A. Cobb! After completing his doctorate in Germany he spentsome time at the Zoological Research Station, Naples where he col-lected nematodes, mounted them in balsam (some of the first perma-nent slides of nematodes) and described his first genus, the marinenematode, Tricoma. Agricultural nematology in Italy began in the 1950sat the Entomology Station of the Ministry of Agriculture in the labora-tory of A. Marinari. Other research, undertaken at the Osservatorio perle Malattie delle Piante, Pescara, was oriented towards extension nema-tology under the guidance of A. Scognamiglio, and at the PlantPathology Institute, Bari University, on fumigant nematicides and nem-atode virus vectors in viticulture by A. Ciccarone. By the early 1960s,plant parasitic nematodes were recognized as being serious crop pestsin Italy and, in 1964, G. Martelli and F. Lamberti reported on the distri-bution of Xiphinema index in declining vineyards in Southern Italy.However, it was not until 1970, that the Istituto di NematologiaAgraria was established, in Bari, with Lamberti as Director. The mainfocus of the Institute was to carry out national surveys of the nema-tode fauna in cultivated and uncultivated areas and research on nema-tode taxonomy, biology and epidemiology. The Institute grew anddeveloped strong research collaborations with the private sector. In1973, the journal “Nematologia Mediterranea” was founded and, in1974, Lamberti linked up with C.E. Taylor and J.W. Seinhorst to organ-ize a NATO Workshop on Nematode Vectors of Plant Viruses. Thiswas the first of a series of outstanding workshops initiated byLamberti. G. Zacheo was one of the European pioneers who con-tributed to an understanding of the biochemical changes that occur fol-

NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA 45

lowing nematode infection, and as techniques changed, ultrastructure,cytochemistry and immunocytochemistry were used (T. Bleve-Zacheoand M.T. Melillo) to confirm the temporal, spatial localization andfunction of proteins and enzymes in nematode pathogenesis.

Nematological research started in Poland in the 1950s. At theInstitute of Plant Protection in Poznan, A. Wilski (who was suc-ceeded by S. Kornobis) focused on nematodes on crops, their biolo-gy and control and especially on mechanisms of resistance of thepotato to G. rostochiensis (J. Giebel). The Laboratory of AppliedEntomology of the Department of Ecology in the Polish Academy ofSciences (PAS) in Warsaw, was headed by H. Sandner, a specialist inentomopathogenic nematodes. In 1971, the Institute of Ecologyreplaced the Department of Ecology and was moved to DziekanowLesny, near Warsaw. In the 1960s, this research group (H. Sandner,J. Kozlowska, A. Fedorko. K. Domurat and L. Wasilewska) wasmainly faunistic, concentrating on nematodes parasitizing differentcrops, and over time, developing a strong ecological interest.

By the 1970s, A. Fedorko, S. Stanuszek and M. Kamionek hadextended their research activities to entomopathogenic nematodesand biological control. In 1975, Kamionek joined E. Pezowicz and A.Bednarek at the Warsaw Agricultural University under Sandner, toconcentrate on entomopathogenic nematodes.

The Institute of Ecology, comprising L. Wasilewska, J. Kozlowska, E. Dmowska, K. Domurat and S. Stanuszek, continuedas a major research center and, after 1990, K. Ilieva-Makulec,focused for many years on the abundance, diversity, structure andfunction of nematode communities in different habitats – natural orman-transformed. M. Brzeski, concentrated on nematode taxonomyand systematics at the Research Institute of Vegetable Crops,Skierniewice and, later, at the Museum and Institute of Zoology ofPAS. Research of A. Szczygiel and A. Zepp, at the Fruit ExperimentStation, Research Institute of Pomology and Floriculture Brzezna,was focused on nematodes of fruit crops, especially strawberries.

Unfortunately, these research groups split up as many personnelretired or left nematology. The nematological research interests ofPoland remain now in the hands of a few e.g., K. Ilieva-Makulec(nematodes in the soil food web) and M. Tomalak (pinewood nema-tode).

Spain, like other countries, had isolated nematological contributionsin the early part of the twentieth century and then, starting in the 1950s,

46 NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

E. Gadea, at the University of Barcelona, initiated a stronger nationalfocus on nematode research. However, it was F. Jimenez-Millan who, inthe 1960s, pioneered the creation of a nematological research centre atthe Instituto Español de Entomologia, Madrid, a center that later movedto the Instituto de Edafologia y Biologia Vegetal, and more recentlycalled the Centro de Ciencias Medioambientales. Whilst there, Jimenez-Millan trained a generation of very successful nematologists, namely A.Bello (criconematids, biocontrol and quarantine), M. Arias (longidoridsand trichodorids), M.D. Romero (cyst nematodes) and A. Gomez-Barcina (biology and control). Jimenez-Millan moved to the Universityof Granada in 1970 and, together with Gomez-Barcina, establishedanother generation of nematologists. Except for A. Ocaña (freshwaternematodes), they eventually radiated to other Andalusian institutions; D. Jimenez-Guirado (free-living dorylaimids and mononchids), R. Peña-Santiago (plant parasitic nematodes) and P. Castillo (plant nematode-mushroom interactions). Since then, the “academic tree of Spanish nema-tologists” has continued to assist growers facing nematological problemsand to research on aspects of nematode biology.

There are some exceptional examples of international collaborationin nematological research, often funded through the European Union,that involve plant parasitic nematodes (e.g., molecular basis of root-knotnematode interactions) and insect parasitic nematodes (entomopathogen-ic nematodes as biological control agents of insect pets) International col-laboration among nematological researchers has not been confined toEurope but has extended to, among others, Israel (e.g., Volcani Institute)and the USA and to key research centers in countries worldwide. Theextent of the research interaction between Europe and the USA hasincluded not only focused research projects and exchange studies butalso the relocation of several distinguished scientists e.g., R. Sikora toGermany from the USA and P. De Ley to the USA from Belgium. Theirinsight, energy and expertise has been infectious among fellow nematolo-gists, and their legacy, through the printed word and direct influence onothers, exemplifies the value of international flow of people and ideas innematology.

Genealogy of nematology in the USA, 1907–1980

In the United States the first organized nematology research programwas in the U.S. Department of Agriculture (USDA) Bureau of Plant

NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA 47

Industry, Washington D.C. led by the “Father of Nematology”, N.A.Cobb, who joined in 1907. It is Cobb’s professional genealogy at theUSDA that is the platform for the current day history and science ofnematology and the nematology research and training centers in theU.S.A. as well the theme of this section of the chapter. Cobb devel-oped a nucleus of scientists who became the architects of nematol-ogy in the U.S.A. (Genealogy Chart 1, Fig. 7) and to whom mostnematologists for the next seven decades could trace their profes-sional 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 them-selves in plant nematology. Many of his colleagues and studentsbecame the leaders and teachers of nematology during the 1930s,1940s and 1950s. By 1980 it becomes difficult to continue thisgenealogical approach to the history of nematology in the U.S.A.

Cobb’s 1889 PhD thesis focused on nematodes from whales andon some free-living nematodes at the University of Jena, Germanywhile studying with Haeckel, Hertwig, Lang and Stahl. His interestin free-living nematodes was derived from the publications ofBütschli and de Man. He became familiar also with marine nema-todes at the Naples Zoological Station. In 1889, he became a plantpathologist in the New South Wales Department of Agriculture,Australia. After joining the USDA in 1907, he published his firstnematology paper in the United States, in 1913, and became theundisputed leader of the USDA Nematology Division until hisdeath, in 1932. Early in his career he started the movement toremove the free-living and plant parasitic nematodes from the sci-ence of helminthology (Helminthological Society of WashingtonDC), and eventually established them in a separate field of“Nematology”. During his career Cobb served as President of theAmerican Society of Parasitology, Helminthological Society ofWashington DC, American Microscopical Society, and WashingtonAcademy of Sciences. He contributed major discoveries in nema-tode taxonomy, morphology, and methodology. His laboratory man-ual “Estimating the Nema Populations of the Soil” (1918) formed thebasis for many of the methods and apparatus used in nematologytoday. As an aside, he and Chambers, an artist and microscopist,were intensely interested in gadgets (Fig. 6) and they producedsome of the finest illustrations of nematodes that have ever beenmade. In administrative style, “his way” was the only way to do

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things and it may have been his quirks of personality that resulted inhis having only two employees who remained, at field stations out-side 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) andChitwood (1929) joined Cobb’s team at the USDA. After Cobb’sdeath, in 1932, Steiner became the director of the USDA Division ofNematology. He led the nematology crusade and hired and directedthe 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 nematologistseventually moved on to universities to provide formal training offuture nematologists. Some early students desiring to obtain trainingfrom Steiner, Chitwood and Christie did their research under thedirection of USDA scientists at the University of Maryland. Theirgenealogy reflects the “Eastern Branch of Nematology” (GenealogyChart 2, Fig. 12) which had a broad focus on taxonomy and mor-phology of plant parasitic and freshwater nematodes. The Thornegenealogy reflects the “Western Branch of Nematology” (GenealogyChart 3, Fig. 13) which focused primarily on taxonomy, morphologyand control of important plant parasitic nematodes. Thorne not hav-ing the artistic talent of Cobb relied on “scratchboard” for his nema-

NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA 49

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

Beginning in the 1950s some major academic training centersbegan to develop; Mai (see Fig. 8) at Cornell University, Sasser (seeFig. 17) at North Carolina State University, Christie and Tarjan atUniversity of Florida, Cairns at Auburn University, Courtney atOregon State University, Thorne at University of Wisconsin andAllen (see Fig. 10) at University of California. The first formal aca-demic nematology class in the USA was taught by Allen at theUniversity of California Berkeley, in 1948, and then by Chitwood, atthe Catholic University in Maryland, from 1949–1952. These earlynematology training centers were associated with Departments ofPlant Pathology, Entomology or Biology, and continue today asdepartments that offer PhDs in nematology. Detailed histories ofnematology along with some general international history of nema-tology with photographs of nematologists can be found at theUniversity of Florida websitehttp:flnem.ifas.ufl.edu/history/nem_history.htm

50 NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

Fig. 7. Geneology Chart 1. Early nematologists employed by the USDA from1907–1964. Dates in parenthesis are the periods they worked for USDA and thentheir moves to other employment.

NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA 51

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 websitehttp://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.

52 NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

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.

NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA 53

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

Genealogy Charts 2, 3, 4, have attempted to trace the origin ofnematology training, dates that individuals joined the training centers asfaculty members and dates that their students graduated and the institu-tions at which they were first employed, from 1922 to 1980. We apolo-gize for omissions or misrepresentations. Chart 2 continues from USDAChart 1, representing the genealogy of Steiner and Chitwood who con-

54 NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

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

tributed to the nematology training centers at the University ofMaryland, Auburn University, University of Florida, North CarolinaState University, Rutgers University, University of Illinois and TexasA&M University. Chart 3 represents the Thorne Genealogy that gaverise to the nematologists at Oregon State University, University ofArizona Field Station, Kansas State University and the University of

NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA 55

Fig. 14. Geneology Chart 4. A collection of independent nematology training centers not directly related to either the Eastern or Western branches of nematology.

California. Genealogy Chart 4 (Fig.14) represents the genealogy of mostof the independent nematologists at Cornell University, University ofNebraska, University of Iowa, University of Illinois, University ofKentucky, Purdue University and Louisiana State University.

During this early period of USA nematological development,the European Society of Nematologists was formed, in 1954-55, fol-lowed by the Society of Nematologists, in 1960-61, and theOrganization of Nematologists of Tropical America, in 1967. ManyUSA nematologists belonged to all three societies. The Society ofNematologists grew rapidly reaching a total of 680, in 1980.

Other forms of nematological training during the 1950s includ-ed a series of Shell Chemical Workshops throughout the USAaimed primarily at educating farmers on the importance of nema-todes. Federal funding supported the formation of RegionalNematology Research Groups (Northeast, Central, Southern andWestern) to encourage collaboration and communication between

nematologists in each region. For example in the Southern RegionalNematode Project (S-19) Sasser (see Fig.17) and Cairns formed andheld nematology workshops at North Carolina State University, in1954, and at Alabama Polytechnic Institute, in 1955, for professionalnematologists. In the Northeast Region, Mai held nematology work-shops at Cornell University in the late 50s. Sasser and Jenkins con-ducted a summer course in 1959. Since 1968, California has heldannual, statewide nematology workshops for faculty students,extension advisors, regulatory and industry nematologists to improvecommunication and to foster collaboration. These workshops andresearch groups helped promote the importance of nematodes andthe need of funding for research and new faculty positions.

Some of the other early driving forces that contributed to therapid growth of nematology in the USA were, 1) the discovery ofnematicides (D-D, EDB) in the 1940s that demonstrated to farmersthe crop losses caused by nematodes, 2) the designation of nematodessubject to quarantine and regulation: stem and bulb nematode in 1926,golden nematode in 1941, the burrowing nematode in 1954, 3) the dis-covery of crop losses due to ectoparasitic nematodes and 4) theimportance and role of nematodes in plant disease interactions. Thesedriving forces spawned a soil fumigation industry, federal and stateregulatory programs, and the attention of the agricultural industry to

56 NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

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

provide special funding for nematology teaching, research, and agricul-tural extension programs. For example, the discovery of the burrowingnematode in Florida and its potential threat to California’s citrusindustry stimulated the California agricultural industry and theCalifornia Department of Agriculture to propose legislation to recog-nize nematology as a separate and distinct science from entomology,plant pathology and parasitology. Funds were provided in the statelegislation to support the establishment of a separate StatewideDepartment of Plant Nematology at University of California Davis, in1954, under the direction of Raski. Within 5 years the department hadgrown to 12 faculty members on the Davis and Riverside campuses.The name was changed to the Department of Nematology in 1962 . In1965, Davis (headed by Raski) (see Fig. 17) and Riverside (headed bySher) (see Fig. 9) became separate departments of nematology andhave continued as such up to the present time. By 2000, the twodepartments had hosted 90 visiting scientists and postdoctoral scien-tists and trained 140 graduate students.

Another significant advancement of nematology in the USA wasthe 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 onroot-knot nematodes of economic food crops in developing nations. It

NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA 57

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.

attracted scientists from 60countries and brought with itcollaboration with many scien-tists in the USA. In the processof conducting the projectNorth Carolina State Universityhad the unique opportunity toassemble a vast amount ofinformational observations andresearch on Meloidogyne specieswhich led to important publi-cations.

Currently, applied nematol-ogy in the USA during the1990s and 2000s has fallen on a

period of reduced funding from state and federal resources, resultingin reductions in faculty positions, graduate students and support staffin the universities as well as other trained researchers working in thefield of nematology. There also has been a mass retirement of seniornematologists who started in the 1950s and 1960s and have not beenreplaced. Concurrent with these changes there has been a consolida-tion in chemical companies along with a reduction in the use ofnematicides and a search for new nematicides for the control ofnematodes in important agricultural crops. These reductions inapplied nematology are reflected in the December, 2005 SON mem-bership of 604, which was down from its peak membership of 680members, in 1980.

So even though the crop losses due to nematodes in agriculturecontinue to be as important or even more important than in the1950s, 60s, and 70s, the emphasis and breadth of nematologicalresearch has shifted from a strong, applied agricultural emphasis to astrong biological emphasis on systematics/evolution/morphology,ecology/biodiversity and classical and innovative genomics along withan administratively driven increased emphasis on teaching in thebiological sciences. This biological research shift in nematology washighlighted, in 2002, by the Nobel Prize in medicine being awardedfor research on the Caenorhabditis elegans model system suggestingthat basic nematode research is important, strong and growing.

58 NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

Fig. 17. Dewey Raski and Ben Lownsbery,University of California Davis and Joe Sasser,North Carolina State University at APS meetingsin Estes Park, 1954.

3.FIRST CATCH YOUR NEMATODE! – THE DEVELOPMENT OF METHODS FORRECOVERING NEMATODES FROM SOIL

DAVID MCNAMARA

Formerly: East Malling Research Station, Kent, UK andEuropean and Mediterranean Plant Protection Organization, Paris, France.

Introduction

It has often been said that there is no such thing as a fat plant nema-tologist. And this is strikingly obvious if you attend a nematologysymposium, where you will be surrounded by fit and athletic lookingpeople. The reason for this is obvious: unlike their colleagues inplant 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 obtainsamples of their organisms, nematologists must dig up quantities ofheavy soil, carry it back to the laboratory and then engage in com-plicated and difficult procedures involving lots of water, before theycan get a sight of their little creatures. This is all heavy work. (Ofcourse, the above description of life style does not apply to nema-tologists who have reached a higher position of authority and whohave a team of assistants to do the heavy work for them (Fig.1). Nordoes it apply to the group of younger molecular nematologists whowork only with tiny samples of macerates and extracts of nema-todes, presumably given to them by the slim, fit nematologists.)

Finding nematodes in soil

The earliest success at seeing soil nematodes was performed by directobservation of plant roots using magnifying glass or microscope. Thismethod was used in the second half of the 19th century to demon-strate the presence and the impact of cyst nematodes and root knot

FIRST CATCH YOUR NEMATODE! 59

nematodes. It could also beused to find free-livingectoparasitic nematodes butwas a very inefficient means ofdetermining their presence orpopulation densities.

In the early years of the20th century, two men provid-ed crucial insights into thephysical relationship of nema-todes with other soil compo-nents. This led to the develop-ment of a range of efficientextraction methods whichenabled the science of nema-tology to advance our under-standing of the quantitativeeffects of parasitic soil nema-todes on plant growth.

The first insight, on theextraction of nematodes, wasthe exploitation of the factthat nematodes are aquaticanimals and that they are(usually) mobile; this led theDutch physician, G.

Baermann, working in Java in 1917, to develop the method, nowknown as the Baermann funnel technique. By putting soil containedin a muslin bag into a funnel filled with water for several hours ordays, he discovered that nematodes (e.g. larvae of Ancylostoma)tended to migrate downwards out of the soil and through themuslin, and then could be seen in the water at the stem-end of thefunnel. Unfortunately, the water resulting from this method wasgenerally rather murky (because of the presence of small soil parti-cles that would leach out of the soil through the muslin, for exam-ple, colloidal clay) and the nematodes could only be seen with diffi-culty – but they could be seen, and counted, and transferred tomicroscope 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 FIRST CATCH YOUR NEMATODE!

Fig. 1. R.S. Pitcher (right) instructs David McNamara on the methods for capturing the world’s largest soil nematode, Ersatzonema gargantua.

narrow layer of soil needed to be traversed by the nematodes; thesieve is, preferably, not constructed of metal, in order to avoid therelease of toxic ions into the water. Furthermore, a flat dish is gen-erally used instead of the funnel so that the nematodes do not stayfor many hours in the narrow base of the funnel where the oxygensupply is limited (Whitehead and Hemming tray).

A major advantage of the Baermann funnel method is that it canalso 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 suspen-sion in water would sink more slowly than soil particles of a similarsize, based on the fact that the organic material of which the nema-todes are composed has a lower specific gravity than the rock whichconstitutes the inorganic fraction of the soil. By setting the soil sam-ple into suspension in water, waiting for a predetermined period oftime (i.e., a period just less than the time needed for biologicalmaterial to sink), and then passing the supernatant liquid through asieve of appropriate size, he was able to separate the nematodes,and other organic matter, from the smaller soil particles still in sus-pension. The pore size of the sieve was chosen so that the small soilparticles would pass through, whereas the nematodes would notpass and could be collected from the surface of the sieve. Sieves ofdifferent pore sizes can be used in order to selectively extractspecies of different body size.

Cobb’s decanting and sieving method produced a relatively cleansuspension of nematodes for examination under the microscope,provided that the original soil contained little organic material, oth-erwise it would be necessary to search through much distractingdebris in order to find the nematodes. This problem could be partlysolved for samples from richly-organic soils by combining themethod of Cobb with that of Baermann; a very clean sample couldbe obtained by putting the product of decanting and sieving onto asieve suspended in water, thereby allowing the nematodes them-selves to move away from the other organic debris. However, not allnematodes can, or will, move out of the organic debris, so thatmany nematodes (species or individuals) will fail to be detected.Good advice to anyone who wishes to become a successful fieldnematologist is to choose to do your research in an area where thesoil has little or no organic material!

FIRST CATCH YOUR NEMATODE! 61

These two methods, of Baermann and Cobb, contained theessential principles of nematode extraction from soils, on which thevast majority of later methods would be based. These principlesranked nematodes as being just one of many components of the soilwhich could be considered separately for the purposes of nematodeextraction, and which could be separated from each other by theirspeed of sedimentation, their size and, in the case of nematodes, bytheir motility

The methods that refined these principles and mechanized theextraction of nematodes were invented in the second half of the20th century. For example, so-called “elutriators” used an accuratelycontrolled, upward current of water in place of simple sedimenta-tion. The force produced by the upward current could be preciselyfixed depending on the diameter of the tube through which itflowed and the rate of water flow into the tube. The earliest exam-ple was the elutriator developed by Mike Oostenbrink, in 1954 inThe Netherlands. This was an upright metal funnel filled withwater, into which a fixed flow of water entered from below. Thesoil sample was washed into the top of the funnel. The heaviest soilparticles sank to the bottom of the funnel while lighter particleswere held in suspension at different levels of the funnel (dependingon the diameter at these levels). The upward flow of water was soestablished that nematodes would be carried up and over the top ofthe funnel to be concentrated onto a sieve. Even the cysts ofHeteroderidae species can be recovered by this method by increas-ing the water flow rate.

Oostenbrink’s compatriot, Wim Seinhorst, invented a simplersystem of two glass Erlenmayer flasks, connected to each other butwith one inverted above the other. Both flasks are filled with waterand the soil sample is contained in the upper flask. As the soil parti-cles fall from one flask into the other, an upward current of water isproduced that retains the nematodes in the upper flask. Seinhorstalso invented a more sophisticated elutriator, in 1956, which wascomposed of an upright glass column with several sections along itslength of different diameters, therefore allowing nematodes of dif-ferent sizes to be separated at different levels. After a defined periodof operation, the contents of the different sections could be drainedoff through side tubes, to be directly examined or to be further sep-arated by the Baermann funnel technique (depending on the organiccontent of the soil sample).

62 FIRST CATCH YOUR NEMATODE!

It was not surprising to other nematologists that Seinhorst andOostenbrink should be competing to produce the most effectiveextraction procedure. They were known to be deadly professionalrivals and, even though they started their university educationtogether and they worked in the same town in The Netherlands,Wageningen, throughout their careers, they certainly could never bedescribed as true good friends!

Other versions of the elutriator were subsequently invented. Forexample, the elutriator often called the “Trudgill Tower”, developedby David Trudgill and his colleagues in 1973, is a plastic cylinder,without any variation in diameter, in which different sizes and typesof nematodes can be extracted by varying the flow rate comingfrom the base of the cylinder. Byrd and his colleagues producedsemi-automatic elutriators, in 1972.

Although, cysts can be recovered with the elutriators discussedhere, they are very often extracted by means of specialized cyst extrac-tion techniques. The most commonly used apparatus is that develo-ped by Fenwick in 1940, the so-called “Fenwick Can”, which extractscysts most effectively from air-dried soil. The effect of the air-dryingis to cause the cysts to float to the surface where they overflow thecan and are collected on a sieve. The Schuiling centrifuge, developedin 1982, is a more sophisticated technique which is semi-automatedand can process soil samples very rapidly.

Centrifugal flotation is a technique, used in other areas of biolo-gy, which was adapted by Caveness and Jensen, in 1955, for theextraction of nematodes from soil. The principle of the method isthat nematodes will be held in suspension in a solution whose spe-cific gravity is greater than that of the nematodes, whereas soil par-ticles will sediment. In practice, solutions of sucrose, zinc sulphateor magnesium sulphate are used as the suspending solution. It isinteresting to note that, in the different publications concerning thistechnique, the specific gravity of the suspending solution (particu-larly sucrose) is given either as 1.15 or 1.18. This might suggest thatresearchers had calculated different values for the specific gravity ofthe different nematodes they were studying, but, in fact, it ratherindicates that nematologists are not very skilled as laboratorychemists: the original recommendation was that a solution of 484 gsucrose/litre should be used, which would produce a specific gravi-ty of 1.18, but some nematologists misunderstood this to mean 484 gadded to one litre of water!

FIRST CATCH YOUR NEMATODE! 63

Centrifugal flotation is considered by many to be a quick andefficient method of extraction, possibly more efficient than decanti-ng/sieving or elutriation, and it has the advantage that it can recovernon-motile stages, including eggs, but the disadvantage is that it onlyoperates on small soil samples. In addition, as with other methods, itis not suitable for soils rich in organic matter.

In the 1990s, efforts were made to use DNA probes to provideinformation on the presence of certain nematode species in soilsamples, but, so far, such probes can only be used to detect but notyet to quantify.

When examining the history of the development of extractionmethods, the first, and most striking thing, is the number of thesemethods that retain the names of their inventors when being dis-cussed (e.g. Fenwick Can, Seinhorst elutriator, Oostenbrink elutria-tor, Trudgill Tower, Baermann funnel etc.). The take-home messageis: “If you want to be remembered as a nematologist, invent anextraction technique”!

The second noteworthy observation is that, since the early yearsof the 20th century, the methods have become more sophisticated,more automated, less time-consuming and, presumably, more effi-cient. You would, therefore, think that different nematological labo-ratories would move towards using the same, and most efficient,methods. This does not seem to be the case; when you visit differ-ent laboratories, you notice that every one has a different, favouriteextraction method. Every nematologist tells you that the methodthey use is the one that works best for them. As my late Ph.Dsupervisor, R.S. (“Pitch”) Pitcher once said: “Extraction of nematodesis like sex: everyone does it and everyone is confident that they arequite good at it, but very few comparative tests have been carriedout”. In fact, a few comparative tests have been carried out (onextraction, not sex), usually when laboratories are collaborating on aparticular project and need to align their extraction techniques, sothey compare the extraction of nematodes from similar soil samples.The results of such tests, if ever published, usually reveal significantdifferences in the numbers of nematodes recovered. Clearly, morecomparative tests are needed.

64 FIRST CATCH YOUR NEMATODE!

Collection of field samples

As the methods to extract nematodes from soil became more effi-cient, it became obvious that the sampling methods for obtainingthe soil also should be improved. There is little point in employinglaborious methods to give an accurate picture of the types andnumbers of nematodes in soil samples, if the soil samples them-selves do not represent the total soil in the field.

The first efforts to try to get an understanding of the accuracyor otherwise of field populations were related to potato cyst nema-todes (Globodera spp.). In post-war Europe, many countries wereapplying legal measures to try to reduce the damaging effects ofthese nematodes (by, for example, prohibiting the growing of pota-toes on infested land) and it was, therefore, important to try to learnwhether they were present in fields and, if so, at what populationdensity. B.G. Peters and Freddy Jones, in the UK, were probably thefirst, in the early 1950s, to publish statistical analyses of the errorsinherent in taking samples to detect or estimate populations. Otherstatistically-minded nematologists followed. It was recognized that aPoisson distribution of nematodes could be assumed when trying todetect whether a field contained cysts and, therefore (and rathercounter-intuitively), the probability of finding an infestationdepended more on the quantity of soil in the sample than on thenumber of sub-samples. To try to determine the population density,on the other hand, it was shown that cyst distribution in the fieldconformed more closely to a negative binomial distribution. This isbecause nematode populations are generally patchily distributed, fora number of reasons (e.g., plant root distribution, spread due to agri-cultural activities, distribution of initial soil contamination etc.); inthis case the number of sub-sampling points becomes of moreimportance. These principles are still used to decide soil samplingstrategy for most types of nematodes for research purposes as wellas for statutory reasons.

Microscope examination of nematodes

Once they have been extracted from soil, it becomes necessary toexamine the nematodes, mainly for the purpose of correct identifi-cation. For this, nematologists have taken advantage of the progress

FIRST CATCH YOUR NEMATODE! 65

of development of microscope technology, not only different typesof electron microscopy but also the improvements in lightmicroscopy throughout the 20th century. Examples of developmentare the higher quality optics and light sources of high power micro-scopes, and such methodologies as phase contrast, interference con-trast, camera lucida and drawing tubes, microphotography andvideo, computer aided imaging, electronic measuring.

To take full advantage of microscope technology, it was neces-sary to ensure that the nematode specimens were in the best condi-tion for visualization. They first needed to be “fixed” to avoid decay,and Courtney, Polley and Miller, in 1955, developed a chemical mix-ture of formalin and triethanolamine (TAF) which did not distort ordiscolour the nematodes and which has become the most widelyused fixative. In 1949, Franklin and Goodey published a method formounting the specimens in lactophenol and glycerol which “cleared”the optical interference from the contents of the intestine, and alsoallowed for long-term storage of the specimens on microscope slides.Baker, in 1953, and Seinhorst, in 1959, produced alternative methodsfor mounting and clearing in glycerol. Such methods, with some latermodifications, continue to be used for preparing nematodes for bothdirect observation and for permanent slide collections.

With the arrival of molecular methods to identify nematodes, itwill be interesting to see whether molecular technology will com-pletely replace current slide preparation methods for nematodeidentification.

Conclusion

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

66 FIRST CATCH YOUR NEMATODE!

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 tocontrol Pratylenchus in his beds of Lilium auratum. I was more for-mally introduced to them in 1963 when Wallie Clark visited theUniversity of Canterbury from DSIR in Nelson. He gave us a fewnematode lectures and labs in invertebrate zoology. I was hooked.The next year I spent more time in botany labs looking at, andtreasuring, nematodes among algal filaments than at the algae them-selves. Marine zoology field trips yielded ironids from intertidalsands, and Wallie was on hand to classify them. The zoology depart-ment had a strong ecological base and a 1965 honours course in lim-nology (with a class of one) introduced an isotopic method of meas-uring processes. In 1964/65 and 1965/66 there was the opportunityfor summer work in the Antarctic, assessing the breeding success ofAdelie penguins; it was a change from working in the slaughterhouse. Although my supplementary efforts to collect juvenile stagesof nematodes from seals were unsuccessful I did manage to recoverPlectus and Eudorylaimus from clumps of moss; an ecosystem withlow nematode diversity. [Plectus murrayi continues to cause taxo-nomic discussion but “sinking” Antholaimus into Eudorylaimus isaccepted.]

February 1966 saw me starting my PhD studies with WallieClark in the Zoology Department at Massey University, NewZealand. In 1966, Wallie was striving to get nematology launched atMassey. A Leitz Ortholux arrived and Wallie suggested that JudyKillick, who had earlier emigrated from Rothamsted to work forhim in Nelson, come over at lunchtime and view it. At lunchtimeWallie was nowhere to be seen so I met Judy over an Ortholux;

THROUGH NEMATODE DIVERSITY TO LIVING SOIL PROCESSES – HOLISTIC STUDIES AID PROGRESS 67

nematology has since taken us to many places together. However, Ihave been converted to Zeiss optical systems.

Wallie Clark considered sand dunes to be a relatively simple systemwith a simple nematode fauna (some 40 years later, in 2006, thesewould be hypotheses to be tested, and rejected). I sampled a site atHimatangi Beach extensively. That over 50 species were recorded,including 41 proposed new species from a range of sites, was an earlylesson in the richness of nematodes and in our level of ignorance.Everything that moved in samples was counted, leading to a classicillustration of the relationship between Nygolaimus and their enchy-traeid prey [see: Pedobiologia 8: 173–207, 1968]. Later, similar countsshowed the relation between predacious tardigrades and their nema-tode prey in Hestehave, Denmark. In 1967, Wallie returned to theUniversity of Canterbury and I moved with him. The work on threebacterial-feeding species in culture on agar was completed, includingobservations on the facultative predation by Diplenteron (diplogasterid)and initial attempts at reviewing nematode feeding types. The thesiswas completed right on the two-year minimum.

In their wisdom, the New Zealand University GrantsCommittee provided a “no-strings” postdoctoral fellowship of £650that enabled me to work at Rothamsted where, in particular, F.G.W.Jones and David Hooper influenced my work. Judy worked in theBiochemistry Department, partly for John Clarke. Although FredJones “required” me to look at the possibility of Heterodera spp.interbreeding he was also emphatic that I should be allocated newplastic bags for sampling at Wicken Fen; many samples were stillbeing collected in cloth bags and there was a washing machine inthe header house dedicated to cleansing them. Wicken Fen yielded37 species of nematode. Access to the Nematode Collection, loving-ly cared for by David Hooper, enabled a comparative study of dory-laimid morphology that, in turn, led to reflection on passage of foodthrough the oesophago-intestinal junction. Ideas on nematode diver-sity and feeding types were committed to paper and sent toPedobiologia – the main reaction being a concern that I was publish-ing behind the “iron curtain”.

In late 1969, I accepted an amanuensis position in the Danish IBP(International Biological Programme) beech forest programme as oneof the group assessing energy flow through soil animals. This wasbased in the thatched farmhouse at the Mols laboratory (Denmark)where Christian Overgaard-Nielsen had done his pioneering work

68 THROUGH NEMATODE DIVERSITY TO LIVING SOIL PROCESSES – HOLISTIC STUDIES AID PROGRESS

that launched nematode ecology. In the previous year, at Rothamsted,I had picked up Alan Whitehead’s tray extraction method and used itin the Hestehave beech forest. It yielded good numbers of tardigradesand, according to an expert in their ecology group, proved reliablealso for enchytraeids. Once it was realised that much of ecology wasconcerned with “active’” nematodes Whitehead & Hemming traysbecame widely applied around the world. Although the energy-focussed approach proved to be inappropriate, the intensive 12month sampling programme in Hestehave provided good basic infor-mation on nematode populations and biomass, linked to a range ofother biological studies. There were at least 76 nematode speciesfound in this habitat. Mounting, with Judy’s paid assistance, andmeasuring over 100 specimens from each of three depths eachmonth gave me an appreciation of differences between successivedevelopmental stages. This, together with the wide range of taxadescribed during my PhD, still underpins my work with nematodes.Further, the IBP established links between dedicated workers thatstill continue today, even though some are now third generationpractitioners. For example, I currently do much of my work withDavid Wardle who trained under Denis Parkinson, a member of aCanadian IBP group, and with David’s students. Denmark also pro-vided my first contact with Nordic nematologists including KnudLindhardt, Bengt Eriksson and Björn Sohlenius.

In late 1970, DSIR paid my fare to return to New Zealand – forthe second time in Judy’s case. As in 1969, I chose an “ecological”rather than “taxonomic” position – not that I have ever actuallybeen formally interviewed for a job – and went to the Soil Bureau,the national soil survey organisation. The Soil Bureau not only useda genetic (processed-based) soil classification but also had sectionsfor agronomy, soil analysis, soil biochemistry, soil biology, soil chem-istry and mineralogy, soil engineering, and soil physics. Soil surveyorsand colleagues collected samples from soils of known properties andsome samples came from overseas, so I had plenty of help! InDecember 1970, my Director advised me “Gregor, you are a secondgeneration scientist. Go and get on with your science”. I followedmy nose, but it was not until 1987 that I was able to address one ofthe questions on the list I drew up in 1970 (collecting drilonematidnematodes from the body cavity of 30 cm long native New Zealandearthworms – but only after the earthworms had festered in thefridge at home for several days).

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In national terms my efforts were justified when, in the early1970s, we identified and later unravelled the problems with andimpact of Meloidogyne and Heterodera on white clover in NewZealand pastures. These nematodes significantly reduce symbioticnitrogen fixation by Rhizobium in nodules on white clover – thenitrogen supply fundamental to the nation’s pastoral industries. In2006, Chris Mercer and colleagues are continuing efforts to incorpo-rate resistance to these nematodes in commercial white clover culti-vars. Little do the plant breeders realise that clover roots withoutHeterodera or Meloidogyne will be heaven for Pratylenchus!

One of the greatest ecological advances came after I had collect-ed monthly samples from normal, grazed pastures for 13 site-years(seven sites, some with different treatments or for 3 successiveyears). Total nematode abundance was positively correlated withpasture herbage production (i.e., plant growth that could be used bysheep or cows). While this was contrary to conventional wisdom,the counts included bacterial and fungal-feeding nematodes andpredators as well as plant-feeders, and were recognised as, in someway, reflecting below-ground processes. The effects of irrigating pas-ture or of year-to-year differences at a site were of lesser influencethan was the underlying soil. Also, the diversity of the nematodeassemblage differed among the seven soils – the greatest diversitywas found in a soil derived from material that had erupted from avolcano about 100 years earlier. Soil was more important thanmonth, year or management practices in determining the composi-tion of the nematode fauna. All this was achieved with nematodesessentially being identified to genus (sensu J.B. Goodey, 1963) [see:Soil Biology and Biochemistry 16: 95–102, 1984]. I was fortunate to bein a country with fairly uniform, grazed pastures on a diverse rangeof soil types and at a time when soil fertility was still driven by biol-ogy rather than by what comes out of a bag.

As one who was ploughing through numerous samples notknowing what would result, I was amazed when Diana Freckmantelephoned and asked me to talk at the Society of Nematologistsmeeting in August, 1977. Apart from giving “A view of nematodepopulations and their role in ecosystems” [see: Journal of Nematology11: 213–229, 1979] there was a fantastic pre-conference tour. Duringthis I was amazed to hear “nematode” “nematode” “nematode” men-tioned in the bus – for many years only Judy and I had used theword conversationally. Visits to laboratories in Riverside, Davis, Fort

70 THROUGH NEMATODE DIVERSITY TO LIVING SOIL PROCESSES – HOLISTIC STUDIES AID PROGRESS

Collins and Laramie were just great, and I am still in touch withpeople I met on that visit.

The positive correlation of plant yield with nematode popula-tions was integrated into the wide ecological literature [see: Advancesin Ecological Research 17: 61–113, 1987]. Also, my interest in morpho-metrics, which began during my PhD, was strengthened during eco-logical studies because in some way it helps explain how so manynematode species can co-exist in a single soil. Earthworms, throughtheir effects on decomposition pathways and on soil structure havebeen found to influence nematode abundance and diversity.

Two laboratory studies are critical in understanding how thepositive correlation arises. It must be remembered that as animals,nematodes are ultimately dependent on energy fixed by primaryproducers. In the soil food web nematodes either feed directly onplants (“grazing foodweb”) or in the “detritus foodweb” (as bacterial-feeders, 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, undernutrient-poor conditions, grazing of nematodes on bacteria increasesthe turnover of plant nutrients and thus increases plant growth[Ecological Monographs 55: 119–140, 1985]. We had to wait until1998 before the Howard Ferris” group at Davis demonstrated whyfungal-feeding produces a lesser response [see: Plant and Soil 203:159–171, 1998]. The loop was completed when I, transmuted toLandcare Research in Palmerston North, with Chris Mercer, RichardBardgett and others, and applied Surinder Saggar’s pulse labellingtechnique. We grew white clover plants with various nematodespecies infecting the roots, let the plants photosynthesize with 14Clabelled CO2 and determined the distribution of the 14C label after14 days. In the presence of nematodes, more of the new photosyn-thate was found in the soil microbial biomass, being available forplant uptake as it cycled in the rhizosphere. The degree of “leakage”varied, generally reflecting the degree of root damage by the nema-tode [see: Nematology 1: 295–300, 1999]. Thus even plant-feedingnematodes can make a positive contribution to soil nutrient cycling;this may be a mechanism by which small populations stimulateplant growth, but large populations may lead to pathogenicity.

We now knew that the soil type controlled the composition of thenematode fauna and that the activities of the nematode fauna as a

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whole contributed to the productivity of a site. Several other questionswere worrying nematode biologists and ecologists. What resources donematodes actually use? How many nematode taxa occur at a site?How does the composition of the nematode assemblage at a siterespond to various stresses? How does all this fit into the global ecolog-ical picture? Such questions are neither trivial, as nematodes are themost numerous animals on the earth, nor simple, as the limitedprogress since Overgaard Nielsen’s 1949 work has shown.

At the Second International Congress of Nematology inVeldhoven (1990) a group met and agreed to pool their combinedknowledge on resources used by plant and soil nematodes. The widelycited “state of knowledge” synthesis on nematode feeding types arosefrom this meeting [see: Journal of Nematology 25: 315–331, 1993].Although there has been some skirmishing around the edges, the onlysignificant subsequent advance has been the confirmation that severalTylenchidae 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. In1984, I was talking to Carol Morley, a student at NREL, aboutmononchids and casually mentioned that I had one in culture appar-ently feeding on bacteria. So did she! [See: Ecology 70: 1127–1141,1989]. Around 1992, I was working through hundreds of tubes ofsamples collected from a field trial some months earlier when Inoticed that the technician had failed to fix one of the samples – itcontained solely mononchids and protozoa (at 50 xs). These, andsimilar, observations have been reported but still people regardmononchids as solely predacious. The similarity of the behaviour oftheir populations to those of omnivores is not coincidence – it isreal, reflecting food resources and our ignorance.

Our marine colleagues, such as John Tietjen and JohnLambshead, lead the way in assessing nematode species diversity insamples. For soils, most of us have a great “taxonomic impediment”(that is, we do not know what species we have) and must work atgenus level, although some nations have species lists. The addedcomplication of compensating the “taxon count” for the effort (i.e.specimens identified) is too often forgotten, but Brian Boag and Itried to bridge the gap in carrying out a global review [see:Biodiversity and Conservation 7: 617–630, 1998]. Mike Hodda hasprobably been the most successful in looking at diverse habitats andallocating nematodes to nominal species by working on an English

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grassland and tropical Cameroon forests. Anton Šály’s study inSlovakia counted species in a mosaic of habitats. The latter twostudies add to the complication in that they looked at a series ofsites in a region, determining regional or ψ diversity rather than α orcommunity diversity. While the diversity of nematodes in adjacentcores from the same locality can be seen as a sampling problem itdoes show habitat patchiness, and is very useful in comparing patch-iness in neighbouring habitats with similar underlying soil types.Christien Ettema and I looked at this, comparing adjacent pastureand forest, on a soil in New Zealand [see: Soil Biology andBiochemistry 35: 339–342, 2003]. It was good to have someone elsecome up with the same list of genera as I would have (except herAporcelaimellus was more up to date than my Aporcelaimus). It wasalso entertaining to be told by the farmer’s wife how she dug underrows of potatoes to “tickle” potatoes from the tenant’s crop. I thinkthat there is now an appreciation among nematologists that all thevarious diversity measures are valid; the problem is finding the rightframework with which to interpret the results.

Christien Ettema’s working with me was the latest in a series ofexchanges that began when Tom Bongers had me talking aboutnematode ecology in Wageningen in 1987. I was amazed to be takenhalfway around the world to the home of nematology, and to findthat my eight lectures, “Resource utilization by nematodes”, whichincluded the ecology of plant, soil and marine nematodes, wentdown well. In Wageningen, Ron de Goede and Gerald Korthalswere eager learners. When he was in New Zealand in 1990-91, Iasked Marco van Étagère to measure specimens of Longidorus tani-wha and sort out the juvenile stages. [Wallie Clark had describedthis nematode earlier, taniwha being the native name for a monster.]A very concerned student approached me next morning to say thathe could find only three juvenile stages rather than four. We lookedat his tabulation and confirmed his interpretation. I commented thatthe nematodes had not read the textbook and that Marco wenthome wiser on those days when he had more questions after work-ing than before. This was the first record of only three juvenilestages 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 workon movement of nematodes through sand etc., packed into rings,finding differences with texture. Consensus was that nematodes were

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best able to migrate when soils are at field capacity. By 1980, weknew that changing the structure of a soil of a given texture affectedthe nematode fauna. With soil physics colleagues I set up a laborato-ry trial using cores of undisturbed soil with differing degrees of com-paction. Hypotheses were clear, replication good. However, the threenematode species had not read Wallace (1963). Populations of allthree bacterial-feeding species increased at all moistures until every-thing died out at around wilting point. There was a suggestion thatpopulation increase was greater when the water films were thinner –presumably bacterial concentrations were higher and nematode feed-ing easier [see: European Journal of Soil Science 53: 355–365, 2002].When I first presented these results in an invited paper at CallawayGarden, 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 theNew Zealand Society for Parasitology and it serves as a meeting placefor nematologists sensu lato. In the 1980s, when scanning electronmicroscopes became simpler he used to spin yarns to complementimages of the magnificent copulatory spicules of nematode parasitesof insects. Initially, I had to curtail my “free-living” interests and talkabout plant-pathogenic nematodes. However, at a joint meeting withthe Australian society in Adelaide I was asked to look at the possibleeffect of Duddingtonia flagrans (= Arthrobotrys flagrans to some) onnon-target soil nematodes. Duddingtonia flagrans is perhaps the firstnematode-trapping fungus to have been shown to have a significanteconomic impact [see: Veterinary Parasitology 126: 199–315, 2004].When fed to sheep or cattle it can pass through the gut to be deposit-ed in the dung pat. The chlamydospores germinate and the hyphalnetwork effectively traps the bacterial-feeding juveniles of tri-chostrongylid nematodes that would otherwise crawl up blades ofgrass to be ingested by, and infect, livestock. Having identified per-haps 100,000 soil nematodes from various field trials in Australia,Wales, The Netherlands, Denmark, Sweden and, just free of embar-go, New Zealand, it can be said that adding the fungus to stock foodhas no effect on the abundance and diversity of soil nematodes, andthus no effect on nematode-mediated soil processes. Whew, what arelief. However, the soil nematodes had not read our site plans. NearUppsala, the trials were on a “flat” area adjacent to a river. There wasa barely detectable slope towards the river; soil texture changed down

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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 sam-pling the nematodes were able to show us that the soils differed. Thissubtle soil effect was greater than any effect of the nematode-trappingfungus. Isn’t habitat diversity marvellous!

Largely in response to a requirement of a funding agency, TomBongers, in 1990, proposed the Maturity Index that weights nema-todes as an indicator of environmental disturbance [see: Oecologia83: 14–19, 1990]. It is not a diversity index, rather an index of theperceived population reproductive strategies of the various taxapresent. It seemed to be a convenient way to summarise (i.e., index)typical data found after various disturbance events – yes, evenadding powdered cow dung to soils! Although widely cited [318times by 10 March 2006, according to ISI if you care for citationrates] and highly influential, I am on record as doubting that thepostulated c-p groups are robust. In an effort to move indices tomore reflect ecosystem function Howard Ferris and others proposedthe Structure and Enrichment Indices [see: Applied Soil Ecology 18:13–29 , 2001]. To me these confound the “ignorance” embedded inthe feeding group paper with the doubts about c-p classes.Questioning is the basis of science. I get to referee many papers thatuse Tom’s and Howard’s indices – as long as the sites and soils arewell described and the work generally well planned, as applicationsof current tools, they pass.

In a way, these indices are a return to the IBP philosophy inwhich the flow or flux through nematodes was the important thing;we have moved on from the IBP philosophy. Ecosystem ecologists, asopposed to nematologists, seem to be getting strong relationshipsbetween the abundance of nematode functional groups and measuresof ecosystem processes. It reminds me of a colleague who, 20 yearsago, conceded that rotifers were much more abundant than bacteria-feeding nematodes in a set of samples, but they were “ignored”;another colleague said that, as he worked in a nematology depart-ment, he could not count the enchytraeids in his samples. I hope thatwe have moved on from there. Both bacterial-feeding nematodes androtifers share the same, bacterial resource; enchytraeids, if nothingelse, are prey for Nygolaimus. Analyses that I have done with bothDavid Wardle and Wim van der Putten indicate that abundance of“predacious” and “omnivorous” nematodes correlate with the samevariables; they both use similar, diverse resources.

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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 herbi-vores, 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 identifysoil nematodes to useful taxa and calculate indices – even someindices that ecologists as a whole use and understand.

3) 50 years after the establishment of ESN (European Society ofNematologists), soil nematodes as a whole are being treasured andthere 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 arange of techniques to better understand:

1) Nematode diversity (with 100+ nematode taxa, each with 4 juvenile stages, females and sometimes males, in each parcelof land).

2) Nematode use of, and interactions with, food resources toprovide ecosystem services (aggregating nematode information bysome functional groups and across patches).

3) Nematode biology by drawing on morphological, develop-mental and ecological information from all habitats that the variousstages of nematodes utilise.

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Fig. 1. David Hooper andJudith Killick (now JudyYeates) in their respectiverooms in the NematologyDepartment at RothamstedExperimental Station, about1961. This was the size oftheir space. Techniciansworked on Saturday morn-ings, with one of their tasksbeing to blacken the bench-tops with boot polish.About this time they werehelping Basil Goodey withhis book, and David wasdoing his work onLongidorus 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; onright 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; thatin the right darkened for microscope use.

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Fig. 4. The wet lab for Nematology Section, Entomology Division, DSIR, Nelson in1962. To left of bench is a vibromixer; along shelf flasks with rubber adaptors forthe 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 rightof bench, a Baermann funnel.

Fig. 5. Gregor Yeates sam-pling nematodes beneathmarram grass in coastal sanddunes at Himatangi Beach(New Zealand) in 1967.Bags of samples to left ofshovel; in trench up to hisknees as the corer was usedhorizontally for sampling atup to 90 cm depth.(Courting was a subsidiaryactivity).

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Fig. 6. Russ Ingham sampling in the prairie whiledoing his PhD at NRELColorado State University.Russ comments “Too badthe amphibian in my handdoes not show up better.We were in the field sampling nematodes when anema toad came by”.

Fig. 7. Conference dinnerscan be great for exchange ofideas. Diana Wall and TomBongers at such a dinner.

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, inthe waters of hot springs and in polar seas where the temperature isconstantly below the freezing point of pure water…enormousdepths in Alpine lakes and in the ocean…sometimes the eggs andlarvae are so resistant to dryness that if converted to dust theyrevive when moistened…diversity of habitat…inconceivably abun-dant”. Cobb’s (1914) (Fig. 1) assertions, the validity of which hasstood the test of time, demand reflection on the physiologicalamplitude and unique characteristics that would facilitate such arange of habitats and activities.

Nematodes parasitize vertebrates, invertebrates, and plants; manyare not parasites but are sustained by feeding on other organisms.Yet, there are recent discoveries of nematodes from the benthos thathave no mouths but a rudimentary gut filled with chemoautotrophicbacteria (Ott et al., 1982; Miljutin et al, 2006). Even in some soilnematodes, a reduced mouth and esophagus has led to speculation ofdiffusion across the cuticle of nutrients in solution (Bongers, 1990,Fig. 2). Early developments in nematology were strongly driven byrecognition of the causal organisms of the maladies of man and

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domestic animals. Commanded by their size and obvious humanimpact, vertebrate-parasitic nematodes have been, for centuries, afocus of investigation (W.P. Rogers, Theodor Von Brand, DonaldFairbairn and many others). Consequently, Ascaris and other verte-brate-parasitic nematodes have been models for understanding nema-tode physiology, embryology, and development (Wright, 1998).

Advances in microscopy enabled observation of the micro- andmesofauna of soil and water. Fascination with the biology of thebacterial-feeding nematodes led to an understanding of their lifecycles, including the existence of an alternative life stage by such19th 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 scien-

NEMATODE PHYSIOLOGY: SIGNIFICANT DEVELOPMENTS 81

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

tific endeavors and advances surrounding Caenorhabditis elegans, thenematode 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) inCalifornia and Warwick Nicholas (Fig. 6), Sydney Brenner (Fig. 7) andothers in England, particularly the recognition of C. elegans as a poten-tial tool for unravelling the mechanisms of gene expression, are the basisfor the enormous importance of C. elegans in recent advances in biology(Riddle & Bird, 1985). A landmark in genomics was the completion ofsequencing of the genome of C. elegans in 1998 by the cumulative andcollaborative efforts of scientists in laboratories worldwide, particularlyby Jonathan Hodgkin (Fig. 8), the Nobel Prize winning Cambridge car-tel of Brenner, John Sulston (Fig. 9), Robert Horvitz (Fig. 10), and themany others whose activity they spawned. Incredibly painstaking cell-lineage studies, mainly through observation of cell division and activityin the nematode egg, by Sulston and colleagues, revealed that 671 cellsresulted from divisions that take place in the egg, and most of the restof the divisions necessary to make up the 959 cells of this nematodeoccur in later juvenile stages. Among the insights fueled by these studiesis a better understanding of the mechanisms and importance of apopto-sis, programmed cell death, in development (Riddle, Fig. 11). The tissueand organ functions associated with movement, sensory functions, andreproductive functions are better understood because of the structuraland mechanistic insights from nematode model systems. The genomeinformation on “the worm” has stimulated investigations on its plant-and animal-pathogenic relatives.

The classic studies on nematode ecology and migration through soils(Harry Wallace, Fig. 12), chemosensory attraction to mates, food (NoelGreet, Cliff Blake, Chris Doncaster), moulting (Donald Lee, Fig. 13; KenDavey; Alan Bird, Fig. 14), survival and adaptation (Adrian Evans; ChrisWomersley; Seymour Van Gundy, Fig. 15), and on pathogenesis and cellular changes (Alan Bird; Glenn Bergeson; Victor Dropkin, Fig. 16), ofthe 1940s to the 1970s, provided an important platform for the advancesin understanding of plant-nematode interactions.

In this chapter, we will highlight, somewhat anecdotally, someof the important advances made in energetics, vertical gene transfer,moulting and osmoregulation, physio-molecular interactions ofnematodes and plants, and ecophysiological adaptation for fitnessand survival. While recognizing the names of the contributors andpioneers, we have not been exhaustive in the completeness of our

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review or in providing citations to sources of the subject mattersdiscussed. In some cases, we are guilty of resorting to familiarity byciting our own papers rather than providing an extensive list of ref-erences; we intend no slight and wish to be judged as expedientrather than arrogant in our economy of approach.

Energetics and function

The C. elegans developmental studies illuminate the resource con-servation dilemma of the plant-feeding nematodes. Assuming thatthe cell numbers of most plant-feeding nematodes are within a rea-sonable range of that of C. elegans, all of the metabolic energy asso-

NEMATODE PHYSIOLOGY: SIGNIFICANT DEVELOPMENTS 83

Fig. 7. Sydney Brenner Fig. 8. Jonathan Hodgkin Fig. 9. John Sulston

Fig. 10. Robert Horvitz Fig. 11. Donald Riddle Fig. 12. Harry Wallace

ciated with those cell divisions, the differentiation of tissues andmost organ systems, hatching, movement, detection of a host, and,in the case of the endoparasites, penetration of the host before feed-ing commences, is fueled by the resources deposited in the initialsingle egg cell as it progresses through the oocyte developmentprocess. Those resources are finite and must be substantially deplet-ed when partitioned among several hundred cells by the time ofemergence from the egg. Thus, energy management strategies thatcause the nematode egg, or hatched juvenile, to remain metabolical-ly active prior to access to its food may reduce infectivity.

The metabolic and respiratory energetics of soil-inhabitingnematodes have been studied for populations of several species.Nematode respiration rate per individual decreases with size accord-ing to the power dependence of basal metabolism and body weightobserved in many organisms, R = a Wb, where R is the respirationrate, W is the fresh weight of the individual, and a and b are regres-sion parameters such that b is close to 0.75 for nematodes and otherinvertebrates (Klekowski et al., 1974; Nicholas, 1975; Apple &Korostyshevskiy, 1980; Atkinson, 1980, Fig. 17). The formula pro-vided by István Andrássy (1956, Fig. 18), in one of the most fre-quently cited but least read papers in all of nematode ecology,allows calculation of the weight of a nematode as a function of itswidth and length. It has been an invaluable tool for stepping fromthe individual to the population and community in studies of nema-tode energetics.

Estimates and measurements of the coefficients of the metabolicpower function have been adopted, with some modifications, forcalculating growth and energetics requirements in plant-parasiticnematodes (Melakeberhan & Ferris, 1988; Melakeberhan &Webster,1992; Reversat, 1987). The calculations highlight the issue of feedingrates and resource partitioning. Unless they have a reliable and sus-tained food source, as in modified plant host-cell structure, largebodied nematodes often have a smaller gonad:body volume ratiothan smaller nematodes. More energy resources are committed togrowth and metabolic activity of the somatic tissues than to produc-tion of oocytes and eggs. Consequently, populations of larger nema-todes that do not have a specialized feeding site grow at a slowerrate than those of smaller-bodied organisms.

Respiration rates of adults range between 1.25 and 8.80 nl O2 h-1at 20°C among several species studied. Metabolic rates of adults

84 NEMATODE PHYSIOLOGY: SIGNIFICANT DEVELOPMENTS

range from 1.15 nl O2 µg (f.w.)-1 h-1 for Rhabditis cucumeris to 4.43nl O2 µg (f.w.)-1 h-1 for Mesorhabditis labiata, at 20°C. At eachtemperature, metabolic rates of nematodes of similar size vary withthermal adaptation of the species. Metabolic rates of Cruznema tri-partitum and Cephalobus persegnis were more sensitive to tempera-ture change than were those of Acrobeloides bodenheimeri, A.buetschlii and Panagrolaimus detritophagus. Cephalobus persegnisexhibited the greatest total metabolic activity across a range of tem-peratures, and P. detritophagus the least. Observed differences inthermal adaptation may contribute to the predominance of speciesin the nematode community at different times during the year or atdifferent depths in the soil (Ferris et al., 1995).

That nematode species endemic, and apparently successful, in

NEMATODE PHYSIOLOGY: SIGNIFICANT DEVELOPMENTS 85

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

the same environment may have different thermal optima (Ferris etal., 1995) is in concurrence with the suggestion of Anderson &Coleman (1982) that temperature-niche breadth mediates competi-tion among species. Differences in temperature-niche breadth deter-mine the predominance of coincident species at different times dur-ing the year, or at different depths in the soil. Temporal predomi-nance patterns determine the relative contribution of the coincidentspecies to nitrogen mineralization in managed agricultural systems.

Vertical gene transfer and life history

A.C. “Tasso” Triantaphyllou (Fig. 19) at North Carolina StateUniversity has made pioneering contributions to nematode cytogenet-ics (Evans, 1998). Vertical gene transfer in nematodes is achievedthrough several mechanisms, including mitotic parthenogenesis, mei-otic parthenogenesis, amphimixis, and hermaphroditism. Significantadvances have been made in our understanding of how genetic infor-mation is transferred and which taxa are amenable to mating experi-ments that will allow inference of gene function. Mechanisms of sexdetermination in both parthenogenic and amphimictic species, andsex reversal, were revealed and explained through the studies ofTriantaphyllou. It seems likely that sex is determined by the effect ofenvironmental conditions on the degree to which genes from the Xchromosome regulate genes on other chromosomes. This would allowfor environmentally-mediated sex determination, which is frequentlyobserved. For example, since in sexually reproducing species femalesare XX and males are XO, one might infer that more X product isrequired to up- or down-regulate the genetic pathways that result infemales than for those that result in males. Consequently, if the signalstrength from the X chromosomes is suppressed at high temperature,more males might result. Current studies in the laboratory of CharlesOpperman, one of Triantaphyllou’s successors, may shed some light onthe genetic and molecular basis of sex determination in nematodeshttp://www.cals.ncsu.edu/plantpath/.

Hermaphroditism, an interesting alternative in vertical genetransfer in some members of the family Rhabditidae, was anotherrevelation of the in-depth studies on C. elegans. In sequential her-maphroditism, the gonad first produces sperm, which are stored in aspermatheca. The gonad then produces oocytes, which become fer-

86 NEMATODE PHYSIOLOGY: SIGNIFICANT DEVELOPMENTS

tilized eggs as they pass through the spermatheca. In C. elegans,about 150 sperm are produced in each arm of the gonad and storedin each spermatheca. The number of sperm produced apparentlylimits the number of offspring produced by the nematode to around300 (Gems & Riddle, 1996). True males also occur in a population,but are rare (around 1:1000). However, the frequency of males isenhanced at elevated culture temperature. When a hermaphroditicfemale is mated with several males, as many as 1400 progeny maybe produced (Kimble & Ward, 1988), suggesting that productivity inthis form of hermaphroditism, is sperm-limited.

Excretion and osmoregulation

Excretory products of metabolic activity differ in animals of differ-ent habitats. Nitrogenous waste products, usually in the form ofammonia, result from metabolic pathways that involve proteins andamino acids. Since ammonia is toxic, terrestrial animals, includingarthropods and vertebrates, generally bind the –NH3 group intoeither urea or uric acid, which are accumulated prior to excretion(Campbell, 1973). Such organisms are termed uricotelic. Nematodesare aquatic organisms, inhabiting marine and fresh water and thewater films of soil environments. Like most aquatic organisms, theycontinually excrete ammonia into the environment as it is pro-duced, thus avoiding the toxic storage problem. Such organisms aretermed ammonotelic (Perry, Fig. 20 and Wright, 1998). The excre-tion of waste nitrogenous products into the soil environment maybe a significant contribution to nitrogen availability to plants (Ferris,et al., 1998; Chen & Ferris, 1999). A major difficulty in studyingosmotic and ionic regulation of soil-inhabiting and plant parasiticnematodes is their small size, and larger nematodes like ascarids areoften used as model systems (Wright, 1998).

Physio-molecular interactions of nematodes and plants

In an era of increasing environmental awareness, it is necessary tofind economically and ecologically sustainable nematode manage-ment alternatives through an understanding of the physio-molecularand genetic bases of plant nematode interactions from the sub-cellu-

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lar to the ecosystem level. Studies of the physiological changes andformation of specialized feeding sites associated with sedentary plantparasitic nematodes by Alan Bird in Australia and of cellular changesby S.G. Myuge, Glenn Bergeson and Victor Dropkin in the 1960s and1970s have been the foundation and inspiration for many host para-site studies at the cutting edge of science. The recent activities ofseveral research groups merit high recognition. All are importantresearch programs that are integral to longer-term trajectories fromwhich will emerge novel approaches in nematode management.

First, research spearheaded by Richard Hussey (Fig. 21) at theUniversity of Georgia has characterized the molecular and function-al nature of glandular secretions of the root-knot and cyst nema-todes, which have very specialized and complex feeding relation-

88 NEMATODE PHYSIOLOGY: SIGNIFICANT DEVELOPMENTS

Fig. 19. A.C. Triantaphyllou Fig. 21. Richard Hussey

Fig. 20. Roland Perry Fig. 22. Jaap Bakker Fig. 23. Valerie Williamson

ships with their host plants. In collaboration with Rick Davis, NorthCarolina State University, Thomas Baum, Iowa State University, andJaap Bakker (Fig. 22) and Arjen Schots at Wageningen University,comprehensive profiles have been developed of genes for parasitismthat are expressed in the esophageal gland cells of the nematodes(Davis et al., 2000). The potential application of these studies is totarget the genes in intervention strategies that will disrupt the hostparasite relationship http://www.plant.uga.edu/faculty/hussey.htm.

Second, the pioneering work of the Plant Nematode GeneticsGroup comprising David Bird, Charlie Opperman and collaboratorsat Rothamsted Research (Rothamsted Experiment Station) inEngland, which sequenced the Pasteuria penetrans (nematode-para-sitic bacterium) http://www.cals.ncsu.edu/plantpath/. An impor-tant goal of that group is to understand the molecular basis of nem-atode-plant interactions, using Meloidogyne, Heterodera andGlobodera spp. as models and employing cellular, genetic andgenomic approaches.

Third, Valerie Williamson (Fig. 23) at the University ofCalifornia, Davis, has, over several decades, made major contribu-tions to our understanding of the molecular and genetic basis of theMi-gene (present in most commercial tomatoes) and other forms ofresistance against root-knot nematodes and to the associated hostand parasite recognition mechanisms. Isgouhi Kaloshian, now at UCRiverside, and Kris Lambert at the University of Illinois, did theirpostdoctoral and/or graduate work in Williamson’s laboratory andhave expanded and extended her studies.

Sterols are among the specific nutritional components thatnematodes need from their hosts, and identifying ways to disruptthe sterol supply has been a major focus in the research of USDepartment of Agriculture’s David Chitwood (Fig. 24). Recently,Chitwood & Skanter (2006) identified two genes in Heteroderaglycines that code for products similar to the 17â-hydroxysteroiddehydrogenases and are involved in the synthesis of steroid hor-mones in mammals.

Harry Wallace’s (1973) soil-nematode interface and Alan Bird’s(1974) and Victor Dropkin’s (1980) nematode feeding behaviouranalyses have led to more applied studies. Starting from his graduateresearch with John Webster, the second author of this chapteracknowledges that the classic work of Harry Wallace on the soil-nem-atode interface provided the basis for his own work on the manipula-

NEMATODE PHYSIOLOGY: SIGNIFICANT DEVELOPMENTS 89

tion of soil nutrients to adversely affect nematode infective and devel-opmental behaviour (Melakeberhan, 1999). Moreover, VictorDropkin’s (1980) categorizing of plant parasitic nematodes intodestructive (host cells killed, Pratylenchus), adaptive (cells modified,Heterodera), and neoplastic (cells modified and undergoing newgrowth, Meloidogyne) feeding behaviors generated interest in whethernematodes of different feeding behaviour affect host physiology dif-ferently. Similarity of effects leads to the possibility that the damageof several nematode species may be offset by a single managementoption (e.g., nutrient amendment). If effects are different, more situa-tion-specific options will be required (Melakeberhan, 2006).

Ecophysiology: physiological adaptations for fitness and survival

Nematodes occupy many trophic levels and perform many servicesin the soil food web (Bongers & Ferris, 1999). Plant and soil nema-todes have evolved a suite of adaptations that confer fitness in avariety of spatio-temporal niches and that enhance their probabilityof survival under adverse conditions. The physiological basis andmechanisms of nematode survival, including omnivory, dauer stages,cryptobiosis and dormancy, are reviewed by Womersley et al.(1998). Plant-feeding nematodes are primary consumers of incomingresources. They, in turn, constitute a resource for many other organ-isms in the soil food web through predation by fungi, bacteria, and adiversity of mesofauna. Besides providing ecosystem services ofnutrient cycling, the predation involved in the transfer of carbonthrough the soil community may result in top-down regulation ofthe primary consumer species, particularly where food resources arelimited due to seasonal host phenology or competition at high nem-atode densities. A brief review of some mechanisms of nematodesurvival and fitness provides insight into the observational and infer-ential powers of the scientists who have studied these aspects ofnematode biology.

OMNIVORY: Although the feeding habits of many plant and soilnematodes have not been determined, a great deal of information isavailable from experiments, observations, inferences based on feed-ing structures, and on organism associations. The classic paper of

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Gregor Yeates (Fig. 25)and colleagues (Yeateset al., 1993) summa-rized feeding habitinformation availableto that time. They cat-egorized six feedingtypes among soil andplant nematodes: feed-ing on vascular plants,feeding on fungalhyphae, feeding onbacteria, feeding onanimals, feeding onunicellular eukaryotesand omnivorous feed-ing. They also recog-nized two other cate-gories, the ingestion ofsubstrate incidental tofeeding by openmouthed morphotypessuch as bacterial feed-ers and certain preda-tors, and dispersal orinfective stages thatmay not be feeding,often in phoretic relationships with insects. The term “omnivore“ isusually applied to certain nematodes of the Dorylaimida for whichomnivory has been observed or for which feeding habits areunknown. Clearly, some dorylaims are plant feeders (e.g., Xiphinema)and some are predators (e.g., Labronema). True omnivory is an adap-tation to unreliable food sources that may be seasonally or spatiallysparse. Besides its occurrence in the Dorylaimida, it has beenobserved in Mononchida where juveniles may be sustained on bac-teria (Yeates, 1987) in certain Aphelenchina (e.g., Aphelenchoidesspp.) and Tylenchina (e.g., Ditylenchus spp.) where survival betweenplant hosts by feeding on fungi is of obvious adaptive significance.

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Fig. 24. David Chitwood Fig. 25. Gregor Yeates

Fig. 26. David Viglierchio Fig. 27. Neil A. Croll

DAUER STAGES: Many soil nematodes, particularly bacterial feed-ers of the Rhabditidae, Panagrolaimidae and Diplogasteridae, have ametabolically-suppressed specialized survival stage. Schneider (1866)reported the existence of a life stage of rhabditid nematodes with acuticle differing from that in other stages; he considered this form tobe a moulting stage but was uncertain of its role. According toMaupas (1899), Pérez (1866) recognized an “encysted” stage inRhabditis teres and indicated that larvae easily encysted at the end ofthe second stage. Experimentally, Maupas (1899) determined thatalways the same life stage entered encystment when nutrients werelacking. He showed that emergence from the encysted stageoccurred with enrichment and noted that encysted nematodes sur-vive for weeks and are often a dispersal stage. Later, Fuchs (1916), inhis description of rhabditids associated with bark beetles, coined theterm “dauerlarva” for the persistent or enduring stage of these nema-todes. Many of the nematodes that have phoretic relationships withinsects are in a dauer stage during the phoresy. Likewise, ento-mopathogenic rhabditids await their insect hosts in a dauer stage.Dauerlarva induction in C. elegans is mediated by the ratio betweena dauer-inducing pheromone, which is constantly produced by thenematode, and the magnitude of a carbohydrate signal from the bac-terial prey (Riddle, 1988). The ratio provides a measure of popula-tion size in relation to food availability. When the dauer-inducingpheromone is significantly greater than the food signal, dauer forma-tion commences (Ferris & Bongers, 2006).

CRYPTOBIOSIS: An attribute (literally, hidden life) of certainnematodes that enables their survival without detectable metabolicactivity. The most commonly recognized forms of cryptobiosisinclude anhydrobiosis, cryobiosis, anoxybiosis and osmobiosis inresponse to dehydration, cooling, low oxygen, and osmotic shock,respectively (Womersley et al., 1998). The first record of anhydro-biosis, although not recognized as such at the time, was that byNeedham (1744), when he opened the seed galls of Anguina triticion wheat. Anhydrobiosis is a common attribute of nematodes thatare successful in habitats subject to seasonal drying and to those thatfeed on the above ground parts of plants. For example, fourth stagejuveniles of Ditylenchus dipsaci enter anhydrobiosis, usually in largemasses, on or below the surface of plant tissue, and the term “eel-worm wool” describes the appearance of these dried nematodes.

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Similarly, the second-stage juveniles of Aphelenchoides besseyi enteranhydrobiosis under rice hulls. Many nematode species are capableof anhydrobiosis when subjected to slow drying. However, if thedrying is rapid, there is insufficient time for the necessary physiolog-ical and membrane structural changes to take place.

DORMANCY: The term is applied to the condition of loweredmetabolism and various categories have been recognized in nema-todes (Womersley et al., 1998): Facultative quiescence: dormancyunder unfavorable conditions with development readily resumed asconditions become favorable. Obligate quiescence: required dorman-cy for a life stage with development readily resumed under favor-able conditions. Facultative diapause: dormancy initiated by environ-mental factors with delayed resumption of development underfavorable conditions. Obligate diapause: dormancy initiated byendogenous factors with delayed resumption of development underfavorable conditions after specific requirements are satisfied (e.g., inMeloidogyne naasi). Zheng & Ferris (1991) described the delayeddevelopment of some eggs in Heterodera schachtii despite favorableconditions. They recognized eggs in four categories: the non-dor-mant condition of eggs that hatch rapidly in water; eggs that hatchrapidly in host root diffusate; eggs that hatch slowly in water over along period of time; eggs that hatch slowly over a long period oftime in host root diffusate. The combination of these categories ofegg development results in distribution of hatch over a considerableperiod and enhances the probability that some of the emerging juve-niles will encounter a host plant under conditions conducive toinfection, and thus species survival.

HOST RANGE AND HOST RECOGNITION: Many plant-feedingnematodes that are successful in annual crop agriculture have widehost ranges. There is a high probability that they are able to feed ona variety of the plants provided in cropping sequences. However,there are other successful strategies. Some nematodes with quitenarrow host ranges are successful because they remain in a dormantstate until stimulated to emerge by root exudate signals recognizedfrom a host plant (e.g., Globodera rostochiensis). The non-feedingdormant stage might technically be considered a dauer stage withoutthe 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 93

stage juvenile retained in the egg (e.g., Heteroderinae) whereas, inother 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 ofmany 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 alwaysebullient and insightful Rolo Perry (1996; 2006) of RothamstedResearch points out, many information gaps remain when explain-ing the physiological basis and mechanism of the interactions.

In a career cut short by his untimely death, the debonair NeilCroll (Fig. 27) synthesized the available information on a range ofphysiological aspects across the Nematoda in several books. Withregard to nematode behavior, Croll recognized that taxes, directedmovement towards or away from a stimulus, and kineses, change inthe 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 com-bination of taxes and kineses (Lee, 2002; Rodger et al., 2003; Young etal., 1998), and electrophysiological analyses indicate that reducedactivity or more frequent turning can result in aggregation near thestimulus (Perry & Riga, 1995). Taxis and kineses are characterizedaccording to the nature of the stimuli, which may include CO2, pH,temperature gradients and root diffusates. CO2, expected to be inhigher concentrations in the rhizosphere than in bulk soil, is a strongattractant 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 anolfactory tube, bacteriophagous entomophilic nematodes moved toplant roots (Boff et al., 2002). However, prior to invading the host,nematodes must sense additional factors to differentiate between thesources of general signals (Rühm et al., 2003). CO2 may provide adirectional stimulus and stimulate a taxis response. However, once thenematode is near the resource, plant signature compounds, such asflavonoids or alkaloids, may precipitate kinesis responses by the nema-tode resulting in their localization of individuals around food sources.

Although both attraction and repellency of host plants to nema-todes have been the subject of several investigations, only a fewhost- or nonhost-specific compounds have been identified that

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mediate the responses (Chitwood, 2002). In a critical assessment ofthe spatial and temporal nature of the chemoattraction and nema-tode orientation in soil, Perry (2006) concluded that CO2 and rootdiffusates followed by temperature are major nematode behaviourmodifying factors.

Outlook

In a relatively short period, significant advances have been made inour understanding of the biology and physiology of nematodes, andwe are on an exciting trajectory. Three of the driving forces are evi-dent. First, the success of each new set of researchers in stepping offfrom the platform erected by earlier workers, in effect embodyingthe Chinese concept of “standing on the shoulders of the great man”.Second, the wonderful advances in technology that have allowed thescaling-down of sensors and the amplification and conversion of thesignals necessary for the equipment (developed for rats and guineapigs) to measure the physiological processes and secretions of thenematodes. Third, the serendipitous selection of Caenorhabditis ele-gans, of all the organisms in the world, as the model system fordevelopmental biology and for genomic characterization. If, asasserted by Lorenzen & Platt (1994), four out of every five multicel-lular animals on the planet are nematodes, they provide the poten-tial for providing model and assay organisms for advancing science ata multitude of levels, from subcellular biology to the monitoring ofglobal 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.

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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-para-

sitic 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 Soil203: 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 nema-

todes. 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: 136–142WALLACE, H.R. 1973. Nematode ecology and plant disease. Arnold, London. WOMERSLEY, C.Z, WHARTON, D.A. & HIGA, L.M. 1998. In: The physiology and bio-

chemistry 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-para-sitic 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

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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 ofmolecular data. Molecular approaches have proved especially useful forsorting out the identification and relationships in those taxa for whichmorphological data have always presented difficulties. In that respect,nematodes represent a good example since they are remarkably con-strained morphologically. The two congeneric sibling species of free-living nematodes, Caenorhabditis elegans and C. briggsae, well illustratethis fact. Although their discrimination represents a challenge to mosttrained nematode taxonomists, the recent availability of their completegenome sequence shows that these two species diverged 100 millionyears ago, i.e., between 5 and 45 (25?) million years before the splittingof the mouse and the human lineages. In addition, these two nematodespecies have been shown each to bear at least 2000 genes (ca. 10% ofthe genome) that are not found in the other species.

The “molecularisation” of nematode taxonomy

Before these recent advances, the first attempts at molecular taxon-omy in the field of nematology occurred more than 30 years ago andthe major focus of molecular analyses has been the diagnosis of eco-nomically important species and their infraspecific forms in agricul-tural, medical and veterinary sciences. Among plant parasitic nema-todes, root-knot and cyst nematodes have been central to this typeof molecular-based research. The molecular biology approach alsohas been of increasing importance in other economically importanttaxa, such as Pratylenchus, Xiphinema, Bursaphelenchus and theentomopathogenic nematodes Heterorhabditis and Steinernema.

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The “molecularisation” of nematode taxonomy has been success-fully developed for use in diagnosis. Thus, any difference in proteinor isozyme phenotype, affinity for antibody, DNA polymorphism,DNA probe or sequence data for any gene or region of the genomehas been used as a potential diagnostic character. In the same way,studies have used molecular data in phylogenetic analyses, and inthose in which it has been applied a phenetic approach has beenadopted using distance measures or relationships between specieswithin genera or between genera.

One of the first examples of the power of molecular data wasgiven by Antoine Dalmasso in a complete study of allozymes in theMeloidogyne genus. This approach was applied to M. incognita, M. are-naria and M. javanica, a complex of species reproducing by obligatorymitotic parthenogenesis. In terms of identification, these clonal speciespresented special difficulties which cannot be accommodated by aspecies definition based upon reproductive isolation. The examinationof their isoenzymes revealed their degree of genetic diversity and indi-cated that they are true species. From that point, enzymatic polymor-phism has been widely used on plant parasitic nematodes. In theframework of the International Meloidogyne Project, exhaustive sur-veys of the main root-knot nematode species were undertaken byAnastasios Triantaphyllou in the late of 1980s based on esterase andmalate dehydrogenase phenotypes. In general, however, the most com-monly studied enzymatic system is that of esterases. Multilocus studieshave been used to investigate nematode dispersal, to trace their originand for species identification. In principle, several enzymatic systemscan be resolved from single individuals, but diverse studies have reportedlow polymorphism of the resolved loci. Although this approach hasimportant limitations (e.g., the biological material must be kept alive orbe frozen until used, and, at a given locus, the technique reveals only afraction of the actual genetic variation) the alloenzymes represent a useful marker, and the method is still largely in use in many laboratoriesworldwide, more than 25 years after the original publication.

The major focus in molecular taxonomy over the last 20 years hasbeen DNA analysis, and the first data were mainly provided in theearly 1980s by the group in John Webster’s laboratory in Canada forboth plant-parasitic and entomopathogenic nematodes. At that time,techniques to visualize DNA sequence differences were based onbasic approaches such as detection of restriction fragment length poly-morphism (RFLP), DNA probes and DNA sequencing. In general,

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most DNA studies for species and pathotype separation have madecomparisons between RFLPs in total DNA of unknown nematodepopulations against species and pathotype standards. Obtaining thenucleotide sequence has provided access to the ultimate detail of vari-ation in the DNA. Sequencing DNA fragments was quickly devel-oped by techniques that have now become routine and economic.DNA sequences were collated and stored in data libraries (e.g., EMBLand GenBank) and have been universally usable, powerful data. Thesehave also provided search facilities which allow unknown sequencesto be identified by similar search engines, such as BLAST. Sequencingwas done directly or after cloning into plasmid vectors.

Foremost among the factors which have contributed to therapid increase of molecular data sets in taxonomy is the develop-ment of the Polymerase Chain Reaction (PCR). Taxonomists wereno longer constrained by the size of the organisms for obtaining asufficient amount of biological material for analysis. PCR amplifica-tions were conducted upon a single juvenile nematode or an indi-vidual egg in a relatively crude assay. The concept of universalprimers has reduced the need to initiate each taxonomic projectwith a time-consuming hunt for primer sequences that will amplifythe desired product. These universal amplification primers areoligonucleotide sequences which anneal to genomic regions of suchhigh evolutionary conservation that they serve as amplificationprimers for a wide taxonomic range of organisms.

The random amplified polymorphic DNA (RAPD) techniqueuses the PCR principle for random amplification of DNA sequences,and does not require any preliminary sequence information of thegenome under study. Amplification is performed using a singleprimer with a very short sequence (8–10 base pairs) under annealingtemperature conditions (usually low) that enhance multiple bindingat sites scattered throughout the genome. Several DNA fragmentsare usually amplified and some of these may be present in a propor-tion of the individuals in a nematode population. Using a large set ofprimers has the advantage of screening the entire genome. However,the interpretation of RAPD data is sometimes limited by poorrepeatability of the results, with the problem aggravated by thesmall size of nematode stages (larvae) in which the quantity ofDNA obtained per individual is reduced, thus preventing accurateassays of DNA concentration. Another limit of these markers is thatthe RAPD patterns display dominance, preventing identification of

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heterozygotes. Nevertheless, the RAPD technique has been oftenused as the first source of information because results can be genera-ted quickly and easily.

A useful approach was developed in the middle of the 1990sbased on amplified fragment length polymorphism (AFLP). Thistechnique selects, by PCR amplification, restriction fragments genera-ted from a total digest of genomic DNA. Using this method, sets ofrestriction fragments can be visualized by PCR without prior knowl-edge of the genome of the target nematode species. As for other fingerprinting-based methods, such as RAPD, they give access to analmost unlimited number of genetic markers. However, in the AFLPtechnique, heterozygote genotypes cannot be easily distinguishedfrom other heterozygote genotypes. Significant examples of applica-tions of such methodology were given by the group of Fred Gommersin The Netherlands on the gene pool similarities in potato cystnematodes and by Philippe Castagnone-Sereno on the virulence andmolecular diversity in the parthenogenetic root-knot nematodes.

Most popular markers

Different genomic regions have been analyzed depending on theproblem examined, in particular those regions that have differentrates of evolution and/or different modes of inheritance (maternalvs Mendelian). Rapidly evolving genes have been shown to be usefulfor comparisons of closely related taxa, and slowly evolving genesfor comparisons of distantly related taxa. Two of the most popularmarkers used in molecular taxonomy have been mitochondrialDNA (mtDNA) and nuclear ribosomal DNA (rDNA).

The mtDNA represented a logical choice in molecular nema-tode taxonomy. This entire circular molecule is present in high copynumber and consists of rapidly evolving genes that has allowed largetaxonomy application. Several universal primers which bind to high-ly conserved regions of the mitochondrial genome were demonstrat-ed to amplify DNA from organisms ranging from insects to humans.Ironically, these same primers have not been useful for the amplifi-cation of nematode DNA. Nonetheless, several other primer setshave been published that work for numerous nematode taxa. In asurvey of mitochondrial genes such as cytochrome oxidase subunitsI and II (COI, COII) and the large 16S ribosomal gene, Vivian Blok

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and collaborators showed extensive variation that distinguished themajor species of tropical root-knot nematodes. In addition, mito-chondrial genomes have been entirely sequenced in different nema-todes species including the plant parasitic nematode genus,Meloidogyne. Considering the estimated rate of ribosomal mitochon-drial RNA (16S), it was demonstrated that the ancestral lineages ofCaenorhabditis and plant parasitic nematodes diverged during orbefore the Cambrian period, over 500 million years ago.

Nuclear ribosomal DNA still provides one of the most completetools for a multitude of molecular tasks in nematode taxonomy. Themain ribosomal locus in eukaryotic organisms consists of three genesencoding the 18S, 5.8S and 28S subunits of the ribosome. Betweenthese genes are the internal transcribed spacers 1 (ITS1, between the18S 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 groupare the intergenic spacers (IGS). Virginia and John Ferris showed theusefulness of these markers as taxonomic tools in extensive surveysof cyst-forming nematodes. Early examination by Barry Honda andcolleagues of these ribosomal repeats in Meloidogyne was phylogene-tically interesting because they demonstrated that a 5S rRNA gene islocated in the IGS region between the 18S and the 28S genes. Thisgenomic structure is unusual in higher eukaryotes, and differs fromthat of Ascaris and Caenorhabditis. The reiteration of these genes inthe genome makes their detection considerably easier than for singlecopy genes. In addition, ribosomal loci exhibit different degrees ofconservation along the sequence. rDNA has a general trend of sequenceconservation, tending to sequence divergence from 5” to 3” in tran-scribed regions. With these properties, by studying one single gene, itis possible to cover many taxonomic levels, which has rendered thistype of gene one of the most popular in nematode taxonomy.

Phylogeny

In addition to the accessibility of molecular data and to techniquesthat contributed to the rapid increase of molecular data sets, anoth-er feature that has stimulated research in nematode phylogeny hasbeen the availability of software packages for computational analy-sis. Programs for data base searching, sequence editing, data conver-sions, alignment, and phylogenetic analysis have been obtained for

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many computer configurations. Methods for estimating genetic dis-tance as well as several methods for phylogenetic inference, includ-ing UPGMA, neighbour-joining, and maximum parsimony, havebeen shown to be very helpful at resolving nematode phylogenies atdifferent taxonomic levels.

ITS2 and COI together have provided a powerful tool for phylo-genetic studies of closely related species, and different researchgroups have characterized the relationships between species in vari-ous genera of entomopathogenic nematodes (Heterorhaditis andSteinernema) and of plant parasitic nematodes as diverse asMeloidogyne, Heterodera and Globodera, Pratylenchus, Xiphinema andBursaphelenchus. The 18S rDNA was useful for phylogenetics at theother end of the nematode taxonomic spectrum. A paper of MarkBlaxter and collaborators in 1998 described, for the first time thephylogeny of the whole Nematoda phylum. However, a potentialdanger of studying a single gene or region of the genome is that thederived phylogeny may reflect the evolution of the gene rather thanthe organism. Care is needed also to ensure that only the homologoussequences are compared and that the problems of pseudogenes, geneconversion and duplication are considered. Another problem is that aset of markers appears to be lacking for use at intermediate taxo-nomic levels, i.e., between the genus and family. To overcome thesetwo limitations, one solution has been to sample a larger gene set;any biases presented by a single gene with a history not reflectingthat of the species is hopefully offset by the larger selection of genesthat reflect that proper relationship. Candidate nuclear protein-cod-ing genes that have proved to be useful in insect studies, such aselongation factor 1a, glucose-6-phosphate dehydrogenase, phosphe-nolpyruvate carboxykinase, are now being used for nematode phy-logeny studies. In a broader approach, David Bird and collaboratorsvery recently selected a set of orthologous genes initially identifiedfrom genome-wide EST analysis to describe the most robust phylo-genies for nine nematode species belonging to the order Tylenchida.

Diagnosis and species discrimination

The small size of most nematode species of agronomic importanceimplies that a limited number of morphological characters are avail-able for identification. In addition, identification methods must

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deliver accurate, rapid, reliable and affordable results. Molecularapproaches are becoming more widely applied to meet thesedemands, in part because of the relative simplicity of their applica-tion in the laboratory. In nematode diagnosis, significant progress hasbeen made for major plant parasitic nematode groups. As pointedout by Antoine Dalmasso and subsequent researchers, isozymes pro-vide a means of identifying the four most economically importantMeloidogyne species. Species-specific monoclonal antibodies whichallowed nematode quantification were developed by Arjen Schots inthe late 1980s for the separation of Globodera pallida and G. ros-tochiensis. Both types of approaches are routinely used by officialPlant Protection Services throughout the world. Subsequentattempts to develop molecular diagnostic methods have been basedon DNA hybridization techniques. The first studies were based onanalysis of RFLP. Although RFLP analyses allowed clear discrimina-tion at both interspecific and intraspecific levels, they could not beused routinely because they were time-consuming and requiredlarge amounts of DNA. Species-specific DNA probes were thendeveloped using repetitive DNA, among which are the highly reit-erated sequences known as satellite DNA (satDNA). The twoauthors of this chapter have illustrated its power as a diagnosticmarker in the plant parasitic nematode genera Bursaphelenchus andMeloidogyne. In particular, they developed a reliable and rapidyes/no system to discriminate quarantine species such as B.xylophilus and M. chitwoodi from congeneric species. In assays usingsatDNA as specific probes, an unambiguous separation of specieswas obtained with the main advantage of avoiding time-consumingDNA extractions. Because of the highly repetitive nature of satelliteDNA, 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 allowedthe selection of primers that lead to a specific amplification signal inPCR experiments. Beside satDNA, universal primers which bind tothe highly conserved regions present in the genes of all taxa, areavailable (e.g. mtDNA or rDNA). They have been used extensivelyin PCR technology for diagnostic purposes for most plant parasiticnematodes of agronomic interest.

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Current research and future prospects

New molecular approaches that are currently under investigationshould enable routine diagnosis. The real-time (quantitative) PCR(qPCR) technique relies on continuous monitoring of amplicon syn-thesis, and allows a faster detection and quantification of the targetDNA, without the need for laborious post-PCR gel electrophoresis.Very recently, rDNA-based qPCR was successfully applied for thedetection of the pinewood nematode, B. xylophilus and the cystnematodes, H. schachtii and G. pallida. The DNA microarray tech-nology provides a promising alternative for high-throughput geno-type-based diagnostics. The distinct advantage of this detectionapproach is that it combines powerful DNA amplification strategieswith subsequent hybridization to oligonucleotide probes specific formultiple target sequences. Briefly, hundreds to thousands of 30–50nt probes for specific targets are arrayed onto a single glass micro-scope slide, to which fluorescently labelled PCR product or genomicDNA to be tested is then hybridized and detected. Recently, a largeresearch project was initiated in the EU to investigate the feasibilityof a microarray-based method for the detection of the quarantinepathogens and pests of potato, including the quarantine nematodespecies M. chitwoodi.

Molecular markers have been successfully used in moleculartaxonomy for decades. However, the long-term goal of moleculardiagnostics is to develop protocols for the accurate and rapid identi-fication of all nematode species. The need for a speedier system ofidentifying species arises from the fact that only a small proportionof nematodes have been taxonomically described and that there aremany more nematode species to be identified. DNA barcoding is ataxonomic method which uses a short DNA sequence from a par-ticular region of the genome to provide a “barcode” for identifyingspecies. The novelty of the DNA barcoding proposal resides in itsenormous scale and proposed standardization. An unknown organ-ism may be identified from its sequence of a target gene. Fast neigh-bour-joining cluster analysis will link the unknown sequences withsome species in the database, usually its closest relatives. Molecularbarcode system projects have been developed in nematology mainlyfor soil and marine nematode identification. The ideal genetic mark-er for barcode development would combine ease of measurementwith a mix of conserved and rapidly evolving segments. In nema-

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todes, the message emerging from diagnostic research is that the ITSregion has sufficient information to identify most nematode species.Nevertheless, in some cases (e.g., mitotically parthenogenetic speciesof Meloidogyne and other species of recent hybrid origin), ITSwould misidentify species. For improved confidence in a successfulbarcoding system, the mitochondrial COI gene would be one goodcandidate that would complement ITS as a locus unlinked to thenuclear ribosomal gene region.

However, DNA barcoding has met with spirited reactions, somebroadly in support but many against. For the latter they see DNAbarcoding 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, characterdelineation, character state identity), in other respects both areinherently subjective (homology/alignment, divergence metrics).Sequence divergence in standard gene(s) is an extremely crudemethod for determining species limits, and more appropriate mar-kers that are potentially directly linked to species criteria, such asreproductive isolation, should be used. Therefore, it is worth per-sisting with the plurality of genetic, anatomical and ethological crite-ria 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 thefirst person to notice the developing females of potato cyst nema-todes (PCN) on a potato crop expressed his feelings, but perhaps itshould have been. The year was probably 1881 and the severe dam-age that cyst nematodes can cause was already known from themanner in which the German sugar beet crop had been devastatedby beet cyst nematodes, which were first noted on sugar beet in1859. Indeed, for quite some time, damage to potatoes was attrib-uted to the beet cyst nematode. However, the story begins earlierthan this, from the time at which the potato crop was first broughtto Europe from the New World following the Spanish conquests.The first imports of this new crop almost certainly came from Peruand reached Spain in the form of just a few tubers that had survivedthe long sea voyage and were perhaps left over from ships” storesfor the journey. These tubers had probably been handled quite a lotso it is likely that they carried no adhering soil and thus were free ofany cysts of PCN. The most likely date for the arrival of potatoes inEurope is 1570 but they were certainly not introduced by eitherFrancis Drake or Walter Raleigh, as believed by some people. Thetubers proved viable when they arrived and were bulked up suffi-ciently for potatoes to be offered for sale in Seville by about 1573.At first, yields were low in Europe because the potatoes from Peru

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were adapted to short-day growing conditions and tuberised poorlyin the long days of European summers. When tuber formation even-tually began in autumn, there was little time left for tuber bulkingbefore the frosts of winter arrived. Eventually, however, selectionswere made for lines of potatoes that would initiate tubers underlonger day growing conditions. This allowed the potato crop tospread further north in Europe and eventually to become a staplecrop throughout the region. Conditions for potato production werefound to be ideal in Ireland, with its plentiful rainfall and virtualabsence of frosts where the coast is warmed by the Gulf Stream, sothat reasonable yields could even be obtained from lines stilldependent on short-day conditions for tuberisation. The excellentgrowth of the crop in Ireland led eventually to a complete depend-ence on it as a staple foodstuff, encouraged by the confidence thathere was a crop that could not be destroyed by fire, a practice usedby tyrannical landlords wishing to keep the peasant population onthe brink of starvation, and therefore subdued, by burning some oftheir wheat crops.

The situation took a dramatic turn in the early 1840s whenweather conditions allowed late blight, Phytophthora infestans, tospread throughout the Irish potato crop. This resulted in two con-secutive years (1845/6) of crop failures with much resultant starva-tion and death of the population. The population of Ireland was fur-ther reduced when large numbers of people emigrated to the NewWorld to escape famine. A consequence of this tragedy was theimportation of new potato lines from South America in an attemptto identify sources of blight resistance that could be used in potatobreeding. It is almost certainly because these tubers carried soil con-taminated with cyst nematodes that led to the introduction of PCNinto Europe. Cyst nematode infestations initially are cryptic butbecome obvious when their populations reach densities largeenough to cause plant damage and crop loss. Depending on the fre-quency with which host crops are grown, this often takes about 20years, so field damage to potatoes was first noticed in the 1870s fol-lowing introduction of the nematodes in the 1850s. This c. 20-yearperiod is mirrored, for example, in the first record of damage byPCN confirmed on Long Island in 1941 (see Brodie, 1998, pp.317–331 in Marks & Brodie, Potato Cyst Nematodes, CABInternational) following movement of military equipment contami-nated with infested soil from Europe to Long Island, at the end of

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World War I. Similarly, an infestation appeared in upstate NewYork in 1967 following the use of contaminated farm machineryfrom Long Island.

The means of spread of cyst nematodes is diverse, and includesthe examples given above plus others such as in contaminatedguano from Peru (Inagaki & Kegasawa, 1973, Applied Entomology andZoology 8: 97–102) and birds” feet to the new polders of TheNetherlands. The possibility that birds may move cyst nematodes intheir guts and deposit them in their droppings was tested by Brodie(1976, Journal of Nematology 8: 318–322), who found that the pas-sage through the gut of some bird species was rapid (so unlikely toallow long distance transfer) and that cyst contents died after expo-sure to birds” excreta. This also made it unlikely that the guanofrom Peru was contaminated per se, rather that it was the use ofsacks previously used to carry potatoes that contaminated the guanowith cysts. Further weight was given to this theory of transport byFranco (1977, PhD thesis, University of London) who traced old lad-ing bills for guano imported into the UK. Most of the imports weremade through the ports of Liverpool, Hull and London, and PCNinfestations seem to have been centred on these three ports.Interestingly, most of the infestations that developed around Londonproved to be of Globodera rostochiensis, most of those round Hullproved to be of G. pallida whilst those around Liverpool containedboth species. This suggests that perhaps the numbers of cysts intro-duced to the UK were quite small and that the species passedthrough genetic bottlenecks.

As potatoes gained in popularity, and as improved cultivarswere produced by plant breeders, it became increasingly commonto ship large quantities of tubers between countries as seed.Inevitably the tubers carried some soil with them, and the soil fre-quently contained cysts in the days before the importance of PCNwas recognised. It is this means of spread that has resulted in theappearance of infestations in so many countries around the world,with Europe acting as a secondary distribution centre, and thespecies found depends on the species infesting the land in which theseed crop was produced.

It has become normal, at least for British people, to blame thestomach upsets they frequently suffer when travelling to warmercountries in the Americas on “Montezuma’s revenge”, and it wouldperhaps not be unreasonable for Montezuma to wish for revenge

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following the treatment of some of the Mexican people duringCortes’s conquest. However, the Incas of Peru suffered similar treat-ment 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 particu-larly difficult to control and to this day threaten the success of theEuropean potato industry, so perhaps might be regarded asAtahualpa’s revenge.

Proper recognition and description of the problem

Infestations of PCN have been discovered and reported from morethan 65 countries around the world, usually as developing femaleson small patches of heavily attacked plants to which attention hasbeen drawn. Such patches were quaintly referred to as “nematodenests” by O’Brien and Prentice (1930, The Scottish Journal ofAgriculture, 415–433), slightly less quaintly as “lenses” by Wood et al.(1983, New Zealand Journal of Agriculture 11: 271–273) and perhapsmost 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 werea species distinct from those found on sugarbeet, and which hedescribed as Heterodera rostochiensis, research was able to beginproperly on the characteristics of this species. O’Brien and Prentice,although still confused over the identity of the species, were able todescribe in detail the symptoms shown by attacked potato plants(being the first to use the term “feather dusters” for the appearanceof heavily attacked plants). They also described the PCN life cyclein detail, the interaction with the fungus Rhizoctonia solani, thedepth distribution of infestations, the relationship between diseaseseverity and soil pH, the rate of spread of infestations from an initialfocus (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 byapplication of farmyard manure.

World War II was probably a factor responsible for PCN com-ing to the fore as a pest in the UK. The home production of foodbecame of prime importance during the war and, because potatoeshave such a relatively high energy production per unit area of land,

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the total area of potatoes grown almost tripled during and immedi-ately after this war. The increase in area grown was accompanied byan increase in the frequency of cropping with potatoes and thisallowed PCN populations to multiply greatly, to the extent thatcropping frequency had to be reduced in certain parts of the coun-try. From data available at the time, Jones (1970, Journal of theRoyal Society of Arts 118: 179–199) estimated losses due to PCN inthe UK during the late 1940s and early 1950s at about 12% of pro-duction. Such an important problem clearly demanded research intoways of reducing these losses.

The burgeoning petrochemical industry provided an importantlead when it was shown that what was essentially a waste product,dichloropropane-dichloropropene (DD), was an effective soil steri-lant and would limit or even almost eliminate the damage caused byplant parasitic nematodes. For the first time, this allowed the dam-age caused by nematodes to be quantified, and the damage causedby PCN to be directly controlled. At the same time, the potential ofvarious collections of potato germplasm to provide genes for resist-ance to PCN was realised, and Ellenby (1948, Nature 162: 704)quickly found several important sources of resistance to PCN in theCommonwealth Potato Collection. One of these genes was tobecome known as the H1 resistance gene and was seized upon byplant 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, andfirst released in 1966. It quickly became widely grown, not onlybecause of its nematode resistance but also because it was liked byconsumers, a fact reflected in its current position in the potato salescharts of the UK – still top after more than 40 years. Such confi-dence did these developments promote that Nollen and Mulder(1969, Proceedings of the 5th British Insecticides and FungicidesConference, 671–674) presented a scheme using resistant potatoesand soil fumigation to control PCN that would allow potatoes to begrown three times every five years and still lead to the eventualextinction of the nematode. Yet another important development atthis time was the intensive research for better nematicides withmore specific action than that of a simple soil sterilant. This searchled to the release in the late 1960s of two extremely importantproducts in the fight to control nematode damage, namely aldicarband oxamyl, granular oximecarbamate nematicides.

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Nollen and Mulder realised that their scheme would only work ifthe nematode infestation was of what was then known as pathotypeA, and the scheme quickly became unworkable as pathotypes otherthan A came to predominate almost wherever H1 resistant cultivarswere grown. A further problem was that DD did not always achievethe degree of kill expected, due to soil conditions not being ideal atthe time of application, and excessive use of this fumigant quickly ledto build-up of undesirable residues in the soil. The oximecarbamatenematicides seemed to overcome this particular problem, althoughthey did have the disadvantage of high mammalian toxicity and beinghazardous to handle. They have remained a cornerstone of PCN man-agement programmes to the present day, although aldicarb has beenfound in groundwater on Long Island and tougher registration require-ments in the EU have threatened its future.

The availability of PCN-resistant potato cultivars led to therecognition of resistance-breaking pathotypes. Each time that newexamples were found they were given code letters. Pathotype A wascontrolled by the H1 gene and the next type of resistance found wasa gene, referred to as H2, discovered by Jack Dunnett (at theScottish Plant Breeding Station) in the diploid wild species Solanummultidissectum. This led to the identification of pathotype B as oneable to break the new resistance. Dutch workers quickly foundpathotypes they designated as C and D, and pathotype E was able toovercome all resistance, including H1.

The agricultural advisory services of the UK and TheNetherlands played important roles in the PCN story in that mem-bers of these services worked closely with farmers growing the newPCN-resistant cultivars. Colin Guile reported differences in thecolours of the “cysts” of pathotypes A, B and E but Freddie Jones atRothamsted dismissed these findings when neither he nor one of us(DLT) could detect these differences. Only later did it become clearthat Guile was referring to developing females rather than cystswhen he published his findings in 1967 (Annals of Applied Biology60: 411–419), an episode that underlined the importance of precisionin the use of scientific terms. His findings were supported by thoseof Derek Webley, another worker from the National AgriculturalAdvisory Service (NAAS, later to become the AgriculturalDevelopment and Advisory Service, ADAS), who found morpho-metric differences between the second stage juveniles of the patho-types (1970, Nematologica 16: 107–112). Further work, using disc

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electrophoresis on the so-called pathotypes, by Trudgill andCarpenter (1971, Annals of Applied Biology 69: 35–41) showed thatthe juveniles had important differences in their protein make-up, afinding taken much further in the two-dimensional electrophoresisstudies of Bakker et al. (1992, Fundamental and Applied Nematology15: 481–490). Overwhelming proof that we were dealing with tworeproductively isolated species of PCN rather than one with patho-types 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 betweenmales and virgin females of what she thought were five pathotypeA and five E populations. She found she was working with tworeproductively isolated groups, one consisting of three of the A pop-ulations and the other of seven populations, the five E populationsand two of the A. It transpired that the potato clone originally usedto identify the pathotype of the populations had extra resistanceeffective against two of the E populations used. Consequently, thesetwo populations were misclassified as A rather than E.

These observations and findings opened the door to a newdescription of potato cyst nematodes, as two species rather thanone. The person assigned to this task was the new boy atRothamsted, Alan Stone (1974, Nematologica 18: 591–606), who waslater to succeed Freddie Jones as head of the NematologyDepartment at Rothamsted in 1979. The new species (Heteroderarostochiensis and H. pallida) soon became known as G. rostochiensisand G. pallida, when Edda Behrens pointed out the value of theGlobodera genus, first suggested as a sub-genus by Skarbilovich in1959 (Acta Parasitologica Polonica 7: 117–132). Also, the problem ofpathotype classification assumed greater importance. Alan Stone,together with colleagues in Europe (John Kort, Hans Ross, JürgenRumpenhorst) presented their scheme, in 1976, at a EuropeanSociety of Nematologists (ESN) meeting in Dublin. It used a rangeof potato clones with different levels of resistance to classify popu-lations into five pathotypes of G. rostochiensis and three of G. palli-da. The classification was not absolute and depended on a thresholdmultiplication rate of > or < 1 in a standard pot test. The schemewas subsequently recognized as being flawed as it did not takeaccount of the effects of environmental factors on PCN multiplica-tion rates. Also, values apparently related to one pathotype could

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also be generated by mixtures of two other pathotypes within apopulation. Nevertheless, the scheme was seized on as a means ofclassifying the virulence of field populations of PCN and is still usedby some people to this day. A similar scheme was developed byCanto and de Scurrah (1977, Nematologica 23: 340–349) at theInternational Potato Center (CIP) in Lima, Peru, initially using thesame differential clones and only differing from the Europeanscheme in the code numbers given to the pathotypes. As more dif-ferentials became available from the germplasm held at CIP andfrom breeding exercises, this second scheme eventually differedslightly from the European scheme. Perhaps the most rationalscheme is that offered by Nijboer and Parlevliet (1990, Euphytica49: 39–47), who recognised three pathotypes of G. rostochiensis andonly 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 tofocus 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 difficultto breed from, and little was known about the variation in virulenceof field populations of G. pallida towards this resistance. A collabo-rative project between several European countries showed thatthere were considerable differences in virulence of G. pallida popu-lations and, coincidentally, the Plant Breeding Institute in Cambridgewas using a particularly avirulent G. pallida population to screenthe progeny of their breeding. Much effort over more than 30 yearshas gone into breeding for resistance to G. pallida but few commer-cially acceptable cultivars have been produced, and none has fullresistance or the acceptability to make substantial inroads into thecommercial market – only 8% of current potato land is plantedwith G. pallida resistant potato cultivars in the UK.

From the mid-1980s, with the production by breeders of culti-vars with resistance from different sources, attention began to focuson the range of virulence in PCN and its possible relationship to theoriginal introductions into Europe. It was already evident that therehad been more than one introduction of G. rostochiensis into Europe

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because all UK populations were avirulent on cultivars with the H1gene 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 fragmentationof the genepool as it spread within Europe. However, comparisonswith populations from South America revealed a much greater rangeof variation in South American populations of G. pallida than in thosefrom Europe, although later studies on the artificial fragmentation ofindividual European G. pallida populations sometimes revealed asbroad a range of virulence as that found in naturally occurringEuropean field populations (Phillips et al., 2002, Nematology 4:655–666). Studies with mitochondrial DNA (mtDNA), which isexclusively maternally inherited, were done by Vivian Blok andMiles Armstrong at the Scottish Crop Research Institute (SCRI)with the aim of identifying the distinct introductions of PCN intoEurope. They discovered that G. pallida has a unique configurationof its mtDNA. In metazoans, the 12 or 13 genes that comprise themtDNA 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 iscompounded by variation in the complement of scmtDNAs foundin different populations of G. pallida, with some populations lackingparticular scmtDNAs while others have more than one variant ofthe same scmtDNA. Hybridisation studies by Armstrong et al.,showed that some mitochondrial genes occurred on more than onering and sequencing of five of these circles (Gibson et al., 2007,Journal of Molecular Ecology, in press) has revealed that three ofthese genomes are mosaics, and share long multigenic fragments.These mosaic structures are likely to be the result of intermitochon-drial recombination (not thought to occur in the metazoa) and this issupported with evidence for recombination in the repeat region ofthe 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 intoEurope. The mitochondrial genome of G. rostochiensis is similar butwith 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 otherrespects. Molecular studies by groups in the UK, The Netherlandsand France, and John Marshall in New Zealand indicated that popu-lations of G. pallida are exceptionally heterogeneous. This is proba-bly necessary as it is a relatively immobile soil pathogen with a nar-row range of hosts, so it needs to be able to respond rapidly toselection pressures imposed by those hosts, many of which possessgenes for resistance. Populations can become extremely large, some-times exceeding 100 eggs/g soil (equivalent to c. 3 × 1011 per ha).Such populations are extremely damaging, and Con Ellenby was thefirst to suggest that the sex of the developing juveniles was influ-enced by their nutrition, an adaptation that probably helps limitpopulations and protects the host from being killed. He suggestedthat when populations are high and the host is damaged, the com-petition for resources means that poorly nourished juveniles becomemale and only the well nourished ones become female. DLT pro-duced persuasive data to support this suggestion but it was left toMugniery and Fayet (1981, Revue de Nématologie 4: 41–45) to pro-duce conclusive evidence. They developed a micro-techniquewhereby almost 100% of the inoculum survived to become adultand showed that the sex ratio varied with inoculum density, themajority (>90%) of juveniles becoming female with only one nema-tode per plant and an increasing proportion becoming male as theinoculum density increased.

PCN is also exceptional in the changes it induces at its feedingsite. The host cells develop into an enlarged, multinucleate syn-cytium that supplies the developing juvenile with all its food (Jones& Northcote, 1972, Journal of Cell Science 10: 789–809). Nutrientsare extracted through a specialized feeding tube secreted from thestylet tip, first recognised in Rotylenchulus reniformis by Rebois(1980, Nematologica 26: 396–405), that acts as a molecular sieve andprevents both the stylet lumen from becoming blocked and the hostcells being killed through the loss of vital organelles. Secretions areinjected that initiate and maintain syncytium development and,with the development of molecular techniques, these have been thefocus of much research. Several studies have reported monoclonalantibodies that recognise secreted proteins but it was not until thelate 1990s that the first proteins were identified and genes cloned.

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These were the secreted cellulases of both cyst and root-knot nema-todes (Smant et al., 1998, Proceedings of the National Academy ofSciences, USA 95: 4906-4911; Rosso et al., 1999, MPMI 12: 585–591).Subsequent work led to the identification of a variety of otherenzymes able to degrade plant cell walls, including pectate lyases(Popeijus et al., 2000, Nature 406: 36–37). Remarkably, the genesencoding these cell wall degrading enzymes seem to have beenacquired by horizontal gene transfer from bacteria. This process ofgene transfer seems to have occurred independently on several occa-sions within the phylum Nematoda; cell wall degrading enzymesand chorismate mutase are present in root-knot and cyst nematodes,a gene similar to NodL from Rhizobium is present in root-knotnematodes and, perhaps most surprisingly, cellulases that appear tohave been acquired horizontally from fungi are present inBursaphelenchus (Jones et al., 2005, Nematology 7 : 641–646). Othersecreted proteins present on the nematode surface that may beimportant in suppressing or neutralising host defence responses havebeen 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 cDNAlibraries from infective juveniles or made from excised gland cellshas led to the identification of a large number of secreted proteins.As information on the proteins present in nematodes continues toaccumulate from genome and EST studies, the challenge for thefuture lies in deciphering the function of these proteins in thehost/parasite interaction. Studies on microarrays of plant genes sug-gest that several thousand genes may be up- or down-regulated as aresult 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 interest-ing features, not least of which was the degree to which they couldvary in their ability to tolerate nematode attack. Up to this point,tolerance had been largely considered simply as an expression ofresistance, but it was then realised that resistance and tolerancewere independently inherited desirable characteristics, with resist-ance determining the degree to which nematode reproduction is

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possible and tolerance determining the degree to which the crop isdamaged for a given level of infestation. This realisation was in nosmall part due to the work that we (KE and DLT) did on a long-term field trial established by Jones at Woburn Experimental Farm.The treatments had resulted in a series of plots with a range of pop-ulation densities of G. rostochiensis on which non-resistant PentlandDell and resistant Maris Piper were grown. There was also theadded benefit that G. pallida had been introduced with the seed onone of the Maris Piper plots. We wanted to elucidate the mecha-nisms behind the expression of PCN damage and found manyeffects, mostly attributable to the effects on water and major nutri-ent uptake. Potassium concentrations and, to a lesser extent, thoseof phosphate plummeted as PCN population densities and cropdamage increased. We hypothesised, incorrectly as would be shownlater, that reductions in K uptake were central to the impact ofPCN on potato growth (experiments with different inputs of N, Pand K showed that it was effects on the uptake of N and, some-times in clay soils, P that were important; the plants were able totake up K in luxury amounts). Perhaps the most important observa-tion to emerge from this experiment, however, was that the resist-ant cultivar Maris Piper was much less damaged at a given PCNdensity than the non-resistant Pentland Dell. This sparked the longerterm interest that we developed in tolerance, an interest shared byArne Mulder in The Netherlands.

Maris Peer is an early maturing cultivar with no PCN resistanceand we both independently (by this time DLT had moved to SCRI)identified it as very intolerant. However, it was not as intolerant asMaris 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 sus-ceptible to invasion by the wilt fungus Verticillium dahliae oncenematodes had invaded the roots. This was in contrast to anotherearly cultivar, Pentland Javelin, also with the H1 gene, which wasvery tolerant of PCN attack and, because of basic differences in rootstructure, did not suffer massive invasion by the fungus.

Our work on tolerance showed the importance of environmen-tal effects when studying the relationship between nematode densi-ties, crop yield and PCN multiplication rates. This type of work wasalso beloved of Wim Seinhorst from The Netherlands, who had pre-sented an early version of his theories at the ESN meeting in

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Antibes (1966), the first ESN meeting attended by DLT. In responseto a question from DLT, Seinhorst maintained that proportionalyield loss was independent of external factors. At a NATO work-shop several years later in Martina Franca, Italy (1985), he stillnursed this idea, admitting that he had fixed one of the parametersin the equation for his relationship from the results of his ownexperiments done under carefully controlled conditions. The mainfeatures of practical relevance of Seinhorst’s curves are T, the toler-ance limit or threshold nematode population density beyond whichdamage occurs, m, the minimum yield achieved by the crop as nem-atode density increased, and the constant z, which determines therate of yield reduction with increasing PCN density. It was the valueof z that Seinhorst eventually fixed. Support for Seinhorst’s theorycame at the same meeting from a young Ed Caswell, who had datafrom three sugarbeet fields in California. Ed had measured the pre-planting density of beet cyst nematode in 100 small plots across thefields and had measured plot yields at harvest time. Plotting yieldagainst nematode density produced a scatter diagram but, onHoward Ferris’ advice, Ed had taken the means of yield and nema-tode density in seven or eight error bands and plotted the meansagainst one another. This produced three perfect Seinhorst curves,with slightly different values of T, m and z clearly shown. Since thistime, Seinhorst curves have been produced by many workers, mostfrequently for potatoes by the Italian group that includes NicolaGreco and Mauro Di Vito, among others. Interestingly, however, thevalue 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 theoreticallower limit of determination of PCN population densities unless,like Seinhorst, one is working with carefully prepared levels ofinoculum in controlled conditions.

Seinhorst’s pot studies also yielded an equation for populationdynamics. For the mathematically challenged (e.g. DLT!), this equa-tion was even more impenetrable than that for the effect of PCNon yield. The multiplication rate was at a maximum when popula-tions were small (<T) and the multiplication rate decreased as thepopulation density increased, until the population density reached amaximum. Eventually, if damage was severe, the population at har-vest could be less than at planting as most juveniles that hatchedfailed to develop to maturity.

Seinhorst laid the theoretical base, but his work could not be

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directly used to provide advice in the field. Not least, as Seinhorstpointed out, were the problems of accurately quantifying field pop-ulations. Michiel Oostenbrink (another outstanding Dutch nematol-ogist) proposed that, for practical purposes, the relationshipbetween PCN population density at planting and crop loss was lin-ear and that the parameter T was so small as to be irrelevant in thefield. Eric Brown in the UK also used a linear regression approach toexpress damage relationships, and the way in which this works sowell when comparing the performance of different cultivars wasshown by KE with Mike Russell (Evans & Russell, 1990, Annals ofApplied Biology 117: 595–610), by taking PCN density and yieldmeasurements from many plants of each cultivar grown in a singleplant-plot experimental design.

The arrival of more and more powerful desktop computers madepossible the development of “expert systems” for managing pests andpathogens in the field. As a theoretical basis was available for quanti-fying PCN damage and population dynamics it was an attractiveoption for developing an expert system. However, differencesbetween cultivars in their tolerance of PCN damage, and observationsby an ADAS nematologist, Norman French, that damage was moresevere on sandy than clay soils, presented further challenges. Toobtain 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 patch-work of plots with a wide range of G. pallida population densities.Each trial tested several cultivars differing in their tolerance andresistance. To minimise variation and sampling errors the sample areain each plot was kept as small as practical, and three independentsoil samples were taken, each made up of 40 small (5g) cores. Later,trials in farmers” fields used natural variations in PCN populationdensity to test the model and obtain additional data on the effective-ness of nematicides and PCN decline rates between crops. Theresults confirmed the influence of environmental factors on theparameters in the Seinhorst equations, and an expert system for PCNmanagement has been developed and is awaiting release.

Tom Been and Carrie Schomaker in The Netherlands have alsodeveloped an expert system for PCN management based on Seinhorstequations (Been et al., 1995, pp. 305–22 in Haverkort, A.J. &MacKerron, D.K.L., Current issues in Production Ecology, KluwerAcademic Publishers). In addition, they have studied the intensity of

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sampling required to detect PCN and to obtain reliable estimates ofpopulation densities. This arose from concern to keep the large area ofseed potato land in the Dutch polders free from PCN. Early detectionis essential as part of a strategy to detect foci of infection and minimisetheir spread. However, with any system for managing PCN, the inten-sity of sampling possible is limited by cost, and will almost always beless than that necessary to give the required confidence limits.

The growing of cultivars with partial resistance to G. pallidahighlighted another problem, one that had been foreseen by FreddieJones and referred to in his theoretical papers and made a feature ofthe population dynamics models that he developed with RobKempton (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 thatwhenever a cultivar with partial resistance is grown, the populationof PCN exposed to it is selected for increased levels of virulencetowards 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 carefulselection work with Alan Stone and has since continued it over sev-eral years in Northern Ireland (1990, Annals of Applied Biology 117:385–397). This work has shown that many populations of G. pallidacan be selected to become almost 100% virulent towards cultivarswith partial resistance within less than ten generations. More badnews was in the offing when it was found that, in the absence of ahost crop, populations of G. pallida often seemed to decline muchmore slowly than the 32% per annum average found by Jones (1966,Report of Rothamsted Experimental Station for 1965, 301–316) for alarge number of G. rostochiensis populations in the UK. Some G.pallida populations seemed to decline at rates as low as 10% perannum. Although no PCN populations have been found to showresistance to oximecarbamate nematicides, further bad newsemerged when it was found that G. pallida seemed to be less sus-ceptible than G. rostochiensis to these nematicides. This appeared tobe due to G. pallida second stage juveniles reaching a peak ofhatching later than G. rostochiensis after planting the potato crop(perhaps 6 weeks as opposed to 3 weeks), at a time when the con-centration of nematicide in the soil had declined to relatively inac-tive levels. With the observation that the soil microflora could beselected by repeated application of oximecarbamates to hasten their

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breakdown, and the discovery that G. pallida juveniles carried morelipid reserves than G. rostochiensis (Robinson et al., 1987, Revue deNématologie 10: 343–348) and so were better able to resist the actionof the chemicals because they were able to survive until the chemi-cals had broken down to inactive levels (oximecarbamates are morenematostats than nematicides and nematodes starve to death whilstimmobilised), the bad news was complete. This meant that resist-ance was not as effective against G. pallida as against G. rostochien-sis (because even the best cultivars were only partially resistant andpopulations became more virulent each time a particular resistorwas grown); rotation was less effective against G. pallida because itsdecline rate in the absence of potato crops is less than that of G. ros-tochiensis; and nematicides were less effective against G. pallida. Inother words, all of the “traditional” components of integrated man-agement packages for PCN were less effective than they had beenwhen many field populations were of G. rostochiensis (see Evans,1993, Nematropica 23: 221–231), a consequence of application ofthose very components – Atahualpa’s revenge indeed.

Thus, G. pallida is now predominant in most European fieldpopulations of PCN and will continue to become more so. Themost traditional method of control is crop rotation and UK rota-tions for potato crops average 5.7 years (see Minnis et al., 2002,Annals of Applied Biology 140, 187–95), which is far too short tocontrol this species. In Peru, even with the relatively very pooryields that they obtained, which reflects the relatively small amountof root available for PCN reproduction, the Incas insisted that grow-ers follow a 7-year rotation. They did not know the cause but theydid know that land developed a “potato sickness” if cropped morefrequently with potatoes. New methods for managing PCN popula-tions are, therefore, being investigated at the moment. These includethe development of the sophisticated model referred to above topredict population density changes and crop yields, targeted applica-tion of nematicides so that they are used more effectively, trapcrops to reduce PCN population densities, flooding of land, and bio-control. Effective ways of mapping the distribution of PCN withinfields have been defined and the information used experimentally todirect the application of nematicides to only those parts of the fieldwhere populations exceed damaging densities (Evans et al., 2003,Precision Agriculture 4: 149–162). However, accurate information onPCN distribution requires a heavy investment in sampling, and so-

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called “spatial application” of nematicides is probably only practicalwhen it is intended to use both a fumigant and a granular nemati-cide, with the more expensive fumigant applied in a spatial mannerand the granular nematicide applied as a blanket treatment to pre-vent build-up of troublesome population densities from non-damag-ing 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 pota-toes as the trap crop or, more recently and with promising levels ofsuccess, a non-host of PCN the weed species Solanum sisymbriifoli-um (Timmermans, 2005, PhD thesis, University of Wageningen, TheNetherlands). In certain potato production areas, such as the fen-lands of eastern England, it is possible to build small containmentbarriers around the large flat fields and flood them by pumpingwater onto them. This has proved a highly successful way of con-trolling PCN, with essentially 100% kill after a period of 13 weeksof flooding (Barker, pers. comm.). Unfortunately, only a proportionof the land used for potato production can be treated in this way.Biocontrol of some plant parasitic nematodes (e.g. Meloidogynespp.), using species of nematophagous fungi, may be a commercialproposition but so far has remained an elusive goal with PCN.

Diagnosis of the PCN species

Although G. pallida is becoming the predominant species of PCNwherever management tactics that favour it are deployed, and, ofcourse, where it has actually been introduced, a first step in design-ing PCN control programmes is the determination of which speciesis/are present. Following Alan Stone’s description of G. pallida, theinformation required for identification of the two species using mor-phological and morphometric observations was available. However,the most useful information, on stylet size and shape and the colourof developing females, had already been provided by Derek Webleyand Colin Guile, and formed the basis on which species identity wasconfirmed for a number of years. The disadvantage was that itrequired experience and expertise to make the required observa-tions, and the procedure was time-consuming if sufficient individu-als were to be examined to estimate with accuracy the proportionsof the two species in mixed populations.

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A significant improvement was provided by Fleming and Marks(1983, Annals of Applied Biology 103: 277–281) when they used iso-electric focusing on polyacrylamide gel to separate aqueous extractsof proteins from whole cysts. They showed that the two species ofPCN contained specific proteins of similar isoelectric point (at pH5.7 and 5.9) characteristic for one species or the other. Followingstaining on the gel, densitometer measurements of the bands allowedthe relative proportions of the two species to be estimated, all withno experience or knowledge of nematode morphology and using amethod that merely required the following of a simple procedure.

The two diagnostic proteins were shown to have similar molec-ular weights and Robinson et al. (1993, Annals of Applied Biology123: 337–347) raised monoclonal antibodies to these proteins in anattempt to establish a simple immunoassay for the two species ofPCN, an objective completed by Curtis et al. (1998, Annals ofApplied Biology 133: 65–79). Unfortunately, this potentially usefulassay followed the fate of increasing numbers of promising lines ofwork in science generally when the funding for the work expired.The utility and convenience of immunoassays also prompted aDutch group led by Schots et al. (1992, Fundamental and AppliedNematology 15: 55–61) to develop a system based on three antibod-ies for the determination of the relative proportions of PCN speciesin species mixtures, but again it was never widely taken up.

Perhaps it was inevitable that immunoassays would lose out toDNA-based techniques in the long run, and it now seems likely thatreal-time PCR procedures will provide the standard assay for PCNspecies in the future. Andy Barker and Simon Atkins at Rothamstedhave designed species-specific primers for such a procedure andBates et al. (2003, Molecular Plant Pathology 3: 153–161) at theNational Institute of Agricultural Botany in Cambridge, UK, havepublished a working procedure.

The ultimate goal in assays would be to determine how a givenfield population of PCN will react to a given resistant cultivar. Butas already mentioned, the resistance in the best G. pallida resistantcultivars is polygenic in nature, with the implication that the viru-lence that allows PCN populations to overcome the resistance isalso polygenic. It may be possible to produce, by controlled matings,PCN test populations that have defined levels of virulence to culti-vars with defined levels of resistance, as attempted by Conceiçao etal. (2005, Nematologia Mediterranea 33: 75–85) and in a similar man-

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ner to the avirulent populations of G. rostochiensis produced byJanssen et al. (1990, Revue de Nématologie 13: 265–268). With suchmaterial available it may then be possible to design the necessaryspecific DNA primers.

Hatching factors for PCN

No account of research on PCN would be complete without refer-ence to the work that has been done on hatching factors, pioneeredby, amongst others, Triffitt (1932, Journal of Helminthology 10: 181-2). Few research projects have looked so promising but remained sodaunting. The idea of using the PCN hatching factor as a weapon incontrol programmes dates back at least to 1932, when Triffitt, stillunder the misapprehension that she was dealing with a potato raceof H. schachtii, found that grass root secretions would cause hatch-ing and that a field trial showed that a grass/cereal mixture reducedcyst contents by 23.6% whilst a cereal only treatment affected cystcontent only negligibly.

Triffitt even began work to identify the active hatching factor,and numerous others have tried since. Some success was obtainedwith identification of hatching factors for other species of cyst nema-todes, notably H. glycines, but the goal remained elusive for manyyears for PCN. Clarke and Perry (1977, Nematologica 23: 350–368)produced a schematic for the hatching process in PCN that drewupon many observations made in other studies, and revolved aroundthe concept that the hatching factor induces a change in the perme-ability of the eggshell, probably by displacing internal calcium ions(Atkinson et al., 1980, Annals of Applied Biology 94: 103–109). Thisallows the disaccharide (trehalose) that is found in the perivitellinefluid 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 tobecome active, at which point it cuts open the eggshell and emergesinto the cyst interior and, via apertures in the cyst wall, into the soil.This general scheme remains the accepted theory of the succession ofevents in the PCN hatching process.

Devine et al. (1996, Annals of Applied Biology 129: 323–334)showed that several different components of potato root exudatesmay induce hatching of PCN, and Devine and Jones (2000, Annals of

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Applied Biology 137: 21–29) tested the idea that exogenous applica-tion of hatching factors to soil might stimulate PCN hatch sufficient-ly for it to represent a useful control tactic. One of the best artificialhatching factors found, picrolonic acid, had already been shown toachieve, at best, a 33% decrease in the PCN population, but wasunsuitable for routine use due to its binding to soil particles(Whitehead, 1977, Annals of Applied Biology 87: 225–227). Devine andJones obtained quantities of both potato and tomato root leachates –the potato root leachate from potato plants grown in boxes of graveland the tomato root leachate from the return feed pipe of a com-mercial hydroponic tomato production system. They obtained up to50% reduction in the size of a population of G. rostochiensis, whichthey 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 frompotatoes was finally achieved in a Dutch laboratory when a struc-tural formula was produced for an extremely active hatching factordesignated solanoeclepin (Schenk et al., 1999, Croatia Chemica Acta72, 593–606). The extreme activity of this compound meant thatonly a very small quantity would be required to stimulate sufficientsuicidal hatch in the field to achieve a valuable degree of control.However, the problem remains of synthesis and delivery. Perhapsthe day will yet come when another crop is engineered to producePCN hatching factor and thereby greatly reduce the PCN popula-tion and make it safe to grow potatoes without resort to nemati-cides 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 sci-ence and reflects the work done in the field of nematology general-ly. Many laboratories and individuals have contributed over theyears to the huge volume of research on PCN and it is impossible tomention all of them, so this account has necessarily been a very per-sonal view that we have given. However, two groups perhapsdeserve special mention – the Nematology Department atRothamsted Experimental Station in Harpenden, UK, and thenematologists from Wageningen, The Netherlands. There were true

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leaders of PCN research in both of these centres, most notablyFreddie Jones, David Fenwick and Alan Whitehead at Rothamsted,and Wim Seinhorst, Michiel Oostenbrink and Hugo den Oudenfrom Wageningen. These were the giants on whose shoulders mostof us have stood. Their various research interests came together inthe modeling work of Jones and Seinhorst, investigations of nemati-cides by Whitehead and den Ouden, but perhaps most memorablyin the Fenwick Can originally designed, in 1940, at Rothamsted butgiven the “sloping bottom” of Oostenbrink for convenience of use(Fig. 1), and which remains the main method for routine quantita-tive extraction of PCN from soil. Some of the nematologists whoworked at Rothamsted can be seen in Figs. 2, 3 and 4.

Despite this substantial and sustained research effort, PCNremains a major constraint to potato production in many countries.There is a continuing need for innovative, high quality research toprovide growers with new weapons to tackle this most intractableof pests.

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Fig. 1. The iconic Fenwick Can apparatus for recovering cysts from soil. Althoughnot obvious, the model shown features Oostenbrink’s sloping bottom.

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Fig. 2. Basil Goodeyand Freddie Jonestaking coffee withJohn Webster inthe salubrious cof-fee room of theNematologyDepartment atRothamsted, circa1964.

Fig. 3. DavidFenwick in hisfavourite pose –with the ladies ofthe NematologyDepartment.

Fig. 4. BertieWinslow andFreddie Jones goback a long waytogether! (with alittle help fromChris Doncaster)

8.CEREAL CYST NEMATODE COMPLEX

ROGER RIVOAL

INRA Liason Officer.Formerly, UMR INRA/ENSAR, Biologie des Organismes et des Populations Appliquéeà la Protection des Plantes (BiO3P), 35653 Le Rheu, France.

Cereal cyst nematodes (CCN) form a complex of several closely rela-ted species which are widely distributed, and found wherever gramina-ceous 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. latiponsfrom the Mediterranean area, H. hordecalis from northern Europe,H. filipjevi from eastern Europe and several others, to total morethan 12 species (Wouts, et al., 1995, Nematologica 41: 575–583).

Development of research in this important group of nematodesoccurred after World War II when European cereal production wasincreased to satisfy human and animal needs. Research focussed onthe main species, H. avenae, which infested more than 50% of thecereal fields of Europe. Oat cultivation, with its high host suscepti-bility, was certainly responsible for the large increases in the popula-tions of this nematode, previously called the “oat cyst nematode”.This species reduced yields of spring-sown oats and barley inSweden and Denmark. Even though agriculture and transport mech-anisation had reduced oat production (the main nutrient for horses)by the middle of the 20th century, the damaging nematode popula-tions, developed on this cereal, seriously affected the maize crop ofnorthern France and other parts of Europe. Heterodera avenae wasdemonstrated to be polyphagous on cereals, and was a severepathogen of wheat in south-eastern Australia and in Asian regionscausing “Molya disease” on wheat and barley. Many experiments, indifferent countries, demonstrated that the degree of damage wasdetermined principally by the size of the initial infestation, as modi-fied by soil and climatic conditions and the crop species and culti-var. Synchrony of H. avenae emergence with cereal sowing time

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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 wasobserved with winter sown crops in southern areas. In northernareas damage was more frequently observed with spring varietiessown 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 nema-tode justified the comparative studies mainly between Australianand European populations of H. avenae. Two ecotypes appeared,differing in the induction or suppression of dormancy (diapause)according to temperature conditions. For populations in theMediterranean climate the diapause was obligate, acting when hotdry conditions prevailed and being suppressed when the tempera-ture fell and soil moisture increased. However, populations frommore or less temperate climates had a more facultative diapausefrom July to the end of winter, and this was suppressed by chilling,ensuring emergence of juveniles when soil temperatures increased inthe spring. Reciprocal transfers of northern and southern popula-tions did not alter their basic hatching rythms which resulted cer-tainly 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. ave-nae with chemical nematicides based on the results of experimentsusing fumigants twenty years earlier against nematode pests and othersoil pathogens on a variety of crops. When the delay between nemati-cide application and juvenile emergence was short, an application ofnematicides improved crop production so much that the use of statis-tical analysis was unnecessary to differentiate the treated plots fromthe controls! In Australia, engineering low rate distributors of fumi-gants or systemic nematicides enabled economic control of the nema-todes. In several parts of the world, the use of such chemical nemati-cides was used to demonstrate that these invisible, and frequentlyunknown pests, the nematodes, were responsible for minimizing cropyields (Brown, 1984, Journal of Nematology 16: 216–222).

Fortunately, at the same time, alternative control methods basedon plant resistance were being investigated. The initial research wasdone in northern Europe (Sweden, Denmark) where oat, barley andwheat varieties were found that reduced or inhibited nematode mul-

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tiplication. Quite early, complete resistance was found in the barleycultivars Drost, LP191 and Morocco and in the wheat cultivar Loros.The investigators, being true breeders, demonstrated that the inheri-tance of resistance was based on a restricted number of genes: Ha1,Ha2 and Ha3 for the three barley cultivars, respectively, and Ccn1 forthe wheat, Loros. This research, and the results achieved, was ofgreat importance in nematology as a prerequisite for successful cropproduction. Intensive screening for resistance was further developedin several countries and resulted in the identification of additionalsources of genetic resistance in the various cultivated cereals andrelated wild species. Several of these genes were introduced into thebreeding programmes which produced commercial resistant varietiesof oats, barley and wheat, particularly in northern Europe and inAustralia. Significant progress was achieved, using molecular technology, to identify markers for various types of CCN plantresistance and for developing marker-assisted selection (MAS) topyramid resistance genes to H. avenae in new cultivars in Australiaand western Europe (Nicol et al., 2003, Nematology Monographs andPerspectives 2, 1–19).

Screening for resistance sources using a wide range of popula-tions of H. avenae, and also of H. filipjevi, H. hordecalis and H.latipons, showed variation in resistance efficiency depending uponthe species of nematode and the population tested. Particularlywithin H. avenae, the virulence of populations, determined by theirability to overcome resistance genes, enabled the differentiation ofpathotypes. These pathotypes have been recognised using theInternational Test Assortment of barley, oat and wheat cultivars,with their respective resistance genes, as developed by SigurdAndersen in Denmark. Seeds of the differential hosts were furtherdistributed in different European and Asian countries to enable acomparison of the virulence spectrum among the different nema-tode populations tested. Heterodera avenae populations were divid-ed into three pathotype groups based upon the reactions of barleycultivars with the particular resistance genes (Rha1, Rha2, Rha3),each pathotype group being further divided by reactions of otherdifferentials which led to double integer codes (Andersen &Andersen, 1982, EPPO Bulletin 12, 379–386). This nomenclaturebecame difficult to use because it was based on a simple descriptiveapproach of incompletely understood interactions. A more flexiblescheme, based upon the same differential reactions but using a deca-

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nary notation of differentials, was further proposed in order to giveunambiguous labels to each pathotype. Virulence phenotypes werelabelled by using the sum of the number of susceptible differentialsfor each of the cereal species. Both of these differentiation schemesensured the existence of seven to eleven virulence phenotypesresulting from previous extensive selection pressure or the recentuse 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 raisedthe question as to the true identity of the species involved. Thepathotypes were difficult to distinguish morphologically despite theuse of improved optic and electron microscopy, coupled with auto-matic image analysis processing which enhanced the accuracy ofobservations and measurements of cyst and juvenile features.Controlled matings between pathotypes of H. avenae confirmedthat they belonged to the same species, and testing of the F2 and F3progenies of the pathotypes on barley cultivars confirmed that thisspecies was more complex than previously considered. In the 1980s,and more recently, biochemical and molecular techniques based onthe analysis of proteins and DNA polymorphisms have enabled thereliable identification of most species involved in the CCN complex.These analyses demonstrated, in particular, that several populationsof the “Gotland race” (Sweden) were in fact western European iso-lates of H. filipjevi (R. Holgado and others). Until that time, molecu-lar technology had failed to distinguish pathotypes of CCN andmarkers for virulence traits, but rather had established the newtaxon, Heterodera australis, even though no morphological featuresof the cysts and juveniles differentiated this new species from H.avenae sensu stricto! (Subbotin et al., 2002, Russian Journal ofNematology 10: 139–148).

Procedures for extracting nematodes and preparing them foridentification have not evolved very much over the years. Populationdynamic studies were based on standard sampling (soil cores) andextraction procedures. The Kort elutriator was specifically adaptedfor cyst extraction from wet soils and was demonstrated to be moreefficient than the old Fenwick can. Centrifugation with sugar orMgSO4 solutions improved the extraction of cysts or eggs from soilfor studies on population dynamics and resistance. Significantimprovements have been made in the use of resistance/virulence

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tests. Originally, soils were imported from infested areas and distribu-ted in pots and containers where individual plants were sown toevaluate their capacity for hosting the populations tested. This pro-cedure was replaced by the production of cysts that were to be usedas inoculum being isolated by a plastic net so as to be separatedfrom their progenies. Finally, the increase in knowledge on breakingdiapause allowed us to obtain juveniles in the numbers needed toperform miniaturized tests in tubes filled with sand/kaolin mediumor in Petri dishes with agar. The Petri dish technique was betteradapted for controlled matings between single females and males.Miniaturized tests in Petri dishes were adopted to establish thatpopulations of H. avenae differed in the capacity of the juveniles toproduce females (part of the fitness component) which was impor-tant for designing virulence/resistance investigations and for themanagement of nematode densities (Rivoal et al., 2001, Nematology3, 581–592).

In cereal production from low-value crops, strategies for controllingCCN population densities relied on the adoption of integrated controlmeasures. In several countries in western Europe, manipulation of den-sities by growing crops with different host capacity in field microplotsallowed the determination of damage thresholds for different cerealsand for the population changes during their culture. In the 1980s, inAustralia, a bioassay (SIRONEM) was developed to indicate the poten-tial for damage to occur, and that helped in advising on control optionsusing nematicides, resistant cultivars or agronomic practices (Brown,1987, In: Brown, R.H. & Kerry, B.R. (Eds) Principles and Practice ofNematode Control in Crops. Academic Press, London, UK. pp.351–387).In long-term experiments, monocultures of host cereals led to an unex-pected decrease of H. avenae densities due to biological antagonists(fungi). However, there was a marked contrast in the results betweenthose which developed in suppressive soils and those from the dry landproduction 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 AgricultureResources 1989–1993”. The project, involving scientists from France,UK, Spain and Germany was not approved, even though well appreci-ated. The EU was already producing too much small grain cereal, and tobe successful such an application would have had to have been moreingenious to propose a research programme that decreased crop yield!Nevertheless, long term experiments were initiated on the effects of

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resistance on nematode densities and recolonization by susceptible vari-eties, 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 worldand the wide distribution of the CCN complex there are, unfortu-nately 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 meetingswith the exchange of preliminary virulence results for H. avenae.The complexity of pathotyping in these populations led to theorganization of an EPPO Colloquium on “Cereal Cyst Nematodes”at Rennes (France) in June, 1982. More than 40 scientists, from dif-ferent parts of the world attended this meeting which ended arounda famous St John’s fire, well served with a bottle of fine Bretonapple brandy! Results of virulence tests were further examined anddiscussed every two years at the beginning of each ESN Symposium,until 1998. In addition to exchanging materials, bilateral visits andhosting of Ph.D., Fellows in different countries of Europe (e.g., UK,Germany, Belgium, France), there were overtures to nematologistsfrom developing countries that were made through training coursesentitled “Soil borne pathogens of cereals or wheat” and organized byCIMMYT (Julie Nicol ) in Turkey (2003) and China (2005).

Evidence from these notes and landmarks should be used todemonstrate that the Cereal Cyst Nematode Complex is acknowl-edged as a global economic problem especially on wheat and barleyproduction systems, both in the past and the present. Global warm-ing could enhance dramatically the noxiousness of these pathogensin both dry land and rainfed production of cereals as well as in theintensive production systems in western Europe. Remote sensingtechniques based on thermal-infra red measurements have demon-strated already that H. avenae populations are a major factorincreasing plant water stress in infested areas (Nicolas et al., 1991,Revue de Nématologie 14, 285–290). Although substantial progresshas been made, it is clear that additional studies are needed to eval-uate the economic importance of CCN in developing countries (e.g.,those in north Africa, eastern and western Asia) and also in devel-oped countries (e.g., western and eastern Europe, USA) which faceboth greater climatic constraints and reduced fertilizer utilization. Amajor research challenge is the concern over the genetic diversity ofthese species and populations and their phylogenetic relationships.

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Fig. 1. Attack of Heteroderaavenae on Triticum durumin southern France.

Fig. 5. Below: Scientistswho attended the EPPOColloquium on “Cereal CystNematodes”. June, 13–26,1982, Rennes, France.

Fig 2. Mating between maleand female of Heteroderaavenae produced on wheatcultivated in Petri dish.

Fig 3. Above: Kortelutriator for extractingcyst nematodes fromwet soil.

Fig 4. Left: Resistanceor virulence tests forcereal cyst nematodesin miniaturized conditions.

Researchers must identify and confirm sustainable managementsolutions, and these require a deeper understanding of the popula-tion dynamics of H. avenae and other species in the complex.Active research on resistance sources associated with their molecu-lar characterization is necessary for a more rapid integration intocereal cultivars. A new and important challenge is offered to bothyoung and old nematologists involved in traditional or moreadvanced scientific skills, originating from developed and developingcountries. As happened in the earlier years with the previous CCNresearch group, they should join in efforts to create a critical mass ofscientific capacity to deliver sustainable solutions in applied andtheoretical 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 increasedfrom about one million ha (the first official record in the USA in1924 was 1.8 million ha) to somewhat more than 80 million ha atthe 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 themost important according to research data and testimonials (Fig. 2).

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Much has been learned about the species since its description byMinoru Ichinohe over 50 years ago, but a surprising number ofthings we “know” about the nematode reside in the realm of opin-ion and folklore. (The facts, on the other hand, are well-reviewed inthe 2004 book Biology and Management of the Soybean CystNematode, Second Edition, edited by D.P. Schmitt, J.A. Wrather, andR.D. Riggs). Even more remarkable in view of its known economicimpact is that this fascinating animal receives limited attention fromsoybean farmers, the research community and funding agencies. Outof necessity, the focus of information in this article is largely pre-sented from a North-America-centric point of view because theSCN 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 soybeanis grown, but we do not know the details of the struggles thatnematologists in most other countries are having with SCN research(except through some personal communication). For example, thespecies was described by a Japanese scientist and a couple of paperswere published by Japanese researchers in the 1970s on ultrastruc-ture and ecology, but little other information has been publishedthereafter from that country. From Brazil, a major producer of soy-bean, few publications can be found postdating the one thatdescribed the detection of the nematode in that country (in 1993)even though SCN is an economically important soybean pathogenin Brazil.

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Ancient history, distribution, and economic importance

The story of SCN as a plant parasite began long before the domesti-cation of soybean ca. 6,000 years ago. Numerous genes for resistancein soybean and its wild relatives and for virulence in the nematode,are known or posited. (We define virulence simply as the ability toreproduce 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 resist-ance in the 2002 book Plant Resistance to Parasitic Nematodes (editedby J.L. Starr, R. Cook, and J. Bridge.) There is general agreement thatthe ancient origin of SCN is in Asia. Greg Noel (Fig. 3) has suggest-ed that SCN was unintentionally brought to the West in the late19th century by agronomists who imported soil from Asia for studiesof 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 soy-bean production as it has expanded, and now includes the MiddleEast (Iran), as of 1999, and at least one area in Europe (Italy), as of2001. In the United States alone, economic losses due to SCN arenear one billion US dollars annually. It is somewhat surprising,therefore, that in North America, only a relatively small group ofnematologists and plant pathologists work on the problem. Part ofthe reason for this is historical, and part is lack of demand on thepart of soybean producers. Unfortunately for our ability to reduce

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soybean losses, SCN appears to have adapted so well to life in themidwestern US, where most North American soybean is grown,that it does not usually cause visible symptoms (such as stuntingand chlorosis) even while reducing yields by 5 to 30% (Figs. 4–5). Ascrop management decisions are based strongly on commodity pricesand sometimes aesthetics, with limited attention given to yield lim-iting 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-lim-iting population densities of SCN: left, Missouri; right, Iowa.

Based primarily on subjective assessments (e.g., windshield sur-veys), annual losses due to SCN in the US range from about 0.5% to13%. Some damage function data exist from microplot and fieldresearch that demonstrate the potential yield suppression by SCNalone. However, since most data for yield loss (such as those illus-trated in the graphs below) are not research-based, it is difficult todetermine whether losses are increasing, decreasing, or remainingstable over time. Year-to-year assessments are heavily influenced byenvironmental conditions. In spite of these reservations, there is nodoubt that loss is occurring directly and through disease complexes.

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Actually, the greatest losses are most likely occurring through multi-ple 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) southernUSA; B) USA overall. (Note that soybean production in the southern states hasdeclined to a fraction of its former extent, whereas total soybean production in theUS has increased).

The disease

The disease caused by SCN has been classified under a number ofnames such as “moon night” (a Japanese appellation) and “fire-burned seedlings” (Chinese). These two names reflect the symptomsassociated with severe infestations: oval patches of stunted, chlorotic

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plants in the field (see Fig. 4). The earliest documentation for thedisease was 1881, but it has certainly been a problem for many cen-turies. Greg Noel reported that an Asian post-doctoral fellow work-ing with him described an ancient Chinese character for soybean asa glyph that appeared to have the outlines of H. glycines femalesincorporated in it (Fig. 7). It is difficult to date this glyph, but onecan speculate from other evidence that SCN was causing problems

at least 2,500 years ago. Referring toBook 26 of The Annals of Lü Buweicompiled in China in 239 BC, ruleswere outlined for controlling “threerobbers”, one of which was “the landstealing the crops.” The prescriptionfor this “robber” was “overworkedsoils [need] fallowing.” Since the dis-eased soybeans in these fields weredescribed by the Chinese farmers as“fire-burned seedlings,” the causecould 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 areoften overlooked as a primary cause of disease can be had from thetestimony of Nash N. Winstead (Fig. 8). Upon graduation from theUniversity of Wisconsin, he was employed by North Carolina StateUniversity (NCSU) and stationed at Castle Hayne, NC nearWilmington. He noted that fertility research had been underway forat least 8 years to solve a soybean “leaf yellowing” problem in thearea. Winstead, having just completed his doctoral program and,recalling lessons taught by J.C. Walker and other plant pathologistsat the University of Wisconsin, felt that there must be a root dis-ease. Sure enough, he found cysts on the roots, which he sent toHedwig Hirschmann at NCSU to confirm. This was the beginningof much activity by regulatory agencies to determine the distribu-tion of SCN. A quarantine was actually instituted and maintaineduntil the early 70s, when it became clear that regulatory efforts tocontain the distribution of SCN within the US had failed. SCN wasfound in Canada in the late 1990s.

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Species and infraspecific variation

In 1952, Minoru Ichinohe (Fig. 9) described the species, Heteroderaglycines, in Japan. It is puzzling that it took so long to describe H. glycines, especially when we consider that Heterodera schachtiiwas described about 100 years earlier. Heterodera glycines and H. schachtii are very similar in morphology, but were thought to begenetically isolated by host range (at least). They may not be asgenetically isolated as we think.

In the 1970s, Lawrence Miller from the Virginia PolytechnicInstitute & State University (VPI) in Blacksburg, Virginia, (Fig. 10)reported on a series of unconfirmed experiments in which he madeviable crosses between H. glycines and H. schachtii. His results haverecently been verified by Alison Colgrove, a postdoc working withTerry Niblack at the University of Illinois, Urbana-Champaign,Illinois, with phenotypic as well as genotypic tests. In addition, suchcrosses are occurring naturally in sugar beet – cabbage – soybeanrotations in Michigan, monitored by George Bird of Michigan StateUniversity. 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 researchand management alike. This understatement introduces one of themost controversial and colorful chapters in the history of SCNresearch: the problem of infraspecific variation for virulence on soy-bean (Fig. 11). In the 1960s, even before the release of the firstSCN-resistant soybean cultivar, John Ross, a USDA-ARS scientist,

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Fig. 10. Lawrence MillerFig. 9. Minoru IchinoheFig. 8. Nash N. Winstead

reported on “physio-logical strains” thatdiffered among stateswith known SCNinfestations, accord-ing to differences invirulence on knownresistant soybeanlines. In the 1970s,A.C. Triantaphyllouof NCSU, demon-strated SCN adapta-tion to resistancewithin a few genera-tions. In the 1980s

and 90s, Terry Niblack confirmed that an SCN population adapted toa “new” host in about 6 generations. For example, some populationsadapted to lima bean and tomato, previously considered nonhosts ofSCN; each took six generations to develop into a viable, sustainablepopulation. Similarly, it took about six generations for a populationto adapt to the soybean plant introduction (PI) 437.654, originallyreported as resistant to “all known races” of SCN. In Missouri andIllinois, field SCN populations are able to parasitize (at some level)all known sources of resistance to SCN. Clearly, the relevant genesfor virulence are already present in many SCN populations becauseadaptation occurs too quickly to be explained by mutation.

As indicated in the previous paragraph, physiological variationamong SCN populations was reported by C.A. Brim and John P.Ross in the 1960s before the release of the first resistant soybeancultivar (Pickett). Ross and subsequent researchers observed thatcertain SCN populations could parasitize the three known sourcesof resistance at the time, the soybean plant introductions (PI)Peking, 88788, and 90763 (the PI designation refers to its classifica-tion in the USDA Soybean Germplasm Collection, curated byRandall Nelson), and so it was essential that a framework be devisedto describe these populations so that changes in adaptation to resist-ance could be monitored more easily. The group that first addressedthis 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

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Fig. 11. Earliest known photograph illustrating the effects oftwo different isolates of SCN on the same soybean cultivar (Control CK; Arkansas ARK; North Carolina NC) (courtesy of R. D. Riggs).

R.L. Bernard (USDA-ARS, Fig. 3). From this group, the eminentnematologist Robert Riggs devoted a portion of his 48-year career tostudying physiological variability in SCN. Also from this group, thesoybean breeder, R.L. Bernard, was later responsible (together withnematologist, G.R. Noel) for developing the germplasm that is thebasis for more than 90% of the SCN-resistant soybean cultivars nowused in the midwestern US.

The group solved the problem of terminology by describing thevariation in virulence among four SCN populations in terms ofwhat became known as the “race scheme.” In the years immediatelyfollowing publication of the 4-race scheme, SCN populations werefound and described that did not fit the description — a situationthat was not remedied until 1988, when Riggs and D.P. Schmitt(then at NCSU, Fig. 14) expanded the race scheme to its logicalextent: 16 races.

A digression into classification of SCN resistance

An interesting and unforeseen consequence of the initial develop-ment and publication of the race scheme was that breeders began touse it to classify resistance in soybean cultivars rather than virulencein nematode populations – a very important distinction. In the racescheme, the decision whether to label an SCN population as viru-lent or not depended on its ability to develop into adult females ona resistant line relative to its ability to develop on a susceptible stan-

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Fig. 14. Donald P. SchmittFig. 13. Robert D. RiggsFig. 12. A. Morgan Golden

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

Fig. 15. Illustration of the distribution of resistance to race 3 in soybean cultivarslabeled 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 decisionto hold a special meeting to address the problem of categorizingresistance. At the special meeting, documented by nematologist DonSchmitt and soybean breeder Grover Shannon (then of Delta PineLand, Inc.) in voluminous notes, the consensus of opinion was sum-marized and developed into a manuscript to be published in CropScience. Despite the general agreement at the meeting, some breed-ers rescinded their support for publication of the scheme that wasdeveloped unless their personal opinions were included in the man-uscript. Thus the manuscript began to change form but, fortunately,one of the breeders who had attended the meeting was the finaleditor of the paper. He indicated that the original agreement shouldbe published and so it was! The consensus stood: there would befour levels of resistance based on levels of reproduction of SCN on

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the test line compared to a susceptible standard: if < 10%, the linewas resistant; 10 to 30%, moderately resistant; 31 to 60%, moderate-ly susceptible; and >60%, susceptible. Due to some concern aboutthe interpretation of “moderately susceptible,” the term was laterchanged to “slightly resistant”.

In Illinois, the SCN screening program, which determines thelevels of resistance in 400 to 600 soybean cultivars to five differentSCN isolates each year (conducted by T.L. Niblack, G.R. Noel, andJ. Bond [Southern Illinois University]), a different scale is used: 0 to9%, highly resistant; 10 to 24%, resistant; 25 to 39%, moderatelyresistant; 40 to 59%, low resistance; and > 60%, no effective resist-ance. This change was implemented for three reasons, at least one ofwhich is not scientifically justifiable. First, most of the SCN-resist-ant cultivars in the lower Maturity Groups (4 and below, which aregrown mostly north of 34° north latitude) are derived from PI88788. One of the characteristics of this source of resistance is thatit does not produce a necrotic reaction, but reduces SCN popula-tions by acting on developmental stages after the second stage juve-nile. Sometimes, many females are allowed to develop, but theirfecundity is so limited that (in the field) the ultimate effect is areduction in the SCN population. Without resorting to countingeggs, labelling as “resistant” only those PI88788-derived cultivars thatallow only 10% or less female development would be to undervaluea large number of usefully resistant cultivars. Second, over the 18-year period that data were collected, the 10–25–40–60% thresholdsdefine statistically “natural” categories. Finally, and perhaps indefen-sibly, the word “susceptible” is not used to describe a cultivarreleased 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 andSchmitt, a new, highly-resistant soybean cultivar, Hartwig, wasreleased, developed by Sam Anand, University of Missouri, andnamed after the most legendary, confident, and tenacious soybeanbreeder of the latter half of the 20th century, Edgar E. Hartwig (Fig. 16), USDA-ARS, who influenced SCN research in the south-eastern US in various ways for many years.

Resistance in the cultivar Hartwig was derived from PI 437654

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and “Forrest”. Soon thereafter, three more lines were released withSCN 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 viru-lent on them. The obvious solution, to add them to the racescheme, would have increased the possible number of races to 256,an untenable number for any practical use. Furthermore, the racesystem was not suitable for use in genetic studies (for various rea-sons 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 therace 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 ofMinnesota), D.P. Schmitt (Fig. 14; then of University of Hawaii), J.G. Shannon (University of Missouri), G.L. Tylka (Iowa StateUniversity), and R.D. Riggs (Fig. 13), the latter being the onlyremaining active scientist from the group which had developed theoriginal race scheme.

The solution of this group was called the HG Type Test (aname that only a committee could love), published in 2002. Theframework included an easily-adapted list of all the sources ofSCN-resistance known to have been used in breeding programs.This system could be adapted for use in different countries, and hasbeen adapted for use in making cultivar recommendations in

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Fig. 16. Edgar E. Hartwig Fig. 17. T. L. Niblack

Illinois by including only the three sources of resistance available toIllinois soybean growers. The HG Type Test has been adopted bynematologists and plant pathologists, but has met with resistanceand even hostility from a few public and private soybean breederswho find themselves unable to co-opt it for use in their breedingprograms. The recommendation by the HG Type committee wasfor those who release SCN-resistant germplasm to simply identifythe source of resistance, but until now many private companieshave been unwilling or unable to do so. It will be interesting to seehow this is resolved.

The next permutation of virulence phenotyping for SCN is like-ly to be accompanied by genotype tests, which are not currentlyavailable. Those working on the genetic basis of virulence includelabs headed by K.N. Lambert (University of Illinois), D.McK. Bird,and C.H. Opperman (NCSU). The first genetic map of SCN waspublished in 2005, and genomic analysis of the nematode is in itsinfancy. SCN serves as a model organism for investigation of plantparasitism 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 ofSCN. One of the many tasks is to determine how SCN isolatesmaintain their diversity after 100 to 300 generations of inbreeding.“Random mating” doesn’t really occur; matings in soil are usuallybetween full- or half-siblings. Single-cyst-descent inbreeding shouldresult in SCN populations being fixed for most loci, and yet this isnot what we observe. The imagination is the only limit to unravellingthe genetics and behavior of this pathogen.

Management

Farmers tend to perpetuate their habits and those of their ancestorsin crop management. To survive, though, they must make sufficientprofit to sustain the farming operation with an adequate return tomanagement to support the family. Therein lies the challenge forpest control advisors, whether from the private or public sector.

The most frequently used rotation recommendation that isconsidered to be fundamental to SCN management is a 3- or 4-yearrotation including one resistant soybean cultivar and one suscepti-ble cultivar, as follows: Year 1, resistant soybean cultivar; Year 2,

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corn (maize); Year 3, susceptible soybean; Year 4, corn. Ask anyoneinvolved in making SCN recommendations, and this is what theywill tell you. One would think that such a recommendation, onethat affects so many soybean growers, would have been based onwell-documented field studies. Instead, it was developed in a dis-cussion 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 Carolinaand elsewhere, but in general, research to determine the rates ofmortality and “race shifts” have not given clear and definitiveresults. Rotation to nonhosts clearly reduces SCN population den-sities, but what happens to the genetic structure of the population?The point here is not that the value of rotation should be ques-tioned, but that certain aspects of the long-term effects of nonhostsand resistant cultivars on SCN populations and crop profitabilitystill need to be elucidated.

SCN populations have shown evidence of marked adaptationsto life in the Soybean Belt, i.e. the Corn Belt states, where annualalternation between maize and soybean became the standard in thelatter part of the 20th century. Those adaptations, such as an appar-ent shift to earlier induction of dormancy and an increase in over-winter survival rates, interfered with the portability of managementrecommendations from one region of the US to another, not tomention from the US to other countries. Changes in the diseasecaused by the nematode have also occurred. For example, as men-tioned earlier in this paper, in many of today’s intensive soybeanproduction areas, SCN causes few or no visible diseasesymptoms. Soybean growers, admonished for years to look for stunt-ed and chlorotic plants as evidence of the presence of SCN, are nowhearing that symptoms are no longer characteristic of SCNinfection. This can create frustration for both growers and thosewho craft recommendations for SCN management.

There is purely anecdotal evidence that the parasites and pred-ators of SCN are catching up, geographically, with thenematode. From conversations with nematologists and plantpathologists who were around in the Soybean Belt when SCN wasfirst spreading into the area, it appears that fewer nematodes wererequired to cause significant yield loss several decades ago than arerequired today. Damage thresholds established in the 70s and 80sare no longer applicable today.

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Concluding comments

“Moon night” type symptoms helped us recognize the organismultimately classified as soybean cyst nematode. Today, managementis compromised because the symptoms are not obvious.Nematologists and those scientists working in nematological roleshave to envy weed scientists. Growers will use extreme means tocontrol weeds, even the species that compete very little with thesoybean crop. Many of these same growers will simply ignore SCNbecause 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). Privatepractitioners are rarely educated in nematology and they are few innumber. Will a serious crisis be necessary to get the attention of thepublic and private sector? What we really need are soybean culti-vars genetically engineered to break out in pink polka-dots wheninfected by SCN (not really kidding). Among the generally underap-preciated plant-pathogenic nematodes that cause significant eco-nomic injury to important crop plants, SCN ranks as one of theleast appreciated.

Dedicated to solving the puzzles that SCN has set us, many fineresearchers contributed ideas, data, and opinions; not all of theresearchers or their opinions have been mentioned in this paper, ofcourse. Our editor tells us we must stop somewhere!

THE SCIENCE AND ART OF SOYBEAN CYST NEMATODE RESEARCH 151

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 infor 32 years! My “love affair” with virus-vector nematodes, theirassociated viruses, and the many plant species affected by bothnematode and virus is professionally referred to as my scientificresearch career. Such reference provides no insight into the passion,emotion, commitment, excitement, frustration, intellectual andphysical challenges, and involvement with colleagues and studentsworldwide, many of whom became personal friends. Consequently,this is an unashamedly personal “historical account of plant virustransmission by soil nematodes”.

I take this opportunity to thank everyone – nematodes, viruses,plants and the numerous colleagues and students – and dedicate thischapter to Professor Charles E. Taylor and Professor Franco Lamberti(now, sadly, both deceased) who individually and collectively weremy constant and supportive mentors.

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

152 NEMATODES/VIRUSES/PLANTS: “A 32-YEAR LOVE AFFAIR”

The early beginning: soil-borne plant diseases vs. aerial-borne plant diseases.

In the period 1882 to 1900, several reports demonstrated and con-firmed that some plant diseases were soil-borne. For example,healthy grapevines became diseased when planted in soil taken fromold vineyards in which diseased grapevines were growing. It wouldbe almost another 50 years before the disease was described asgrapevine fanleaf virus (GFLV), and a further 30 years before thesoil-borne vector of the virus, the nematode, Xiphinema indexwould be identified. In 1886, tobacco plants were shown to becomeinfected in a similar manner with a mosaic disease that, in 1943, wasidentified as being caused by tobacco rattle virus (TRV).

Whereas the concept of soil-transmission of plant diseases waslargely ignored until the late 1940s, aerial vector transmissionbecame the vogue in the first half of the 20th century. Quite simplyit was much easier to see, on the crops, insects that had the poten-tial to be the disease carrying agent than to believe that some mys-terious organism present in the soil could transmit a crop disease.The impediment to research that this misconception had is proba-bly best exemplified in the raspberry crop in the UK.

In 1922, in eastern Scotland a devastating disease was recordedthat apparently occurred spontaneously in plantations of cv.Baumforth’s Seedling raspberry. The disease agent was never identi-fied, and disappeared when cv. Lloyd George was planted.Eventually, cv. Lloyd George became infected with a mosaic diseaseand was replaced by cv. Norfolk Giant. In 1941, the original lethaldisease in cv. Baumforth’s Seedling recurred in cv. Lloyd George, andthe disease was named “leaf curl“ because of its resemblance to anaphid-borne North American disease of the same name. Despitecomprehensive research over the next twelve years an aerial vectorfor the disease was never identified. In 1956, the disease now knownto be caused by raspberry ringspot virus (RRSV) was demonstratedto be soil-borne. During the next couple of years several otherviruses, including tomato black ring nepovirus (TBRV) and arabismosaic nepovirus (ArMV) causing diseases in crops in the UK, wereshown to be soil-borne. Thus, during the 1950s, soil-borne viruseshad been identified as being more common and of more economicimportance than had previously been realized. With the develop-ment of new methods to reliably recover viruses from infected

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plants, to transfer them between herbaceous test plants, and toidentify them by their serological properties, research on soil-borneviruses 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 potentialplant disease transmitting agents, thus resulting in early studies fail-ing to establish any nematodes as vectors.

In 1912, in southern England the occurrence of the hop cystnematode, Heterodera humuli, was correlated with the spread of“nettlehead“ disease in hops. A decade later the nematode was exon-erated as being the vector, and it was not until fifty years later thatthe disease was identified as being caused by ArMV and was beingtransmitted by the nematode, X. diversicaudatum. During the 1940s,in the USA wheat mosaic-infected soil was treated with calciumcyanide, carbon disulphide, chloropicrin and methyl bromide andthese treatments prevented transmission of the disease. A decadelater experiments failed to associate nematodes with transmission ofthe virus. Several nematode species in the USA were meticulouslytested as potential vectors of lettuce big vein virus, but with nega-tive results. Subsequently, the virus was shown to be transmitted bythe chytrid fungus, Olpidium brassicae. Meloidogyne spp. were simi-larly tested as vectors of tobacco mosaic and cucumber mosaicviruses as were Helicotylenchus nanus and Pratylenchus spp. as vec-tors of carnation mottle virus, but again with negative results.

Despite several studies testing soil nematodes as potential vec-tors of various soil-borne diseases and plant virus diseases, the inves-tigations all proved fruitless. By the early 1950s, it was concludedthat conducting research to associate nematodes with soil-borne dis-eases was a pointless exercise. However, there were still a few scien-tists who doggedly continued to try to prove that a soil nematodecould vector a soil-borne disease.

154 NEMATODES/VIRUSES/PLANTS: “A 32-YEAR LOVE AFFAIR”

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 around600BC to Marseille, France from where they were distributed, almostcertainly with GFLV and X. index, to Italy and, subsequently, to Spainand Portugal. Grapevines were transported to the New World fromSpain in the 17th century and by the end of the 17th century to AltoCalifornia (California, USA). Here the “Mission“ grapevine, which ishighly susceptible to GFLV, eventually formed the basis of whatbecame an extensive wine-producing industry. It has been speculatedthat GFLV and X. index were introduced into California by ColonelAgoston Haraszthy who imported rooted grapevines from his nativeHungary in 1851. Even more likely, the virus and vector were importedin 1861 when he imported 100,000 grapevines of 1400 cultivars col-lected from many wine producing areas in Europe, where by this timeboth virus and vector were widespread.

It also has been suggested that GFLV and X. index were firstintroduced into the United States in 1900, when imported root-stocks became widely used after the spread of Phylloxera. The evi-dence for this is that GFLV was not present in over 100 clones ofgrapevine cultivars that were still growing at the site of a trial thathad 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-productionvineyards in California being destroyed. Thirteen years later whenthe Act was rescinded there was massive, rapid replanting of vine-yards. This would obviously have provided an excellent opportunityfor the introduction into California of virus and vector and theirwidespread distribution.

GFLV was discovered in California in 1948, and thereafter a seriesof investigations were established in 1954, to identify its soil-bornenature. Firstly, healthy grapevines were planted in containers holding: i)soil from the rhizosphere of GFLV infected grapevines, at sites wherethe disease was spreading; ii) steam-sterilized vineyard soil and; iii) ster-ile soil. The following spring 62 of 70 vines in the untreated soil wereinfected with GFLV, but the virus was not present in grapevines in

NEMATODES/VIRUSES/PLANTS: “A 32-YEAR LOVE AFFAIR” 155

either of the other two treatments. Thus, GFLV had been shown to besoil-borne. The next tests were to prove crucial, as they would finallydemonstrate that a soil nematode was the vector of GFLV.

Soil collected from the rhizosphere of GFLV infected vines waswet screened as 500g samples through a sieve with 75 µm diameterapertures. The debris collected on the sieve, after thorough washing,was poured over the roots of healthy grapevines growing in pots ofsterile soil. Xiphinema index and Criconemoides xenoplax were pres-ent in most, but not all, of the samples. The following year GFLVinfection had developed in 20 of 35 grapevines. In the next set oftests hand-picked groups of nematodes of both species, from GFLVinfected soil, were placed on the roots of healthy grapevines growingin 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 subsequentlyassumed to have been caused by contamination, as subsequentexperiments 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 Goheenconfirming that X. index was the vector of GFLV stimulated thesearch for nematode vectors of other soil-borne viruses occurring inthe USA and Europe. This was the real beginning of virus-vectorresearch and here is presented a brief chronology of scientific land-marks 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 GFLVin vineyards in California, USA.

1959 – Jha & Posnette and Harrison & Cadman report X. diversicau-datum as the natural vector of ArMV in Europe.

1960 – Sol et al. report that tobacco rattle tobravirus (TRV) is trans-mitted by Paratrichodorus sp. (=Trichodorus pachydermus)

– In Scotland the first international symposium is held onvirus-vector nematodes and their associated viruses.

156 NEMATODES/VIRUSES/PLANTS: “A 32-YEAR LOVE AFFAIR”

1961 – Harrison et al. are the first to report a Longidorus sp. as avector 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 distinguish-

able strains of nepoviruses with specific longidorid species astheir vectors.

– Taylor & Raski report that viruses in the vector are notretained through the moult or through the egg.

1966 – Hoof identifies differences in the ability of L. elongatus pop-ulations to transmit TBRV.

1968 – Hoof reports differences in the ability of trichodorid popula-tions to transmit TRV isolates and concluded that transmis-sion occurs only when the virus isolate “suits“ the nematodepopulation (specificity).

1969 – Taylor & Robertson identify “virus-like” particles, based onthe particle morphology, at specific sites within the feedingapparatus of Longidorus.

1970 – Taylor & Robertson identify “virus-like” particles, based onthe particle morphology, at specific sites within the feedingapparatus of Xiphinema and of Paratrichodorus.

– McGuire et al. identify “virus-like” particles, based on theparticle morphology, at specific sites within the feedingapparatus of X. americanum.

1971 – Hoof reports differences in the ability of Xiphinema popula-tions to transmit nepoviruses.

1972 – Yagita & Komuro report the transmission of mulberryringspot nepovirus (MRSV) by L. martini in Japan; the firstrecord of a new vector and virus association reported outsideEurope and North America.

1973 – Salomao reports the transmission of pepper ringspottobravirus (PRV) by P. minor (= P. christiei) in Brazil, South America.

1974 – Harrison et al., establish that RNA-2 of the bipartite genomeof RRSV, which codes for the virus coat protein, is involvedin the recognition between the virus and its vector.

– Siddiqi establishes Trichodorus and Paratrichodorus as twodistinct genera.

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– A NATO Advanced Study Institute “Nematode Vectors ofPlant Viruses“ is held in Italy.

1975 – Yagita publishes evidence that L. martini, the vector ofMRSV, has only three and not the usual four juvenile devel-opment stages.

– “Nematode Vectors of Plant Viruses” by F. Lamberti, C.E. Taylor and J.W. Seinhorst (Eds), published by PlenumPress, London and New York.

1977 – Heath et al., publish a nematological atlas for Britain contain-ing maps of the distribution of Longidorus, Paratrichodorus,Trichodorus and Xiphinema species.

1978 – Trudgill & Brown report the infrequent transmission ofRRSV by L. macrosoma to be associated with an apparentlack of release of virus particles from the site of retentionwithin the vector.

– McNamara identifies sources of potential contamination invirus transmission experiments which could account for sev-eral reports of apparent non-specific associations betweenvectors and viruses.

1979 – Lamberti & Bleve-Zacheo reappraise members of the X. americanum-group, which results in uncertainty in theidentification of X. americanum transmitting NorthAmerican nepoviruses in all previous reports.

1981 – Trudgill et al. report differences in the efficiency of transmis-sion of nepoviruses by longidorid vectors.

1983 – Trudgill et al., propose a set of criteria for assessing reports oflongidorid nematode transmission of nepoviruses.

1984 – Hoy et al. report the differential transmission of strains oftomato ringspot nepovirus (ToRSV) by X. californicum, amember of the X. americanum-group.

1985 – Brown demonstrates differences between populations of X. diversicaudatum in their ability to transmit strains ofstrawberry latent ringspot nepovirus (SLRSV).

1986 – Brown demonstrates differences between populations of X. diversicaudatum in their ability to transmit strains ofArMV and shows that the vector’s ability to transmit virus isinherited.

– Robertson & Henry associate retention of virus particles withthe layer of carbohydrate lining the oesophagus in Xiphinemaand Paratrichodorus.

158 NEMATODES/VIRUSES/PLANTS: “A 32-YEAR LOVE AFFAIR”

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 oftransmission 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 vectortransmissibility are associated with TRV RNA-2.

– Halbrendt & Brown report that several North American pop-ulations of the X. americanum-group nematodes have onlythree, and not the usual four, juvenile stages.

– Vrain et al. report that molecular taxonomy methods sup-port the establishment of several morpho-species in the X. americanum-group from North America.

– “Dorylaimida. Free-living, Predaceous and Plant-parasiticNematodes” by M.S. Jairajpuri and W. Ahmad, published byOxford and IBH Publishing Co., New Delhi, India; includesthe Longidoridae and Trichodoridae.

1993 – Ploeg et al. report that virus coat protein mediated resist-ance, which has been shown to be effective with severalviruses transmitted by insect vectors, is not an effective con-trol strategy for TRV when the virus is transmitted by thenematode.

– “Aphelenchida, Longidoridae and Trichodoridae: TheirSystematics and Bionomics” by D. Hunt, published by CABInternational, Wallingford, England.

1994 – Brown et al. report that several X. americanum-group speciescan each transmit three distinct North American nepoviruses,that differences occur between populations of X. americanumsensu stricto in their ability to transmit North Americannepoviruses and that the specific associations between thesenematode species and the North American nepoviruses is dif-ferent from that which occurs between European virus-vec-tor species and their nepoviruses.

– Jones et al. report Paralongidorus maximus as the vector ofan atypical isolate of RRSV in vineyards in Germany.

NEMATODES/VIRUSES/PLANTS: “A 32-YEAR LOVE AFFAIR” 159

1995 – MacFarlane et al. report that the transmission of tobravirusesby nematodes is not determined exclusively by the virus coatprotein.

– Mayo et al. suggest that flexible peptides on the C-terminusof the coat protein of nematode-transmitted viruses, espe-cially tobraviruses, may be responsible for virus and vectorrecognition.

– Ramel et al. report the first unequivocal evidence of anepovirus, a strain of ArMV transmitted by X. diversicauda-tum, causing a disease in a graminaceous plant, barley.

– Robbins et al. report that several Longidorus spp., includingL. martini (see 1975), have only three and not the usual fourjuvenile stages.

– “The family Trichodoridae; Stubby Root and Virus VectorNematodes” by W. Decraemer, published by KluwerAcademic Publishers, Dordrecht, The Netherlands.

1996– Brown et al. identify the site of retention of TRV inTrichodorus.

– MacFarlane et al. confirm that each of the four Open ReadingFrames (ORFs) contained in PEBV RNA-2, and that flexiblepeptides 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 istransmitted by its vector.

1997 – “Nematode Vectors of Plant Viruses” by C.E. Taylor andD.J.F. Brown, published by CAB International, Wallingford,England.

1998 – Weischer & Brown define terminology for specificity, exclusivi-ty and complimentarity of nematode transmission of viruses.

– Wang & Gergerich using immunoflourescent techniques pro-vide the first unequivocal evidence that “virus like” particlesat specific sites within the feeding apparatus of Xiphinemaamericanum senso lato nematodes, as reported by McGuire et al., in 1970, are indeed nepovirus particles.

2000– Karanastasi et al. develop advanced serological techniquesinvolving immunogold labelling to provide the first unequiv-ocal evidence that “virus like” particles at specific sites withinthe feeding apparatus of Paratrichodorus, as reported byTaylor & Robertson in 1970, are indeed tobravirus particles.

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2001 – Karanastasi et al. report that differences in the body cuticle ofParatrichodorus species clearly separate them fromTrichodorus species, and that similar differences reveal twoseparate groups of Trichodorus species. Chen et al. develop amethod for separating specific nematode species from speciesmixtures using antiserum and lectin-coated magnetized beads.

– Vassilakos et al. identify that the tobravirus 2b protein acts intrans to facilitate vector transmission.

2003 – Karanastasi et al. identify specific retention of tobravirus par-ticles in the feeding apparatus of trichodorids, including non-vector species in which the virus is retained at sites fromwhere it can not be transmitted to plants.

– Chen et al., develop a magnetic capture system for recoveryof specific X. americanum nematodes from mixtures of soilnematodes.

– Wang et al., develop a multiplex polymerase chain reactionusing ribosomal genes for identifying single individuals ofseveral Xiphinema species including X. index.

2004 – Boutsika et al., develop a molecular diagnostic method foridentifying trichodorid virus-vector species and their associated tobacco rattle virus.

With research on nematode transmission of viruses developingrapidly after the seminal paper by Hewitt et al., in 1958, it wasconsidered pertinent to arrange an international forum to exchange information between the various researchers. A symposium washeld in July 1960 at the Scottish Horticultural Research Institute.Most notably, Dutch researchers attending the conference received atelegram from their co-workers in Wageningen, The Netherlands,confirming that Trichodorus (Paratrichodorus) pachydermus was the natural vector of TRV. Evidence that L. elongatus transmitted theScottish 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-transmittedviruses were localized problems of insignificant economic impor-tance in comparison with the soil-borne wheat and oat mosaic virusdiseases occurring over extensive areas in the USA. Thankfully, thisconclusion was ignored!

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The Golden Years

In 1970, I joined the then small Zoology Department at the ScottishHorticultural Research Institute with Charles Taylor as Head ofDepartment. Four years later I was fortunate enough to attend whatwas to be for me the most important meeting of scientists involvedin virus vector research. Franco Lamberti, Charles Taylor and WimSeinhorst were the joint organisers of the NATO Advanced StudyInstitute at Riva dei Tessali, Italy. Eighteen lecturers and 48 dele-gates from numerous countries throughout the world reviewed anddebated all aspects of the biology of virus-vector nematodes. Twoweeks of unadulterated bliss was spent discovering what was knownabout the nematodes and viruses and being able to hone one’sresearch ambitions and ideas. By then much was already knownabout the nature of the association between virus and vector, andabout virus acquisition, retention and transmission by vectors. Thepresentations on virus transmission, taxonomy, morphology, feedingbehaviour, ecology, geographical distribution and control could onlyserve to stimulate my enquiring mind. The contacts made whilstattending this meeting would feature greatly in my career, enablingme to work around the world with some of the very best contem-porary nematologists. Importantly, friendships were established thatwere to endure throughout one’s career, and the memories – theunfortunate scientist who walked through a glass door, luckily notcausing himself any injury; or the colleague who alarmed TeresaLamberti when she looked out of her bedroom window in the earlymorning to be confronted by him a few meters away with binocu-lars dangling, but we all knew that he was a very enthusiastic ama-teur ornithologist!?!

By the 1980s, virus-vector nematodes were an established com-ponent of many nematology teaching programmes. In 1984, theEuropean Society of Nematologists established a Virus-VectorWorkshop as part of each of their biennial meetings, including theSecond and Third International Nematology Congresses. Most per-sonally gratifying were the numerous specialist training workshopsin which I was privileged to be invited to participate and sometimesorganize – University of Coimbra, Portugal; University of Cordoba,Argentina, Zhejiang University, China etc. To give a 3 hour lecturestarting at 06.00 attended by what seemed to be hundreds of enthu-

162 NEMATODES/VIRUSES/PLANTS: “A 32-YEAR LOVE AFFAIR”

siastic Chinese students is unforgettable, and I was still answeringquestions 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 5years. The excitement felt when witnessing my first student achievehis Ph.D., never diminished when my other students achieved thesame distinction. Each one was and remains precious, and it is mostgratifying to me that each has gone on to do even greater thingsthan I could ever have hoped to achieve.

PERSONALITIESNumerous outstanding individuals have been, and many remain,

involved in the various research components that collectively arereferred to as virus-vector research.

Taxonomy and systematicsEarly taxonomy of the virus-vector nematodes was most ably

served by David Hooper at Rothamsted. Stalwarts of virus-vectortaxonomy have been Michel Luc, France, the late Juan Heyns,South Africa, Piet Loof, Netherlands, August Coomans, Belgium,Wilfrieda Decraemer, Belgium, Dieter Sturhan, Germany, FrancoLamberti, Italy, Robert Robbins, USA Luiz Ferraz, Brazil and JingwuZheng, China. Each, undoubtedly, was amongst the most productiveand influential scientists serving the taxonomy of the Longidoridaeand Trichodoridae. Luc producing a definitive taxonomic guide ofXiphinema species, Decraemer a taxonomic guide to theTrichodoridae and Robbins a guide to the Longidorus species.Coomans, Luc and Heyns produced a remarkable compilation onXiphinema systematics, and Lamberti was unflinching in devotingmany years to attempting to clarify the X. americanum complex ofspecies whilst all other taxonomists studiously avoided what mustbe the most difficult group of all. Meanwhile, Charles Taylor andWalter Robertson provided insight into aspects of the ultrastructureof longidorids. From the late 1990s to the present, major advanceshave been made in the molecular taxonomy and systematics oflongidorid and trichodorid nematodes by Maurice Moens and col-leagues in Belgium, and by Roy Neilson, Vivian Blok, Mark Phillips,John Jones and colleagues in Scotland.

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BiologyMany scientists worldwide have contributed to the biology of

virus-vector nematodes. Amazing visual insights into virus-vectorbiology were provided by the series of films showing details of tri-chodorids feeding on plant roots produced by Urs Wyss, Germany,and similar studies were done by Eirini Karanastasi, Greece and withXiphinema and Longidorus spp. by David Trudgill and WalterRobertson, UK.

Mariella Coiro and the author demonstrated the number ofprogeny that a single Longidorus and Xiphinema species can pro-duce, and also the time taken for a single egg to be produced.

The discovery by John Halbrendt, USA and the author thatsome Xiphinema species have only three and not four juvenile stageswas highly controversial. A comment by one of the referees, a highlyrespected systematist in the USA, was that the authors would bebest advised to find new careers as the Nematoda had four andnever three juvenile stages! The research was eventually published,but not accepted by several European based virus-vector taxono-mists, until they themselves identified species that clearly only hadthree and not four juvenile stages. This was followed by the revela-tion that also several Longidorus species had only three juvenilestages. Several years earlier a Japanese scientist published irrefutableevidence that this phenomenon occurred, but his work had beenignored. Perhaps the debate continues!

EcologyAs with the biology of virus-vector nematodes many scientists

worldwide made significant contributions also to our understand-ing of their ecology. An important observation made by DavidMcNamara was that X. diversicaudatum stored in plant-free, ster-ilized soil quickly became translucent and died within a fewweeks, whereas those stored in untreated, plant-free soil appearedhealthy and survived for at least 22 weeks. This observation hasnever been explained, although it was speculated that soilmicroorganisms might be responsible. John Halbrendt and theauthor raised populations from single females taken from naturallyoccurring populations of X. americanum sensu lato and revealedthat little morphological variation occurred between specimenswithin a raised population, but that several morphological variants(species?) occurred in natural soil populations. On the other hand,

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Charles Taylor and the author showed that the occurrence and dis-tribution of virus and associated vector nematodes at field siteschanged very little over 25 to 30 years.

DistributionThe first comprehensive, systematic survey of soil nematodes

was made for the Longidoridae in the British Isles and Italy as part ofa co-operative project between the Istituto di Nematologia Agraria,Bari, Italy and the Scottish Horticultural Research Institute. This wasdeveloped into a European Plant Parasitic Nematode Survey(EPPNS) that eventually produced detailed distribution maps on theoccurrence of many plant parasitic nematode species throughoutEurope. The distribution patterns provided the impetus for numer-ous research studies on the biotic and abiotic reasons for the locationof various species. Also, the distribution patterns, combined withmorphological data provided new insights into the systematics andpossible origins of the species. The original survey was subsequentlycomplemented by additional surveys in the former USSR, theMediterranean region, North America, and Latin America.

Virus and vector associationsThe information in the late 1950s that X. index transmitted GFLV

stimulated many researchers worldwide to seek further virus-vectorassociations, and during the next 15 to 20 years many new associationswere reported. A research team in the former East Germany wasamongst the most prolific, although several of the virus-vector associa-tions appeared to be anomalies. With the more recent development ofnew, stringent testing procedures, not least being the introduction ofexperimental procedures in which individual nematodes are tested fortheir ability to transmit virus, a set of criteria for assessing reports ofvirus transmission by nematodes was developed by David Trudgill,David McNamara and the author. Applying these criteria to all publi-shed reports of virus transmission by nematodes they concluded thatapproximately two thirds of the reports failed to fulfill the criteria.

Charles Taylor and Walter Robertson in the UK and JimMcGuire and colleagues in the USA identified the sites of virusretention in each of the vector genera in the late 1960s and early1970s. Regrettably, to this day the specific nature of the associationand particularly the mechanism of release of the virus from thesesites remains a subject of conjecture.

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Throughout the 1960s, 70s and 80s virologists made significantadvances in determining the genetic structure of nematode-transmit-ted viruses, eventually revealing that vector transmission, and speci-ficity of transmission, was encoded in the RNA-2 segment of thenematode-transmitted virus genomes. The most exciting discoverieswere made during the later 1990s early 2000s by several Ph.D., stu-dents e.g., Ton Ploeg, Nikon Vassilakos, Evangelis Vellios, EiriniKaranastasi, Konstantina Boutsaki, Rodanthi Holeva, Quing Chen,and Cleber Furlanetto at the Scottish Crop Research Institute.Individually and collectively in research teams, these researchershave revealed much of what we now know about the specificity ofvirus transmission by nematodes, the genetic determinants of virusand vector recognition and the distribution of viruses in plant roots.

SWANSONGFor the individual farmer the occurrence of a nematode-trans-

mitted virus disease in a crop is of paramount importance. Forexample, in the UK, when “pick your own” raspberry and strawber-ry crops were being advocated as the potential financial saving ofsmall farms, the occurrence of a virus transmitted disease in a cropsituated in a “suitable” field, at least in part, resulted in the collapseof these family farm enterprises. Similar disease outbreaks aroundthe world in fruit orchards, potato fields, and vineyards result inalternative crops having to be grown. Such crops, especially replace-ment crops in orchards and vineyards, very often have lower marketreturns resulting in an insidious, slow decline of the profitability ofthe farm enterprise, eventually leading to their financial collapse.

Interestingly, the Golden Anniversary, 50 years, of the EuropeanSociety of Nematologists coincided, within a couple of years, withthe publication of the original report of a soil nematode being thenatural vector of a plant virus. Today the Society and this researcharea have each fully matured. Unquestionably, during the last halfcentury virus-vector nematodes, and in Europe potato cyst nema-todes, have been among the foremost stimuli in plant nematology,presenting a plethora of research opportunities to the nematologycommunity. With virus-vector nematodes most of the challengeshave been successfully met and nematode taxonomy, systematics,biology, ecology, interactions, and control, have benefited greatlyfrom the impetus throughout the general area of research. Newmethods, especially those continually being generated in molecular

166 NEMATODES/VIRUSES/PLANTS: “A 32-YEAR LOVE AFFAIR”

biology, provide exciting research opportunities for the global nema-tology community. Many intellectual and practical questions involvingvirus-vector nematodes, and their transmission of viruses, remain tobe answered. These intriguing challenges are available to the currentgeneration 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 theScottish Crop Research Institute in 2002 and relocated to the interna-tional, mountain ski resort of Bansko, Bulgaria where he, his wifeJune, and friends Vlada and Lyubomir Penev, operate a self-cateringfamily hotel (www.penbro.com). Derek was appointed Deputy Rector ofthe College of Tourism in Bansko, where he holds a personal Chair inRural, Village and Eco Tourism. He can be contacted at:

[email protected].

NEMATODES/VIRUSES/PLANTS: “A 32-YEAR LOVE AFFAIR” 167

11.HORTICULTURAL HAZARDS: IN ANDOUT OF HOT-WATER BATHS AND OTHERTRANSIENT 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 ofinterest in nematodes and the opportunities for their management.Many nematologists were recruited to university and research stationpositions, a number of important meetings were held in the US,Caribbean and Central America and we saw the formation of SON in1961 and ONTA in 1967. In 1968, a Caribbean Symposium onNematodes of Tropical Crops was held at the University of the WestIndies (UWI), Trinidad. This was jointly organised by UWI, theCommonwealth Development Corporation (which had interests insugar cane, coconuts and bananas), the Commonwealth Institute ofHelminthology (now part of CABI Bioscience), and FAO. The meetingbrought together nearly 50 scientists including many of the leadingnematologists from the USA, and several from the UK and other partsof Europe. The objectives of this meeting were to stimulate greaterresearch effort in the management of nematodes of tropical crops andto promote teaching and training programmes in plant nematology.One of us (SRG) was to be a direct beneficiary; “I first met NigelHague at this meeting and it was he who encouraged me to do a high-er degree in plant nematology (Fig. 1). Also present were Fred Jonesand David Hooper, and it was through them that I received some spe-cialist training at Rothamsted Experimental Station in England prior to

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

The other of us (PAR) also benefited from specialist training atRothamsted with Fred Jones, David Hooper, Alan Stone and col-leagues, from where a career in nematology research and extensionensued in California. Using two contrasting examples, burrowingnematode on banana and stem and bulb nematode on garlic, werecount some of our experiences and insights on work to developnematode management strategies and tactics for horticultural crops.Although the examples are as different as bananas and garlic, theunderlying themes, experiences, and outcomes are remarkably simi-lar, and we suspect they are much like other nematode-plant prob-lems 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 over-looked by nematological societies, a sad omission! Cobb’s material

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

was collected from some banana plants growing in gardens adjacentto Government House in Suva, Fiji. The same population was therewhen Al Taylor visited, in 1967.

Radopholus similis is thought to be indigenous to the westernPacific, and its pan-tropical distribution is probably a result of themovement of nematode-infested banana suckers from that regionduring this past millennium. Nematode infestations of bananas inthe New World were recorded as early as 1910, by which time thisfruit had become established as an export commodity. A JamaicaDepartment of Agriculture report mentions that a dreaded bananadisease, thought to be caused by bacteria, was suspected to be dueto “eel worm at the root”. The problem had also been reported inFrench West Africa before World War II. Not until the 1950s, didthe disease known as “blackhead toppling” begin to be recognized asthe major banana production constraint (Fig. 2). Two unrelatedissues had contributed to this.

1. When the banana trade began in the latter part of the 19th cen-tury the industry was based upon one variety “Gros Michel”(Musa AAA). Unfortunately, this variety, although popular with

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

consumers was highly susceptible to Panama wilt caused by thefungus, Fusarium oxysporum f. sp. cubense. This was devastatingfor plantation owners and smallholders. With no effective treat-ments to combat the dis-ease the “industry”changed to the“Cavendish” varietieswhich were immune tothe pathogen. What wasnot known at the timewas that the Cavendishvarieties (also MusaAAA) had less toleranceto R. similis than did GrosMichel. This change ofvariety was done over arelatively short period oftime and neither propaga-tion nurseries nor quaran-tine officials were awarethat field-produced suck-ers were likely to beinfested with nematodes.Thus the nematode prob-lem became more wide-spread upon the adoptionof Cavendish as theexport variety. The con-nection between thearrival of the nematode problem and the increase in the cultiva-tion of Cavendish varieties has rarely been recognized..

2. The period after World War II saw bananas return to the inter-national export trade and the drive towards their more intensiveproduction. As the losses from blackhead toppling became moreserious greater attention was given to the cause, creating manycareers in practical field nematology. Contemporaneous withthis was the ascendancy of the agricultural chemical industry.The Shell Chemical Company and the Dow ChemicalCompany had developed soil fumigants that had been shown tobe very efficient in controlling nematodes. One product, DBCP,

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Fig. 3. A pit dug around a banana plant showingthe distribution of roots and highlighting thetechnical problem from a nematode managementperspective.

first described by C. W. McBeth and G. B. Bergeson in 1955, wasnon-phytotoxic and could be applied to established bananaplants. DBCP became the standard field treatment whereverbananas were grown for export. The liquid formulation wasapplied at six points around each plant with special hand-oper-ated injectors, not an easy or particularly pleasant task. At last apartial solution to this hitherto undiagnosed and poorly under-stood problem was available (Fig 3). In the Caribbean and Central America, the United Fruit

Company and Standard Fruit Company and the Jamaica BananaBoard (in association with the major chemical companies) led theresearch on banana nematodes. The research activities moved fromthe descriptive and taxonomic to the investigative and practical. Fora long time these organisations had employed plant pathologists, andthe textbooks Banana Diseases Including Plantain and Abaca byC.W. Wardlaw and Banana Plantain and Abaca Diseases by R.H.Stover were standard references for all banana researchers and pro-vided 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 assistedby a handful of chemical company representatives in the develop-ment of fumigants and the newly discovered non-fumigants for thebanana industry.

It was recognized that treating established bananas in the fieldwas not the only solution and that much of the problem was to dowith the infection on the planting material. The blackhead-topplingdisease was described by R. Leach (also a plant pathologist) inNature 181: 204–205 (1958). At this time C.A. Loos and S.B. Looswere working for the banana companies, and in a series of papershighlighted the problem of blackhead disease and how it might bemanaged (1960, Phytopathology 50: 383–386; 1961, Plant DiseaseReporter 45: 457–461). In Australia, C.D. Blake and R.C. Colbranwere also developing and promoting different “seed” treatments forthe banana farmers in New South Wales and Queensland. All ofthese scientists had concluded that longer-lasting control could onlybe 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 macheteand discard suckers with the severest necrosis (Fig. 4).

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– Heat-treat banana suckers in hot water baths (a method firstrecommended 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 prac-ticed on some banana estates but never became universally adopted.The logistics of the treatment were daunting. Each sucker weighs 1-2 kg and about 2,000 suckers are required to plant 1ha. The equip-ment needed for treating such volumes of plant tissue had to berobust and efficient to heat water tanks and maintain them at atemperature of 50 °C. The treatment was effective but did noteliminate all nematodes in the corm tissue and nematode populationdensities increased to damaging levels after a few crop cycles.

Dipping in nematicide or coating suckers with mud impregnat-ed with nematicide (a technique advocated by T. Mateille, P. Topartand P. Quénéhervé in the Ivory Coast) were also recommended.Eventually, regulations concerning availability or use of the emulsifi-able concentrate formulations of these toxic products were tight-ened and the practice was discontinued.

No matter how successful the treatments of planting materialwere they did not resolve the problem of re-planting “clean seed” inland that was already infested with R. similis. Although DBCP treat-ment was adopted by many banana growers and gave good nematodecontrol with economic benefits, it had to be properly applied, which

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

required good field supervision. Thus, when the granular formulationsof non-fumigant organophosphates and oxime carbamates becameavailable, many growers preferred them because of their relative easeof application. The compounds cadusafos, carbofuran, ethoprophos,phenamiphos, oxamyl, and in some countries aldicarb, were usedwidely as granules sprinkled at 2–3 g a.i. around banana plants two orthree times a year (Fogain & Gowen, S., 1997, Nematropica 27: 27–32).With some changes in their registration and formulation, such com-pounds continue to be used on commercial plantations to this day.However, registration of DBCP was withdrawn in 1977 following thereports of its carcinogenic properties and fumigants have ceased to beused for treating banana fields since that time.

A.C. Tarjan (1961, Nematologica 6: 170–175) found that R. sim-ilis 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 populationthat was peculiar to the citrus growing region of central Florida.However, for dedicated banana farmers, the concept of leavingbanana land free of bananas was not popular and such fallowing wasrarely practiced. In addition, the task of removing all vestiges ofcorm and root from a field is time-consuming and the probability ofleaving foci of nematodes in a field was always high. It is interestingto reflect that at this time most of the detailed studies on R. similiswere done by the nematologists from Florida. Thus R. similis, thecause of spreading decline of citrus received much publicity andnotoriety, occupying significant sections in nematology textbookssuch as Victor Dropkin’s Introduction to Plant Nematology and EliCohn’s chapter in Economic Nematology. As a result, many surveyswere conducted in countries wherever bananas and citrus weregrown together. Radopholus similis was invariably found in thebananas 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 ParasiticNematodes in Subtropical and Tropical Agriculture. 2nd edition,CABI, Wallingford, UK).

By the 1970s, the opportunity to plant bananas in new land nothitherto used for bananas, and thus free of R. similis, was uncommon.The introduction of commercial bananas to Belize was one suchexample. Unfortunately, the companies developing this new area

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

The development of the Belize banana industry came just toosoon for another technology that revolutionised banana propagation,and has made the different corm treatments, including hot watertreatment, obsolete. Micro-propagation or tissue culture of bananameristems on defined media in sterile conditions was first demon-strated in Taiwan in the early 1970s. This has now become the stan-dard technique for mass-producing banana plants and enables themovement of material free of major pests and diseases (some virusesexcepted). Even in non-exporting countries such as Uganda, the useof tissue-cultured plants is becoming an accepted practice with someplant production businesses dedicated to this technology (Fig. 5).One-half of the problem with nematodes on bananas has beensolved by micropropogation. There remains the task of controllingnematodes on the growing crop. In 2006, the only treatments usedby commercial producers are those with the non-volatile nemati-

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

cides. From a sustainability point of view this situation will becomeless acceptable as environmental and human health considerationsbecome higher on the political agenda. In addition, commercial pro-ducers regularly spray their crops with fungicides to control the leafspot disease Mycosphaerella fijiensis, an aggressive pathogen that hasalso spread throughout the banana producing regions.

Molecular biology might provide an answer; H. Atkinson, at theUniversity of Leeds, has demonstrated the possibility of the trans-genic approach (Atkinson et al., 2004, Transgenic Research13:135–142). If the popular cultivar Cavendish were to be modifiedby transformation with cystatin-forming constructs, some of thearguments used by objectors to genetic modification would not berelevant since Cavendish is sterile. However, current public opinion,at least in northern Europe, is resolutely against any form of geneticmodification. In the future, some hard choices will have to be madeon this issue.

A frequently asked question is “are there any banana varietieswith resistance and or tolerance to R. similis?” The short answer isno, but that is not quite true. Between 1970–72 I (SRG) workedwith a banana breeding scheme in Jamaica where K. Shepherd haddeveloped several disease-resistant tetraploid cultivars which alsoshowed good tolerance to R. similis in the field, but were not resist-ant, and for different reasons were not considered suitable for theexport banana trade (Gowen, 1979, Nematropica 9: 79–91). Thenematologists with the banana breeding group at the United FruitCompany laboratory in Honduras (E.J. Wehunt, D.I. Edwards and J.Pinochet) also evaluated the material in their collection and foundR. 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 parentin the breeding programme directed by Phil Rowe. By this time, thelaboratory had been given to the Honduran Government andbecame the Fundación Hondurena de Investigación Agricola(FHIA). Some tetraploid hybrid varieties that Phil Rowe producedare now being field-tested, and in some countries in the tropics arebeing grown for local markets. Unfortunately, as with the new vari-eties from the Jamaican programme, the banana marketing compa-nies do not consider the fruit of these nematode- and disease-toler-ant varieties to be as good as that of the (nematode-susceptible)Cavendish varieties.

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Nematodes, particularly R. similis but also Helicotylenchus mul-ticinctus, Pratylenchus coffeae and Meloidogyne spp. continue to bekey pests wherever bananas are grown in the tropics (Gowen., et al.,2005, In: Luc, M., Sikora, R.A. & Bridge, J. (eds), Plant ParasiticNematodes in Subtropical and Tropical Agriculture. 2nd edition, pp 611–643, CABI, Wallingford, UK). In the future, the system ofintensive production of export fruit maintained with the use ofnematicides might well have to change if consumers and regulatoryauthorities conclude that the practice of continuous re-applicationof nematicides to banana plantations is unacceptable. Not all bananavarieties 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 requirefungicide treatment. The export banana industry has changed itspreferred variety once before; there should be no reason why thiscannot happen again, and if it means growing a diversity of cultivarsso much the better as no industry should be dependent upon onegenotype!

The stem and bulb nematode (Ditylenchus dipsaci) and garlic

One usually associates garlic in hot water with those wonderful sali-va-inducing aromas in the kitchen as soups and other savoury dishesare 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 outof their labs and offices to investigate the source of pungent odorswhen the garlic hot water dip treatments were being made at theUniversity of California (UC) Kearney Field Station each autumnduring the 1980s.

I (PAR) had arrived as a postdoctoral fellow at the UCRiverside campus from Rothamsted Experimental Station where mypractical nematode experience was with the round cyst nematodes(Globodera spp.). Shortly thereafter, I was appointed as aNematology Extension Specialist for California annual field and veg-etable crops. At the time that my appointment was announced Iwas at a meeting of California nematologists that was being held atRiverside, and among the attendees was Dr. Bert Lear, a senior pro-

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fessor at UC Davis (Fig. 6), and the Godfather of the hot watertreatment for disinfecting garlic cloves containing Ditylenchus dip-saci. Dr. Lear announced that all the calls he had been receivingabout how to control this nematode on garlic would now be direct-ed to me, since I had instantly become the expert on control of allnematodes in crops grown annually. “Oh really”, I replied in trepida-tion about the honor that Bert was bestowing upon me. Of course, Iknew absolutely nothing about garlic or stem and bulb nematode(apart from a lecture or two on this nematode from HowardAtkinson at the University of Leeds), and even less about the hot-water treatment regimes. Bert was true to his word; within a fewweeks I was fielding calls about stem and bulb nematode on garlic,and as a result spent the next dozen or so years researching variousapproaches to controlling the problem.

The stem and bulb nematode has been known on garlic in Europesince 1877; in The Nethelands it was referred to as kroefziekte and inFrance 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 ononions from Canastota, New York, and is now found in many statesincluding California. The nematode is a persistent but unpredictableproblem in horticultural bulb crops, including onions and garlic,

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

tulip and narcissus. Typicalsymptoms (Fig. 7) on infectedonion and garlic plants includeerratic stands, stunting, loopingand bending of leaves below thesoil surface, spikkel formation(swelling), and extensive longi-tudinal splitting of cotyledonsand leaves (Newhall, 1943,Phytopathology 33: 61–69;Roberts, 2006, In: Compendiumof Onion and Garlic Diseases.2nd edition. H.F. Swartz and S.K.Mohan, (eds), AmericanPhytopathological Society, APSPress, St. Paul, USA.). Leaves areshort and thickened and fre-quently exhibit brown or yel-lowish spots and bloat (stemswelling). Infected seedlingsbecome twisted, enlarged, anddeformed and, in severelyinfested fields, die (Fig. 8). Asthe season progresses, the foliagecollapses and a softening of the bulb begins at the neck and gradual-ly proceeds downward. Scales become soft and light gray. Symptoms

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Fig. 7. Close-up of garlic plants showingsymptoms of seed-borne infection by the stemand bulb nematode, Ditylenchus dipsaci, in aCalifornia field.

Fig. 8. View of garlic field symptom distribution caused by infection with the stem andbulb nematode, Ditylenchus dipsaci. A. Symptoms resulting from seed-borne infestationshowing 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 ofinfestation from a previous planting.

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

Although D. dipsaci has a large number of host races, it can bemanaged effectively as a soil-borne problem by crop rotation. Theonion-garlic race of D. dipsaci attacks onions, garlic, leeks, chives,shasta pea, parsley, celery, mints, lettuce, hairy nightshade, and salsi-fy. Rotating garlic or onions with 4 years of non-host crops betweenplantings in infested fields will ensure that the subsequent Alliumcrop is not infested. Elimination of volunteer onions or garlic andhost weeds ensures host-free rotations. The nematode can be spreadin infested soil, debris from bulb processing and storage houses, andin other materials and on equipment.

Hot water formaldehyde dip treatments were first developedfor nematode and fungus disinfection of narcissus bulbs in Europeand the USA (Anonymous, 1967, Hot-water treatment of plantmaterial. Bulletin 201. London: Her Majesty’s Stationary Office;Chitwood & Blanton, 1941, Journal of the Washington Academy ofScience 31: 296–308; Hawker, 1944, Annals of Applied Biology 31:31–33). Lear and Johnson adapted hot-water treatment for garlic andreported, in two papers in the 1960s, the protocols that they haddeveloped and the improvements made by various temperature andexposure time treatments, together with the use of chemical addi-tives to boost treatment efficacy (Lear & Johnson, 1962, PlantDisease Reporter 46: 635–639; Johnson & Lear, 1965, Plant Disease

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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.

Reporter 49: 898–899). As with any heat treatment of live plantmaterial, the key to successful hot-water treatment is to expose thenematode to a thermal death regime, but not to injure the “germ”potential of the planting stock. For D. dipsaci on garlic, exposure to49° C for 20 minutes was enough to kill the nematode and notinjure the garlic. However, so close are the thermal injury thresholdsfor plant and nematode that the hot-water treatment alone was not100 % effective in eliminating the nematode infestation. Lear andJohnson experimented with additives to the hot-water dips andfound that formaldehyde as an additive was effective in obtainingcomplete nematode kill.

The standard commercial treatment for managing D. dipsaci ongarlic was developed based on the results of the Lear and Johnsonexperiments. They had found with garlic cloves that temperature-timecombinations 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 clovescan 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 wasfound to activate dormant nematodes and optimize nematode eradi-cation from seed-cloves. The excellent adaptation of D. dipsaci tosurvive extreme drying via its anhydrobiotic capacity and clumpingbehavior, especially in the fourth juvenile stage, makes the dehy-drated nematode highly resistant to chemical toxicity, and thusemphasises the value of a warm pre-soak to hydrate and activate thenematode before the hot dip treatment. An additional beneficialcomponent was to plunge the hot-dipped cloves into a cooling tankfor 10 minutes at 18° C, to ensure protection of the garlic tissuefrom heat damage. Based on these studies and additional industry-based modifications for large scale dipping the following regimenwas used for many years by the garlic industry:

Seed cloves are separated from bulbs using rubber rollers (aprocess called cracking). The cloves are then placed in a warmingdip of water for 30 minutes at 38° C followed by immersion in0.74% aqueous formaldehyde for 20 minutes at 49° C, then 10 min-utes in 0.06% benomyl at 18° C and air dried. The benomyl in thecooling stage is used for surface sterilization to minimise fungal con-tamination. The California garlic industry, which produces themajority of the US processed garlic (used for garlic flavoring of food

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as garlic salt, garlic powder, or minced garlic) utilized this hot-watertreatment of seed-cloves on a routine basis during the 1960s to the1990s, when formaldehyde was banned from use due to its carcino-genic properties and hence risk to worker health.

During this period, two extensive research efforts were under-taken by one of us (PAR) with support from the garlic industry (atthat time organized under the banner “American Dehydrated Onionand Garlic Association”, or ADOGA). During the 1980s, consider-able interest in nematology was focused on the development andimplementation of nematode control programs using plant and soiltreatments with non-fumigant nematicides of both organophosphateand carbamate chemical classes. The chemical companies pressedhard with these products as potential replacements for the soilfumigants such as EDB and DBCP that were banned because ofhuman health and environmental contamination concerns.

While numerous nematologists worldwide investigated thepotential of aldicarb (Temik), phenamiphos (Nemacur), ethroprop(Mocap), carbofuran (Furadan), and oxamyl (Vydate) treatments tocontrol most of the important nematode problems on a wide rangeof agronomic and horticultural crops, in California, we examinedtheir potential for control of D. dipsaci on garlic. Several otherresearchers working with bulb crops in Europe, such as Nigel Hagueand Alan Whitehead in the UK (1979, Plant Pathology 28: 86–90;1979, Annals of Applied Biology 93: 213–220) and William Haglundand Harold Jensen in the USA (1983, Journal of Nematology15: 92–96; 1983, Plant Disease 67: 43–44), had shown that several ofthese nematicides were effective in controlling soil-borne nematodeproblems on bulb crops. However, we were interested in determin-ing whether applications at planting time could successfully control“seed-borne” infestations of D. dipsaci on garlic, as an alternative tothe hot-water formaldehyde dip treatments. In the US, federal andstate agencies were already scrutinizing industrial uses of formalde-hyde and the commercial-scale dip treatments were complex anddifficult to maintain. We summarized results from a series of ninefield experiments in which granular formulations of the non-fumi-gants were applied at planting directly onto garlic seed cloves in theseed furrow to assess efficacy for control of D. dipsaci infested cloves(Roberts & Greathead, 1986, Journal of Nematology 18: 66–73). Thetreatments were compared with the standard hot-water formalde-hyde dip and to non-treated controls. As with many results reported

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in these types of non-fumigant nematicide comparisons, we found a mixed performance of the compounds. Aldicarb and phenamiphosat 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/formal-dehyde 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 (Nemacur15G) was subsequently registered for this use on garlic by BayerCorporation in the mid-1980s, while Union Carbide Co., producerof aldicarb (Temik) at that time, determined that the market poten-tial for registration on garlic was too small. Nemacur was not usedmuch by the garlic growers and did not replace the use of the clove-dip treatment. Many nematologists could recount similar storiesabout researching the potential of these non-fumigant nematicidesfor various nematodes on various crops, and although some impor-tant commercial treatments were developed and used, we are seeingtoday the demise of these compounds due to human and environ-mental health issues. Most such products have disappeared and/ortheir registered use on crops has been reduced. At the time of writ-ing, a 2006 nematology meeting in California was informed thatTemik use on cotton remains the only primary use of non-fumigantnematicides on California crops, and that use is targeted for earlyseason insect control.

A second research effort was undertaken, beginning in the late1980s and early 1990s, in which we assessed options for modifyingthe hot-water formaldehyde dip treatment. During this timeformaldehyde was banned from use in this and other industrial-scaleprocesses. The garlic growers were deeply concerned about maintain-ing clean planting stock. We conducted a series of twelve field exper-iments in which various temperature and time regimes were com-pared using water without additives and with sodium hypochloriteor abamectin as additives (Fig. 10) (Roberts & Matthews, 1995,Journal of Nematology 27: 448–456). We re-affirmed that the differen-tial between thermal tolerances of D. dipsaci and garlic cloves wastoo small to allow any effective disinfection regime based on hot-wateralone. However, we discovered that abamectin at 10–20 ppm in thecool dip following a water hotdip of 49° C for 20 minutes was veryeffective, as was sodium hypochlorite at 1.1–3.3% aqueous solution asthe 20-minute hot dip. These regimes were recommended to the

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industry as viable alternatives to the formaldehyde dip treatments. Asfar as we know, these alternatives were never adopted in any broadscale for garlic industry use. Instead, the industry then moved towarda system of producing nematode-free seed-clove stock. Production ofnematode-free, seed garlic cloves is achieved by meristem tip culture,followed by greenhouse, screenhouse, and isolated production innon-infested soil coupled with hot-water treatment of cloves. Oncenematode-free seed garlic cloves are available, an effective regimencombines periodic testing for nematodes (California has an officialstate seed certification process for nematode-free seed-clove garlic)and planting for seed increase on fields that have not been planted tohost 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 ofhot-water treatment to disinfect strawberry of foliar nematode,Aphelenchoides fragariae. Hot-water treatment of dormant strawberrycrowns to be used for planting has been available for about 70 years(Christie & Crossman, 1935, Proceedings of the HelminthologicalSociety of Washington 2: 98–103; Hodson, 1934, Journal of theMinistry of Agriculture 40: 1153–1161), but a survey of the literaturerevealed a large variation in recommended exposure periods andtemperatures and differences in sensitivity among A. fragariae popu-lations (Qui et al., 1993, Suppl. Journal of Nematology 25(4S):795–799). Becky Westerdahl and colleagues at U.C. Davis pursuedinterest from the California strawberry industry to refine the hot-water treatment to manage this problem for California growers.They assessed various time and temperature regimes on five com-mon strawberry cultivars, and determined minimum-maximum

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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.

exposure periods that killed A. fragariae without reducing subse-quent 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 wascompleted sixteen years ago. Becky Westerdahl has just informed usthat the California strawberry growers, who have not used the newinformation and refined procedures developed for nematode man-agement, had just called her and said they are now ready, in 2006, toimplement and utilize the technology. So one can never know whena research investment will pay off, only that new knowledge gainedand recorded always retains potential value for future application.We certainly live with nematodes as a chronic, persistent problemin horticulture that will always require management approaches.

What did we learn from these experiences? Hindsight suggeststhat we should have developed the nematode-free, seed-clove stockprogram for garlic in the first instance, instead of the time-consum-ing and expensive applied research effort over two decades onchemical and water-bath treatments for nematode eradication orcontrol. The same could be said for the foliar nematode problem instrawberry. We worked with non-fumigant nematicides becausethey were readily available, a “hot commodity” in applied nematol-ogy research, and with plenty of funding and “in-kind” support fromthe chemical companies. We worked with the hot-dip treatmentsbecause the industry was equipped to utilize the technology,because with D. dipsaci hot-water without additives “nearly did thejob” and effective adjustments were feasible, as our results proved.

But how quickly society’s perspective has changed over what issafe or not as a process in the workplace or as an application in thefood production chain. Sodium hypochlorite (common bleach) isfound and used in almost every household. Nevertheless, the garlicindustry 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 garlicindustry) about the stem and bulb nematode problem in garlic,about the infection process, the biology of the nematode, the roleplayed by secondary infections by other organisms of nematodeinfected plant tissues, the efficacy of different nematicide treat-ments, and how to utilize nematicides. We also learned how to

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

Summary

Whether the nematode problem is on leaves, stems, bulbs, corms orroots, and the affected crop is narcissus, bananas, garlic or strawber-ries, nematologists are needed to help the grower find a workablesolution. In a world in which pest management strategies need to bebased upon a more conceptual understanding of pests and theircomplex relationships with plant hosts and other organisms, thehorticultural industry requires specialist practitioners with greaterfield expertise. Unfortunately, today we are training fewer nematol-ogists and applied researchers: as more research effort is devoted tocell and molecular biology. Whilst the new technologies haveadvanced our understanding of species diagnosis and some elegantsolutions to seemingly difficult or intractable problems have beenproposed there is a great danger in forsaking the practical andapplied skills. We hope that any molecular solutions will be aseffective as hot-water solutions and not any more transient.

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12.THE SPREAD OF NEMATOLOGY TODEVELOPING COUNTRIES: A CASE STUDY

MICHEL LUC

Formerly, Muséum National d’Histoire Naturelle, Paris, France

Until approximately 1950, plant nematology had been, almost exclu-sively, a subject of interest to the developed countries of Europe andAmerica, 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 pestsof the tropics (particularly insects and fungi) were so dramatic intheir 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 thepossibility of nematode damage in the tropics, the real importance ofnematodes there began to be revealed. These pioneering studies wereusually made in countries that had previously been colonies, andwere made by scientists from the former colonial powers.

The following account by Michel Luc illustrates how the firstscientists were, rather haphazardly, charged with the task of explo-ring tropical nematology. As in Luc’s case, the scientists in questionwere often not originally specialists in nematology, but needed to bere-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 themain research station of ORSTOM (Office de la Recherche

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Scientifique et Technique Outre-Mer), in Adiopodoumé, nearAbidjan, Ivory Coast, when, in May 1953, the Director ofORSTOM (Raoul Combes, a plant physiologist and Professor atthe Sorbonne, Paris) asked me to change my career direction andto become a plant nematologist. That decision stemmed from thedevelopment of banana cultivation in the Ivory Coast and FrenchGuinea for which nematodes were suspected to be an importantlimiting factor. After having received some “international” trainingin the UK (Rothamsted, J.B. Goodey), Belgium (Ghent, L.A.P. deConinck), The Netherlands (Wageningen, M. Oostenbrink andJ.W. Seinhorst), France (Lyon: V. Nigon; Versailles, M. Ritter) I returned to the Ivory Coast in early 1955 with a simple missiondefined by the Director: “Go to Adiopodoumé, establish a lab ofNematology and decide, on the spot, what needs to be done.” So my research career in nematology was opened in a quasi-virginland (only nine references on plant parasitic nematodes for all ofWest Africa including the ex Belgian Congo!). During the initialstage, I established a list of the materials (primarily, good qualitymicroscopes), chemicals etc. that were considered necessary as thebasic equipment of a nematology lab. I also obtained correspondingpro-forma invoices for these items. When, however, I requestedpermission to buy all that material, the answer from the Directorof ORSTOM was very simple: “ORSTOM has no money for that.Your activity will be relevant to agronomy, so the money has tocome from SARA”. An explanation is needed here: as the financialsupport 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 atthat time) permission to establish a national administrative groupcalled SARA (Section Autonome de Recherche Agricole) manageddirectly by Professor Mangenot and receiving a comfortableamount of money from the colony’s funds. So I met ProfessorMangenot at the Sorbonne (he spent 6 months of every year inParis, and 6 months in the Ivory Coast) and presented him my listof materials. His reply was: “I am awfully sorry, but nobody hasinformed me that you must become a nematologist, so I have nofunds at all for you”. I was so upset at the thought of having toreturn to the Ivory Coast to create a new lab in a new subjectwithout a penny that I became severely ill (with jaundice, for the

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first and only time). When recovered, I flew to the Ivory Coast ina very pessimistic frame of mind.

Fortunately, my plant pathologist colleagues helped me and Icould begin the first surveys. The results were very exciting as agreat number of the records were of new species (but this is outsidethe present subject area). Professor Mangenot had also, in the mean-time, returned to the Ivory Coast and was facing an important prob-lem. He had discovered a new plant in the primary forest of theIvory Coast, a small Moraceae of the genus Dorstenia. However, inthe greenhouses of the station, all the plants became stunted, theleaves became yellow, the stems dried and the plants died. All plantpathologists, entomologists, plant physiologists, soil scientists, agron-omists etc. were mobilized to examine the problem but they didnot detect a probable cause, and the plants continued to die (fortu-nately, after having produced seeds in some cases). So, I took myturn at examining the problem. I stained the root system and – mir-acle! – I could observe small nematodes fixed on the roots as well askinds of small black galls containing females, eggs, juveniles andmales of the same nematode. I identified it as a second species ofTylenchulus (it is now an Ivotylenchulus species). Professor Mangenothad some doubts about the effect of “such a small animal”, but Idemonstrated to him, by using surface sterilised seed placed in auto-claved soil, plus re-infestation on some lots (the usual nematologicalmethodology), that the nematode was, in fact, the cause of theobserved stunting.

Some time later Professor Mangenot said to me: “If my memorydoes not fail me, you presented me in Paris a list of materials youneed in order to develop your lab. Please resubmit the list to meand I’ll see what I can do. I have some funds that could, perhaps beused for that purpose”. I gave him the list and a few days later hesaid to me: “Well, finally I have funds enough to accept all the itemson your list”. Some months later I received many of the requestedmaterials, most importantly an up-to-date Zeiss microscope.

After that unexpected good fortune I had no greater supporterthan Professor Mangenot and I had no, or few, problems in buyingmaterials, recruiting local and French assistants, or buildingglasshouses and a new laboratory. He also facilitated the recruitmentof new nematologists, and provided funds to travel for missions out-side the Ivory Coast and to attend various meetings.

The description (Luc, 1957, Nematologica 2:329-334) of

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Tylenchulus mangenoti (= Ivotylenchulus mangenoti) – that was mythanks to the Director – should have been my first nematologicalpublication. Actually it was the second one as I had to wait for thedescription of the host plant!

Thanks are due to that “small animal” for effectively provokingthe development of plant nematology in West Africa.

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13.CONTRIBUTIONS BY LATIN AMERICANNEMATOLOGISTS TO THE STUDY OFNEMATODE PLANT DISORDERS ANDRELATED IMPACT ON CROP PRODUCTION

R.H. MANZANILLA-LÓPEZ1, P. QUÉNÉHERVÉ2, J.A. BRITO3, R. GIBLIN-DAVIS4, J. FRANCO5, J. ROMÁN6 AND R.N. INSERRA7

1Plant 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 andConsumer Services, Nematology Section, Gainesville, Florida,USA; 4University of Florida/IFAS,FLREC, 2305 College Avenue, Davie, Florida, USA;5Fundación para la Promoción e Investigación de Productos Andinos, IBTA-CIP-COTESU, Casilla 4285, Cochabamba, Bolivia; 6Agricultural Experiment Station, Crop Protection Department, P.O. Box 21360, Rio Piedras, Puerto Rico.

A brief history of plant nematology in Latin America andthe Caribbean Islands

The majority of Latin American and Caribbean countries can begenerally characterized as having tropical climates. However, thetopography of these countries creates a great variety of climatic con-dition that result in the growth of tropical, sub-tropical and temper-ate crops that are favored in most areas by an abundance of rainfall.These different climates favor also the development and establish-ment of a great diversity of nematode pests that have different tem-perature and plant host requirements. The major nematologicalproblems afflicting the agriculture of these countries are caused byboth indigenous nematode species on native plant crops (such ascultivated Solanaceae) and by exotic species that were introducedwith new crops. Exotic nematode pests pose serious problems toLatin American agriculture. These pests include the root-knot nem-atode, Meloidogyne ethiopica, which may have arrived during theslave trade between Africa and the New World, and the burrowing

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nematode, Radopholus similis, introduced with mixed bananagenomes from the Far East by early Spanish and Portuguese immi-grants. The recent introduction of the soybean cyst nematode,Heterodera glycines, into Argentina and Brazil illustrates the continu-ing importance of efforts to prevent the importation of exoticspecies into Latin America.

The exchange of crops and their pests between the Americasand Europe caused also the inadvertent movement of native nema-tode plant pests from Latin America into Europe where some, suchas the potato cyst nematodes, have become established as majorpests. As a consequence of these introductions, many exotic nema-tode species from Latin America were described and studied inEuropean countries long before they were reported and studied intheir areas of geographic origin.

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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.

In the past, a lack of advanced agricultural research in LatinAmerica resulted in an inadequate knowledge of nematologicalplant problems. The first report of a plant parasitic nematode fromLatin America may have been that of C. Jobert, in 1878 in France,of a root-knot nematode infecting coffee in Brazil. Later, root-knotnematodes 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 strongimpetus in the second half of the twentieth century from scientificand financial assistance by European and North American countries.One of the founding fathers of plant nematology in the UnitedStates, G. Steiner (Fig. 1), promoted nematological studies in PuertoRico where he worked and cooperated with L. F. Martorell in train-ing the first young nematologists from Puerto Rico, A. Ayala and J.Román (Figs 2, 3). L. G. E. Lordello (Fig. 4), the founder of plantnematology in Brazil, also was trained by Steiner. Investigations intonematode plant pests were conducted in Peru in the early 1960s byA. Martin and continued by J. Franco (see Fig. 5) and G. Gomezunder the guidance of nematologists, such as J. Sasser and W. Maifrom 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 DominicanRepublic, J. Escobar (Fig. 7) in Ecuador, L. Abrego (Fig. 8) in ElSalvador, A. Kermarrec in Guadeloupe and Martinique, C. Sosa-Moss(Fig. 9) in Mexico, R. Tarté (see Fig. 10) in Panama, A. Martin andC. Bazán in Peru, J. Edmunds (Fig. 11) in St. Lucia, and F. Dao (Fig. 12)in Venezuela amongst others. Many of these pioneer nematologistsfaced not only the inevitable scientific challenges, but also a languagebarrier that isolated them from much of the main stream of nemato-logical research. In most Latin American countries, nematology labo-ratories were developed from scratch by self-taught nematologistswho bought and translated books (in some countries the only libraryof nematology is the one accumulated by the nematology teacher)and trained new staff members.

Several phytopathologists, such as R. H. Stover, involved inmanagement of banana diseases at the United Fruit Company and

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its subsidiaries, promoted nematological studies in Central Americancountries, especially in Costa Rica, Honduras and Panama. A grow-ing awareness of nematological problems on crops and of the dra-matic yield increases obtained by the application of chemicalnematicides were major forces in the development of nematologicalprojects in Latin America. Chemical companies found new marketsfor their nematicides. Private and government funds became avail-able to Latin American scientists for nematological training in uni-versities and in nematology research institutes in Europe and NorthAmerica. Scientific societies were established to promote nemato-logical studies in Latin America. The Organization of Nematologistsof Tropical America (ONTA) was founded by Ayala and Román, in1967, with the initial aim of fostering cooperation between nematol-ogists in Latin America and those in the United States. This organi-

194 CONTRIBUTIONS BY LATIN AMERICAN NEMATOLOGISTS

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

zation has received great support from nematologists in the UnitedStates and Europe. The contribution provided by A.C. Tarjan and R.Rodríguez-Kábana in promoting ONTA’s scientific activity andcohesiveness played a pivotal role in enabling this emerging Societyto obtain international scientific recognition. The Brazilian Societyof Nematologists (SBN) was founded by Lordello, in 1974, to pro-mote nematological research in Brazil. Under Lordello’s leadership,the number of nematologists in Brazil increased dramatically andSBN became the most important and largest nematological societyin South America. Other nematological societies were establishedlater in Mexico and Peru. A cooperative agreement between theVenezuelan and Dutch governments provided funding and expertsto teach plant nematology in Venezuela in the 1970s. Many nema-tologists from Latin America attended these international coursesand received training in Venezuela. This initiative led to the estab-

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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. 13. E. Aballay

Fig. 14. J.C. Magunacelaya Fig. 15. J. Esquivel

lishment of excellent nematological facilities in Maracay andenabled the Central University of Venezuela to offer graduate levelcourses in nematology. Nematologists such as M. Doucet (see Fig. 5)and S. del Toro (Argentina), E. Aballay and J.C. Magunacelaya (Figs13, 14) (Chile), A.R. Monteiro and S. Ferraz (Brazil), J. Escobar andCarmen 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 wassucceeded by R. Crozzoli (Fig. 19) (Venezuela) and many othersplayed an important role in nematological training and research.

The history of plant nematology in Mexico is typical of thedevelopment of the science in other Latin American countries. The

196 CONTRIBUTIONS BY LATIN AMERICAN NEMATOLOGISTS

Fig. 16. From left: F. Franco, K. Evans, I. Cid del Prado (fourth in row) and J. Cristóbal (sixth in row) with growers. Fig. 18. M. Canto-Sáenz

Fig. 19. R. CrozzoliFig. 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.

establishment and development of plant nematology in Mexico hasbeen affected by the political and economic events faced by thatcountry from 1906 until today (Montes-Belmont, 2000). C. Sosa-Moss was the promoter of plant nematology in Mexico, and histeaching and research have received international recognition. Theteaching programs and research were favored by international coop-eration with European and North American universities and by theoil boom that started in the late 1970s. A funding shortfall for nema-tological research occurred in the 1990s, but interest in nematologi-cal studies has renewed as a result of international trade agreementsthat have affected the phytosanitary regulations and Mexican agro-policies in recent years. Graduate and postgraduate courses in plantnematology have been offered at the University of Chapingo andPostgraduates” College since 1967.

Similarly, nematological teaching activities were promoted atthe National Agricultural School, La Molina, in Peru and eventuallyin other universities in countries throughout the region. Theincrease in production and marketing of crops such as soybean, veg-etables, citrus, grapes and other fruit crops that has occurred inrecent years in Latin America has favored nematological researchand teaching programs in the region.

The remainder of this chapter highlights aspects of research on some of the most important phytonematological problems inLatin 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 theworld banana trade. Most exports are based on the triploid dessertbananas, mostly of the Cavendish subgroup. These are all minorvariants of one genotype, and there is no other major fruit or veg-etable that depends solely on one variety. In the 1960s, the triploiddessert bananas completely replaced the cultivar, Gros Michel,which was extremely susceptible to fusarial wilt. However, thereplacement Cavendish variety is more susceptible to the burrowingnematode, R. similis. Research on the effects of nematodes on Musaspp. production in Latin America and the Caribbean began as long

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ago as 1870 when bananas were first cultivated for export to NorthAmerica (Champion, 1968). Today, there is a marked decrease innematological research in tropical American and Caribbean Musaproducing countries due to the reduction in the number of nemato-logists, the shortage of funds for research and the lack of encourage-ment to train new nematologists. Ironically, these recent shortages innematological training and research are happening in a period ofgreat need due to the drastic changes in the nematode-banana management system caused by increased concern for environmentalquality (product, soil, water) and human health related to the use ofchemical 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 speciesAshby (1915), in Jamaica, was the first to describe burrowing nema-tode symptoms in banana rhizomes as “Black-head disease ofbananas”. The burrowing nematode in Jamaica, initially identified asTylenchus similis by N. A. Cobb and later reclassified as Radopholussimilis, was subsequently found, in 1939, in the French West Indiesand in other banana growing areas of Central America and theCaribbean. Other nematode pests of banana, such as Pratylenchuscoffeae, Helicotylenchus multicinctus and Meloidogyne spp. are of lesseconomic importance. However, there is increasing occurrence inBrazil and French West Indies of Meloidogyne spp. parasitizingbanana vitroplants soon after planting in areas where competitionwith the burrowing nematode is low.

2. Biology, damage and economicsIn a series of leading publications on the life cycle and histology, CliveLoos described the root symptoms and pathology of R. similis in bananaroots. Subsequently, the difference in pathogenicity among R. similisisolates 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 longevityand nematode management efficacy. Until recently, it was thought thatR. 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, doesnot support the validity of this “sibling” species.

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The interaction between the burrowing nematode and the fun-gus Fusarium oxysporum cf cubense was important in the expressionof Panama disease, and had devastating effects on the cultivar GrosMichel in banana plantations in Central America and the Caribbean.Research on this interaction led to the replacement of Gros Michelwith cultivars from the Cavendish subgroup, which are resistant toFusarium, but susceptible to R. similis.

The role of plant hosts other than bananas in spreading andmaintaining nematode populations in new plantations was investi-gated extensively for regulatory reasons and as a prerequisite fornematode management. The importance of the burrowing nema-tode as a widespread cause of banana losses was first reported byLeach (1958) in Jamaica. Since then, crop losses have been estimatedin the different producing countries on the basis of yield improve-ment after nematicide treatment. These yield responses varied great-ly from 15 to 275%. Such differences are due to several factorsincluding soil type, plant physiology and climate. Damage can varyfrom a hidden lengthening of the vegetative period to the mostobvious symptom of attack by R. similis – toppling over of theentire banana plant. Tropical storms and hurricanes are especiallyprevalent in the Caribbean and in Central America and result inmuch greater numbers of uprooted plants when compared withother banana producing areas of the world.

At present, the percentage of necrotic roots combined withnematode enumeration is the basis of most banana nematode moni-toring in Latin America.

3. Management measuresThe golden era of nematicides (1960–1990)The implementation of good phytosanitation practices and the useof clean propagative material in non-infested land have been recom-mended since the 1960s. Peeling and steam/hot-water treatments ofrhizomes has been emphasized as the method to sanitize infectedbanana rhizomes. However, applications of fumigant nematicidessuch as 1,2-dibromo-3-chloropropane (DBCP) as dip treatments tosanitize infected rhizomes or as injection treatments in the soilaround the infected banana plants became a common managementpractice for nematodes in banana plantations. Non-fumigant, mostlysystemic, nematicides (organophosphates and carbamates), were alsosuccessful as post-planting treatments.

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The application of non-fumigant nematicides still remains themost widely used control method in Latin America with granular orliquid nematicides applied through a sure-fill system and hand-heldapplicators to ensure safe applications. In the past, these treatmentswere mostly applied on a calendar basis, but are now done aftermonitoring by banana plant uprooting and/or the enumeration ofnematodes in the roots. Threshold levels vary from place to place(from 4,000 to 6,000 nematodes per 100g of banana roots in someplantations in Costa Rica to only 1,000 per 100g of roots inMartinique) reflecting regional differences in R. similis pathogenicityand also cultivation practices (see Gowen et al., 2005).

Resistant cultivars Development of resistant cultivars has been a research priority inLatin America and the Caribbean with many scientists involved.Unfortunately, resistance to the burrowing nematode in manybanana cultivars is difficult to incorporate without non-desirabletraits, thereby resulting in practical difficulties in breeding programs.

Alternatives to chemical controlDuring the last decade, several factors have influenced changes inmanagement of banana nematodes e.g., loss of important non-fumi-gant nematicides, absence of effective alternatives (biological con-trol) 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 thereplant crop systems of the Caribbean (due to a lack of clean prop-agative material and clean land) than in the large plantations ofLatin America where bananas are grown continuously withoutreplanting. As a consequence, research on alternatives to chemicaltreatments has been more intense in the Caribbean. The efforts haveconcentrated on replant practices with vitroplants on cleaned soils.This concept was known for a long time, but its application wasfeasible only after disease-free vitroplants from meristem culturebecame available. The suppression of nematode populations whenreplanting old banana plantations poses difficult challenges. Manycultural practices were attempted in the Caribbean in order to freethe soil from R. similis. They include bare or weeded fallow androtations with Pangola grass, Sudan grass, or with crops such as sug-arcane and pineapple. However, some of these cultural practices

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were not implemented because of the high cost of planting andmaintaining the rotation crop along with an inability to developmarkets for the rotation crops. Some improvement in decreasingnematode populations was achieved by chemical destruction ofinfected banana plant rhizome and root tissue in the soil. Theimplementation of this practice not only extended the longevity ofthe banana plantations in the French Antilles, but also greatlydecreased (by 63 % from 1996 to 2004) the application of nemati-cides (see Gowen et al., 2005).

4. Future prospectsDuring the past 50 years, many (and perhaps the most important)advances in our understanding of banana nematodes and their man-agement have been obtained through research in the laboratories ofthe United Fruit Co. in La Lima, Honduras and in the research plotsof the Banana Board of Jamaica at Bodles. However, due to the emi-gration of the nematologists, shortage of support funds for basicresearch and failure to train new nematologists, the focus of researchhas moved from centers in one country to another. Presently, mostof the research is now done in Costa Rica and the French Antilles.The golden era of nematicides is definitely behind us. Developmentof banana varieties that are not only resistant to black Sigatoka, butalso 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 whileworking at the United Fruit Research Laboratories, La Lima,Honduras. Loos, together with his wife Sarah, also worked with theUnited Fruit Company in Panama and with the Banana Board inKingston, Jamaica. Loos’s studies emphasized the importance ofusing clean banana rhizomes to avoid the dissemination of the nem-atode. Unfortunately, these preventative approaches to exclude thenematode from banana plantations were too late or poorly imple-mented at the time, and the nematode was widely disseminatedwith infected banana rhizomes throughout the major banana pro-ducing areas of Central America.

Jesse Román conducted research on R. similis on banana inPuerto Rico for many years. After obtaining a Ph.D. in nematologyunder the supervision of Hirschmann at North Carolina StateUniversity, he worked as a nematologist at the Agricultural

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Experiment Station in San Juan, Puerto Rico. Román studied R. sim-ilis pathotypes of cooking banana (plantain) and the banana planta-tions of Puerto Rico, with emphasis on the genetic characterizationof the banana race of this nematode. The results of his studies, in1985, indicated that both R. similis citrus and banana races have fivechromosomes rather than five or four. His data were debated amongnematologists because they did not support the elevation of the citrus race to species level. Today his findings are congruent withthe results of molecular and mating studies indicating that both thecitrus and the banana race are the same species.

Many young nematologists from the United States initiated theircareer in the late 1960s and early 1970s working on R. similis patho-types and chemical management in banana plantations of the UnitedFruit Company in Honduras, Costa Rica and Panama. W. G. Wehunt(Fig. 20) and D. I. Edwards found a great variability in the host pref-erences of banana race populations. Wehunt became a researchnematologist at the USDA Experiment Station in Byron, Georgia andlater in Arkansas. Q. Holdeman studied the host range and races ofthe burrowing nematode in both Central America and in Californiawhere he was senior nematologist at the California Department ofFood and Agriculture in Sacramento. R. A. Dunn, ExtensionNematologist at the University of Florida, Gainesville, started hiscareer in Costa Rica working on nematode pests of banana.

The English nematologist, S.R. Gowen, worked for WINBANon banana nematodes in both the Windward Islands and in Ecuador,in the 1970s. He supervised many local students working on nema-tode control and resistance in bananas, and has published extensive-ly on his observations. G. Pinochet also worked for many years atthe United Fruit Company, Division of Tropical Research in Panamaon nematode resistance and control before retiring in Spain.

There has been a very significant French contribution in LatinAmerica 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 FrenchWest Indies since the early 1970s. Today, his studies are continuedby P. Quénéhervé (Fig. 21) who aims to implement non-chemicalmanagement approaches for banana production in the French WestIndies, and is currently working on sources of resistance to banananematodes. This French nematologist acquired experience in tropi-cal Nematology at IRD (ex ORSTOM) in West Africa and moved

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

Coconut palm disorder caused by the red ring nematode

Biology, parasitism and damageThe red ring disease of coconut has been reported only in tropicalLatin America and the Lesser Antilles. This disorder, which alsoaffects other palms such as African oil and date palms, is caused by

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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

the red ring nematode, Bursaphelenchus (= Rhadinaphelenchus)cocophilus and usually involves an insect vector, the palm weevilRhynchophorus palmarum.

The palm weevil transports and deposits B. cocophilus juvenilesinto feeding wounds it causes in the palm leaf axils. Subsequently,the nematodes colonize palm stem tissues causing the decline ofinfected palms. Premature yellowing and senescence of leaves and adistinct band of orangish lesions, appearing as a red ring in cross sec-tions of the parenchymal stem tissues, are characteristic symptomsof this disease in coconut palms (Griffith et al., 2005).

Nematode feeding activity causes serious vascular damage tococonut and oil palms, which are stunted, unproductive and eventual-ly killed by the infection. It is not clear if the association of the redring disease and their causal agents originated on native oil palms inthe neotropics and moved onto the coconut palms introduced fromSouth-east Asia and oil palms introduced from Africa, or developed asa disease complex in the neotropics around the turn of the century.

Direct damage induced by the nematode and associated weevilresults in annual crops losses of 10–15% for the coconut and oilpalm industry in the neotropics. This disorder has also an aestheticimpact by seriously affecting the landscape industry. The red ringnematode and the palm weevil are regulated by many countries toprotect their coconut industry (South-east Asia) or their landscapepalms (USA) from these pests.

ManagementPhytosanitary measures aimed at reducing weevil populations andother sources of nematode infection are the best method to reducethe incidence of red ring disease. Early removal of nematode infect-ed palms followed by herbicide treatment of the stem is a commonpractice in affected coconut plantations. Applications of insecticidesare necessary if weevils are present in the culled palms. The use ofsystemic chemical nematicides can effectively cure palms infectedby B. cocophilus as these chemicals suppress nematode populationlevels and thereby assist palm recovery. New managementapproaches based on mass-trapping weevils with sugarcane and asynthetic aggregation pheromone are very effective in reducing thenumber of weevil vectors. Updated information on red ring diseaseof coconut and oil palms are provided by a recent review of thisnematode palm disorder by Griffith et al. (2005).

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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-LatinAmerican Nematologists such as N. A. Cobb and D. W. Fenwick(Fig. 22). However, C. Salazar, a Latin American phytopathologistreported this disease in Venezuela in 1934. Fenwick was a Britishnematologist working at the Field Station of the London School ofHygiene and Tropical Medicine (1938) and later at RothamstedExperimental Station (1945–1958) in England. He subsequentlychanged his research interests to tropical nematology in Trinidad andTobago, West Indies and became Director of Red Ring Research, atTrinidad and Tobago Coconut Research Limited in Trinidad.Fenwick’s traditional plant parasitic nematology background led himto suggest that the red ring disease of palms was a soil borne disor-der. According to Fenwick, experimental root colonization support-ed his view. However, he also emphasized the role of the weevil invectoring and spreading the nematode and the disease. This hadoriginally been suggested by Cobb and Nowell in 1919.

After Fenwick’s retirement, these studies were continued byseveral scientists in Trinidad, including G. Blair and R. Griffith.Their work did not support Fenwick’s theory and Griffith providedconvincing evidence that the disease was mainly vectored by theweevil rather than it being soil borne. R. Griffith (Fig. 23) was edu-cated in Trinidad where he graduated from the Imperial College ofTropical Agriculture in Trinidad (1960). He later expanded his sci-entific knowledge and training by obtaining an MS degree from theUniversity of Wisconsin and a Ph.D. from the World UniversityRound Table. His studies on coconut diseases made him an interna-tional expert on palm diseases and led to his appointment asDirector of Red Ring Research, Coconut Research, Ministry of FoodProduction, in Centeno, Trinidad. Griffith promoted studies on redring disease through cooperative research with scientists from othercountries. The contributions of K. Gerber to the studies of weevilvectors of the red ring nematode are noteworthy. Gerber (Fig. 24), anematologist from Austria, spent almost 2 years in Trinidad andother Latin American countries working on this subject. She adapt-ed very well to the Caribbean life style, but experienced healthproblems induced by tropical diseases such as, dengue fever. She isnow enjoying her retirement in Austria.

In the 1990s, C. Chinchilla made important contributions to ourunderstanding of the chemical ecology of R. palmarum in Costa

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Rican African oil palm plantations that have led to methodologiesfor mass trapping of the red ring nematode vector that can beimplemented in concert with aggressive phytosanitation.

Nematode coffee disorders Lesion and root-knot nematodes are the most common nematodepests of coffee and occur in almost all coffee growing areas in LatinAmerica. Their damaging effects are influenced by environmentalconditions. Sandy soils are more conducive to root-knot nematodeinfections than heavy soils, which are tolerated by lesion nematodes.

Lesion nematodesPratylenchus brachyurus, P. coffeae and P. gutierrezi are the mostcommon lesion nematodes infecting coffee in Latin America. Otherundescribed damaging species closely related to P. gutierrezi alsooccur.

Symptoms, damage and managementCoffee roots infected by lesion nematodes are yellowish or brown incolor and decay rapidly. The infected plants are chlorotic and stunt-ed. These symptoms may result in coffee seedling mortality in nurs-eries and in tree decline followed by premature death. Poor beanquality is another adverse effect of lesion nematode infections.

The use of non-fumigant nematicides is effective in suppressinglesion nematode populations and the incidence of plant mortality.However, non-chemical management strategies, including the use ofcoffee resistant rootstocks (Coffea canefora cv. Robusta) are the bestmanagement approaches for long lasting protection of coffee planta-tions from lesion nematodes (see Campos and Villain, 2005).

Root-knot nematodes The early studies on root-knot nematode pests of coffee were initi-ated by European nematologists. As mentioned in section 1, Jobertfound, for the first time, these pests infecting coffee in Brazil. A fewyears later (1887), E. A. Goeldi (1859–1917) (Fig. 25), a Swiss zoolo-gist and naturalist, who was working as a visiting scientist in theNational Museum in Rio de Janeiro, Brazil confirmed the observa-tions published by Jobert. The results of his investigation allowedhim to correlate the root-knot nematode with the serious coffeedecline that was occurring in plantations in the Province of Rio de

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Janeiro. In his report, published in 1892 in the National MuseumArchives (Arquivos do Museu Nacional) volume 8, Goeldi empha-sized the potential menace of this organism to the Brazilian coffeeindustry. Goeldi studied the morphology of the root-knot nematodepopulations 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 coffeeplantations of Latin America because it was probably distributedwith nematode infected transplants.

Of the 17 species of root-knot nematode found infecting coffeeworldwide, 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 symptomsobserved in infected coffee roots vary with the species. Rootswellings (galls) are consistent symptoms induced by M. exiguainfections, but inconspicuous galls, peeling, cracking of the root cor-tex and destruction of the feeder roots are commonly observed inroots infected by M. arabicida, M. coffeicola, M. incognita and M.paranaensis. These four species are considered to be the most dam-aging ones on coffee in Latin America. They debilitate and kill cof-fee trees and have a devastating impact on coffee plantations. Yieldlosses are very severe (20% or more) with high mortality of plantingmaterial in nematode infested nurseries. The indirect financial lossescaused by nematode damage to the coffee industry is discussed byCampos and Villain (2005).

The management of root-knot nematodes on coffee relies on: i)phytosanitation and production of certified propagative plant mate-rial to avoid the spread of these pests in non-infested land, ii) che-mical approaches and iii) non-chemical approaches such as resistantvarieties and rootstocks, rotation and soil amendments with organicmatter. Coffea arabica lines resistant to root-knot nematodes havebeen successfully selected in Brazil and provide a very useful toolfor managing these pests (Campos and Villain, 2005).

Contributions of Latin American nematologists to the study of nematode pests of coffeeL.G.E. Lordello (1926–2002) was the “Father of BrazilianNematology”. He initiated his research on nematode pests of coffeeand described a new species of root-knot nematode, Meloidogynecoffeicola, which was found causing severe damage in coffee planta-

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tions in the states of Paraná and São Paulo. Lordello also describedsevere coffee decline caused by M. incognita in coffee plantations inSão Paulo, Espirito Santo and Paraná State, and provided evidence ofthe damage caused by the lesion nematode, P. brachyurus, to coffee.These early studies elucidated the important role of nematodes inthe decline of coffee plantations and fostered research projects thatwere carried out by several Latin American nematologists. The pio-neering work that Lordello conducted on nematode pests of coffeereflects his biological knowledge and background. He was educatedin Brazil where he obtained a Ph.D. in zoology from the Universityof São Paulo, Escola Superior de Agricultura “Luiz de Queiroz“(ESALQ), Piracicaba, São Paulo, where he was already conductingresearch and teaching as professor in the Department of Zoology. Inspite of his reluctance to travel, he went to the United States to betrained by Steiner. In the early 1960s, Lordello established the firstcourse in nematology in Brazil, which he taught at both undergradu-ate and graduate level. Lordello worked extensively on nematodesof agricultural relevance, including many projects on root-knotnematodes on coffee. Lordello was also a well respected taxonomistdescribing approximately 50 new taxa.

A. Jaehn (Fig. 26), who was trained by Lordello, dedicated themajority of his short scientific career to developing methods formanagement of root-knot nematode on coffee in the state of SãoPaulo, which is one of the largest coffee producing states in Brazil.Jaehn became well known for his work on cover crops, organicmatter and pesticides as a means to manage root-knot nematode incoffee nurseries and plantations. Jaehn died prematurely from anincurable disease.

The molecular and morphological characterization of root-knotnematode pests of coffee in Latin America has played an importantrole in elucidating the taxonomic status of root-knot nematode popu-lations occurring on coffee. This work has been conducted almostexclusively by R. M. D. G. Carneiro (Fig. 27), another of Lordello’sstudents. Her studies, concerning the genetic variability of root-knotnematode species on coffee, led to the description of two new root-knot nematode species infecting coffee, M. izalcoensis found ori-ginally on coffee in El Salvador and M. paranaensis in Paraná, Brazil.This latter species had originally been referred to as M. incognita bio-type IAPAR by R. Gomes Carneiro, another Brazilian nematologistinvolved in the management of root-knot nematodes on coffee.

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Currently, M. paranaensis is themost pathogenic root-knot nematode incoffee plantations inBrazil, the second mostimportant being M. incognita.

Additional mor-phological work onroot-knot nematodesusing SEM has beenconducted by J. M.dos Santos (Fig. 28).He teaches courses atthe graduate level, andis involved in manage-ment studies of root-knot nematodesin coffee plantationsincluding biological,chemical, organic andcultural approaches.

Important infor-mation on the effectof biological control agents such as Pasteuria penetrans, Arthrobotrysspp., Paecilomyces lilacinus, and Verticillium chlamydosporium(= Pochonia chlamydosporia), and cultural and chemical methods tomanage root-knot nematodes on coffee have been provided by V. P. Campos (Fig. 29).

The search for coffee rootstocks resistant to root-knot nema-todes has been successful in Latin America. This cooperative workbetween nematologists and coffee breeders (W. Gonçalves and L. C.Fazuoli) in Brazil resulted in the selection of the rootstock, ApoatãIAC 2258 (C. canephora), which is resistant to M. exigua, M. incogni-ta (some races) and M. paranaensis. Other resistant rootstocks wereselected in Central America, such as the rootstock Nemaya that isresistant to M. exigua and M. incognita. The Mexican nematologist,N. Marbán-Mendoza, was involved in these selection studies thatwere planned by the nematologist, F. Anzueto. The early contribu-

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Fig. 26. A. Jaehn Fig. 27. R. M. D. G. Carneiro

Fig. 28. J. M. dos Santos Fig. 29. V. P. Campos

tions (1974) of L. Abrego on the biology and management of coffeelesion nematodes in El Salvador are now continued by the Frenchnematologist, L. Villain, who clarified the species composition ofcoffee lesion nematodes and their role in inducing coffee decline inCentral 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 LatinAmerica not only because of the area of land dedicated to its culti-vation, but also because of its nutritional properties. It is native tothe Andean regions of South America where the Incas cultivated itsome 2000 years ago. Some wild potato varieties are native to NorthAmerica (USA and Mexico) and Central America (SIAP, 2002). Thepotato tuber was called “Popotl” by the Nahoas of Mexico and it isnow known as “papa”. It was introduced to Europe by theSpaniards. From 1600–1845, for example, potatoes were the staplefood in Ireland and Irish migrants took it to the USA in 1719. Manycountries of the old world adopted the crop as a staple food becauseof its high yield and nutritional value.

Potato production has increased in Latin America at an averageannual rate of 2.2% for the last three decades. Cultivation hasexpanded annually in Ecuador (3.0%), Peru (2.0%) and Brazil(1.0%). However, increased area of cultivation does not necessarilyequate to increased yield, especially in areas where growth is poorand yield is low.

Latin America is the only region of developing countries with acommercial deficit (307, 000 tons) in potatoes. This deficit is partlyexplained by the importation of seed potatoes to meet regionalrequirements. However, a significant improvement in productivitycould increase competitiveness in the fresh potato market.

At present, potato production tends to be concentrated in areasof higher productivity where surplus tubers can be sold. Andeancountries such as Bolivia and Peru have traditionally produced pota-toes for local consumption, but production is becoming increasinglylinked to market forces and commercialization. Annual potato pro-duction in the Andean region is estimated as 7.8 million tons froman area of 640,000 ha.

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Commercial production in Mexico occurs all year around in thehighlands (2000–3000 m above sea level), but especially in springand summer. The cultivated area is approximately 67,000 ha with ayield of 1,635,000 metric tons (SIAP, 2002) and an average yield of20.5 tons/ha. Production located in irrigated valleys (high input agri-culture) occupies 17,000 ha, while that in rainfed valleys where sub-sistence farming is practiced occupies 50,000 ha. In Latin America,highland potato cultivation is by slash and burn of forests, the con-sequences of these practices include loss of diversity and geneticerosion, expansion of the agricultural frontier, low yield (4 tons/hain Bolivia vs 20 on irrigated fields), and land being disqualified forseed potato production.

In Latin American countries, including Mexico, major limita-tions to crop production include limited use of certified seed ofgenetic and phytosanitary quality, the many pests and diseases, andpoor practices in production processes. Globodera rostochiensis andMeloidogyne spp. are among the most important phytopathologicalproblems for the Mexican potato industry whereas G. rostochiensis,G. pallida and Nacobbus aberrans sensu lato are the major limitingfactors for the Andean region of South America.

Two major international bodies have been created to addressand solve these problems: CIP (International Potato Center, in Peru)fosters scientific research on edible tuber crops and their pathogensand pests in the Andes; whilst PRECODEPA (Programa RegionalCooperativo de Papa) aims to improve production and technologyfor the potato industry in member countries (Central America,Caribbean and Mexico).

Potato cyst nematodes Most Latin American countries are located between latitude 30Nand 60S. Although considered tropical, subtropical highlands andmountain ranges provide these countries with the conditions togrow temperate crops such as potato. Latin America is the center oforigin of major crops, including potato (Solanum spp.), tomato(Lycopersicon spp.), chilli pepper (Capsicum spp.) and maize (Zeamays). It is likely that a combination of geography, climate and veg-etation have allowed many species of cyst nematode to exist andthrive at levels that can cause damage to crops (Sosa-Moss, 1987).

The potato cyst nematodes are believed to have co-evolvedwith potatoes in the Andean region of Peru and/or Bolivia. Brucher

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(1960), however, suggested that they originated in the mountains ofNorth Argentina, where the pest occurs, apparently naturally, ininaccessible places (CIP, 1978).

Globodera rostochiensis and G. pallida are the most commoncyst nematodes in Latin American potato growing areas. Records forG. rostochiensis began in 1952 in Peru. In 1971, Sosa-Moss identifiedG. rostochiensis in Guanajuato, Mexico (Iverson, 1972). Ironically,these introductions to Latin America probably originated from TheNetherlands rather than from the Andean regions of South America.Globodera rostochiensis and G. pallida occur in potato growing areasin the Andean region from Venezuela to Chile. They occur also inCosta Rica and Panama (Central America). A historical account ofthe 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 popula-tions of Globodera that are similar to G. rostochiensis and are foundon native potato species and on other Solanaceae in Mexico. Manyof these native nematode species remain undescribed, includingHeterodera mexicana (= G. mexicana), a species inquirenda reportedby Campos Vela in 1967. Native Globodera species have specifichost ranges and are of great economic and regulatory importance toMexican agriculture as they are potentially as damaging as the goldencyst nematode (R1A pathoype), which causes an average of 50%crop loss in heavily infested fields. In spite of the presence of manyGlobodera species, however, G. pallida has not yet been found inMexico.

Potato cyst nematode infection adversely affects plant growth,production and tuber quality in all potato growing areas of LatinAmerica where these nematodes occur. Economic losses caused tothe potato industry in Bolivia by G. rostochiensis and G. pallidaaverage 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 con-ducted in South America. Molecular identification of these nema-todes 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 (Viciafaba), butter bean (Phaseolus lunatus), corn (Zea mays), spring andhairy vetch (V. sativa and V. villosa), together with the use ofimproved potato varieties (e.g. Alpha, Atlantis, Atzimba). However,

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these varieties are not nematode resistant. Potential use of nativeresistant or tolerant potatoes in order to develop commercial resist-ant varieties has been suggested (Sosa-Moss, 1987). Evaluation ofgenetic material provided by the International Potato Center, rota-tion with non-host crops, organic amendments and trap crops are allmanagement approaches adopted in South America.

False root-knot nematode or potato rosary nematodeThe false-root knot or rosary nematode, Nacobbus aberrans, is a seri-ous pest of potato and other crops in the Andean region of SouthAmerica. The results of molecular studies of nematode populationsfrom different geographical regions of Latin America support thehypothesis that N. aberrans s.l. is a species complex (Anthoine andMugniéry, 2005; Reid et al., 2003). Populations of this pest infectingpotato occur in Mexico, but their distribution is limited. In Mexico,this species is a common and important pest of other crops, includ-ing chilli pepper, tomato, and beans. Nacobbus bolivianus is found inBolivia and potato pathotypes of N. aberrans s.l. have a wide distri-bution in the Andean region, including Argentina, Bolivia, Chile,Ecuador and Peru. Many different aspects of nematode biology, par-asitism and integrated crop management have been studied (at leastin a preliminary fashion) in Latin America (Manzanilla-López et al.,2002).

In the Andean region this nematode is the main limiting factor topotato production. The motile stages are able to infect potato tubersand are disseminated with infected potato seed. The lack of certifiedseed in many Andean countries has resulted in dissemination of thispest throughout the majority of the potato growing areas of SouthAmerica. Quality and yield of potato tubers are seriously affected andthe value of crop losses in South America averages US $ 53 millionannually. These figures are the results of long-term field studies con-ducted 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 certainBolivian populations of Nacobbus (known as rosary potato nema-tode). Development of varieties resistant or tolerant to the falseroot-knot nematode is a major objective of management studies inSouth America since a chemical approach is not feasible in the sub-sistence potato growing areas of the Andes. Crop rotations are diffi-cult to implement because of the wide host range of this pest.

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Implementation of sanitation practices for the production of certi-fied seed potatoes free of Nacobbus is another aspect of manage-ment research being undertaken. Several control strategies includingthermo-therapy, solarization, cultural management, burning of rootdebris, manipulation of sowing dates, organic amendments (com-post), green and animal manures (e.g. chicken manure 7–10 t/ha),crop rotation, trap crops, integrated crop management and, morerecently, biological control have been used. More details can befound in Brodie et al. (1993) and Ortuño et al. (2005).

Despite the acknowledged importance of Nacobbus in economicnematology, it remains a Latin American problem, as it is only inthat region that it causes significant yield loss in staple crops such aspotato (up to 80%). Other affected crops include tomato (50-90%),and beans (35%), the former being of high export value. Despite theprogress achieved so far in understanding this nematode’s biology,parasitic strategies, ecology and taxonomical problems, an effectivemanagement program is needed and, hopefully with internationalcollaboration, this will be achieved.

Latin American contributors to the study of potato disorderscaused by nematodesMany Latin American nematologists have been involved in studiesconcerning potato cyst nematodes. C. Sosa-Moss discovered andidentified the golden nematode in Mexico. He promoted coopera-tive studies concerning the cyst forming nematodes parasitizingsolanaceous plants in Mexico with the participation of M. Luc, L.Miller, D. Mugniéry, R. Mulvey and A. Stone. His academic accom-plishments left an indelible imprint on the Autonomous Universityof Chapingo and the Postgraduates” College.

Important studies on potato cyst nematodes were started inPeru 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 theInternational Potato Center, Lima. These three Peruvian scientistsobtained their Ph.D. in American and British Universities. They arestill actively involved in teaching and conducting excellent researchin Bolivia and Peru.

J.A. Meredith, a nematologist from the United States, spentalmost 20 years in Venezuela where she taught nematology at the

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Central University of Venezuela and studied, together with F. Dao,the response and tolerance of Venezuelan populations of the goldennematode to elevated soil temperatures. Since her retirement, herwork has been continued by R. Crozzoli.

In Chile, I. Moreno, in collaboration with European nematologists,is assessing potato responses to Chilean populations of G. rostochiensisand G. pallida. In Panama and Ecuador surveys of potato cyst nema-todes were conducted by R. Tarté and J. Revelo, respectively.

Studies on N. aberrans s.l. populations infecting potatoes havebeen conducted in Argentina, Bolivia, Mexico and Peru.

In Argentina, M. Costilla investigated extensively the parasitismof N. aberrans s.l. on potato. He made available useful techniquesfor the extraction of this nematode from soil and potato tubers.Costilla dedicated his life to nematological studies and teaching. Hedied prematurely shortly after providing a valuable contribution to acooperative study on N. aberrans s.l. that was published byManzanilla-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 themolecular and morphological characterization of Argentinean popu-lations of N. aberrans s.l. Additional work was done by G. Cap andE. Chaves (Fig. 5).

In Bolivia, J. Franco is involved in applied and basic research onNacobbus. His research studies were facilitated by the experience heacquired working on potato cyst nematodes in Peru. Franco movedto Bolivia in 1989, and is conducting N. aberrans s.l. managementstudies in open field and field plots at very high elevations in theBolivian Andes.

In Mexico, the work conducted on Nacobbus by R.H. Manzanilla-López (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 frequentand not economically important in comparison with those in theAndean Region. Her molecular work with A.P. Reid and D. J. Hunt(CABI) has emphasized the importance of understanding the rela-tionship and taxonomic status of Nacobbus s.l. populations in LatinAmerica. She is a native of Mexico but currently lives in Englandand works at Rothamsted Research.

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

Concluding remarks

The spectrum of this review has been limited to some of the moreimportant nematological problems that are characteristic of LatinAmerica and Caribbean Islands. There are many other nematologicalproblems important to agriculture in Latin America and theCaribbean that are not mentioned in this chapter. This region isafflicted by serious crop losses caused by root-knot nematode speciesdue to its warm climate. However, Ditylenchus dipsaci is also a majoragricultural pest, but in the cooler regions and at higher elevations.Indigenous nematode species such as Punctodera chalcoensis andThecavermiculatus andinus damage indigenous crops including maizeand oca (Oxalis tuberosa). In recent years, fruit crops, grapes, vegeta-bles, and ornamentals have become major Latin American exportcrops. They are damaged by other plant nematode species and manyof the nematologists listed above are also involved in studying thesenematological problems. Many phytopathologists, such as G. MúneraUribe, M. Pizano and F. Varón in Colombia conduct integrated man-agement programs that include nematodes. The contributions pro-vided by Latin American scientists have had a great impact in devel-oping 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 LatinAmerican agriculture, basic yield production is still below thatobtained in European and North American countries and nematodedamage to staple food crops and vegetables remains very high inmany areas. The banning of certain nematicides due to regulatoryand environmental concerns makes the chemical management ofnematodes more difficult and opens new challenges to scientists.Basic research is still needed to provide alternative managementstrategies to the chemical control of nematodes and to ensure safeand high quality products. However, this need cannot be fully met

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by the nematologists who are working today in Latin Americabecause their number and research funding have decreased, as hasthe awareness of growers and politicians of the impact of thesepests on intensive, monoculture systems. The decrease in researchfunding and the lack of replacement of retiring nematologists willhave a negative effect on the production of tropical crops such asbananas, pineapple and coffee which will not continue to be mar-keted at today’s relatively low prices. Training a new generation of nematologists capable of using newtechnologies for the identification and management of nematodepests in Latin America is not an easy task. However, we are confi-dent that Latin american universities and research agencies in collab-oration with nematologists from more technologically advancedcountries can achieve this goal.

Acknowledgements

Rothamsted Research receives grant-aided support from theBiotechnology and Biological Sciences Research Council of theUnited 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. RevistaLatinoamericana de la Papa 11: 40–66.

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GOWEN, S., QUÉNÉHERVÉ, P. A. & FOGAIN, R. 2005. In: Plant Parasitic Nematodes inSubtropical 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. Nematropica32: 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. FundaciónPROINPA-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, EstaciónExperimental 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.

218 CONTRIBUTIONS BY LATIN AMERICAN NEMATOLOGISTS

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 thesame as those of other types of quarantine pests, that is, a sequenceof 1) ignorance of the dangers of transferring plant pests from oneregion of the world to an other, followed by 2) recognition of thedangers (usually resulting from some major problem caused by anintroduced pest), then 3) early, and largely ineffective, attempts bynational authorities to prevent further introductions, and finally 4)international cooperation to exchange information on quarantinepests and methods to prevent spread.

The story of potato cyst nematodes is probably the earliest andmost dramatic example of what can happen if nematodes are trans-ported from their area of original distribution to another part of theworld. When potatoes were carried from South America to Europe inthe 17th century, to provide a cheap and nourishing staple food, theycarried with them the seeds (in a figurative sense) of their own poten-tial destruction: they were infested with one or more species ofGlobodera. At that time, of course, the people who transported themknew almost nothing of nematodes or other minute pests, and theycertainly did not recognise that the potatoes that they brought withsuch hope to Europe were infested with Globodera spp. which wouldbecome established in certain countries and would soon spread to vir-tually every European country. This resulted, eventually, in it becom-ing increasingly difficult to grow adequate amounts of potatoes.

The financial cost to Europe, for the period between the 17thcentury and the present day, of the introduction of Globodera spp. isincalculable. Certainly it has been an enormous amount, taking intoaccount the loss of potential yield, both quantitative and qualitative,

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the cost of research into the cause of yield loss and into possiblecontrol measures, the cost of applying chemical nematicides, thecost of internationally co-ordinated management strategies, and theloss of potential foreign markets for potatoes and other agriculturalproduce 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 activityfrom one region of the world to another, can very easily, andunnoticed, carry pests such as nematodes and cause their long-term establishment in the new region;

– that it is virtually impossible to eradicate an introduced pestafter it has become established and spread;

– that these exotic pests can have a devastating impact in thenew region, even more so than in their area of origin;

– that, without a knowledge of pests, their geographic distribu-tion, their biology and host range, it is impossible to preventtheir spread to other regions, short of stopping all internation-al trade in plants or other commodities that could possiblycarry the pests.

Early quarantine legislation

Stimulated by several other extremely damaging pest introductions(e.g. Phytophthora infestans, the causal agent of late blight of potatoand the cause of the Irish Famine of the mid 19th century, and Viteusvitifoliae, the phylloxera insect which destroyed European grapevineproduction during the same period), several countries began, in thelate 19th and early 20th century, to put in place specific legal meas-ures to try to prevent the introduction of exotic pests into their ter-ritories. Measures for this purpose were described as “quarantine“measures. The name “quarantine” derives ultimately from the Italianword for forty – quaranta, and means “approximately forty“. Theuse of this word originated in the fourteenth century during the ter-rible human epidemic, the Plague or Black Death, which sweptthrough Europe, killing an estimated 25 million people. Certaincoastal cities in the Mediterranean remained free of the disease and,in order to prevent infection from outside, their rulers required that

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ships and their passengers and crews should be kept in isolation out-side the cities (often on islands) until it was certain that they werefree from the disease. It is not clear why forty days was chosen asthe period of isolation; it was perhaps related to the mystical signifi-cance that forty days appears to have in religious contexts. Or possi-bly, experience of the disease had indicated that people who wereinfected, but in whom the disease was latent, would develop symp-toms within approximately forty days of isolation. The meaning of“quarantine” later expanded to include any measures intended toprevent the introduction of diseases or pests, not only of humansbut also of animals and plants; the word also retains its original senseof keeping any suspect disease carrier in isolation and in secure con-ditions for a defined period of time within which symptoms shouldappear. In fact, the word “quarantine” in its broad sense has beengoing out of fashion in recent time; it has been partly replaced by“phytosanitary” (so that quarantine measures are known as “phy-tosanitary measures”) and, more recently, by “plant health”, but it isstill retained when referring to organisms against which measuresare taken (as in “quarantine pest“ or “quarantine nematode”); it isalso used as a noun (e.g. “a quarantine”), especially in NorthAmerica, to refer to a particular regulation.

The phytosanitary measures established by national govern-ments relied on the expertise of their own national experts in plantpathology and entomology to recognise which exotic pests mightpresent a potential risk and to devise appropriate measures to preventpossible introduction. The consequence of such a system was thatthere were great variations between the measures of different coun-tries, even between neighbouring countries where the climatic andagricultural conditions were largely similar. The reasons for thiswere that different experts inevitably have different opinions aboutthe importance of a particular pest, and each expert tends to exag-gerate the importance of the organism(s) included in his/herexpertise! Different experts are likely also to minimise the danger ofa pest present in their own country, and to exaggerate the risk of apest in another country. (Incidentally, before I became involved inplant quarantine and after many years of nematological research, Iwas under the naïve impression that science was always the pursuitof an absolute truth; my first discussions about Bursaphelenchusxylophilus with nematologists from other parts of the world demon-strated to me that the interpretation of scientific data is relative and

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depends on the nationality of the scientist!). The result of the differ-ences of expert opinion in different countries was that many of themeasures targeted unimportant pests, they were often not effectiveor they were unnecessarily restrictive (and, as such, they constitutedbarriers to trade).

Internationalization of quarantine

To try to overcome these problems, it was recognised that some form ofinternational agreement was needed in order to ensure harmonizationbetween the regulations of different countries. The first attempt at suchan agreement (The International Phytopathological Convention ofRome) was drawn up at a most unfortunate time – in 1914 just at thebeginning of the First World War. It was never ratified. A second attemptwas made in 1929, with the International Convention for the Protectionof Plants, but, although signed by many countries, it did not receivemuch subsequent support and was soon forgotten. It was not until afterthe Second War, when Europe, and some other parts of the world, facedsevere food shortages due to land destruction and lack of a labour force,that a more permanent international collaboration developed. At thattime, it was recognised that it was absolutely essential to avoid any possi-ble threat from plant pathogens to agricultural food supplies. TheInternational Plant Protection Convention (IPPC), was drawn up by concerned countries in 1951, under the aegis of the Food and AgriculturalOrganization (FAO) of the United Nations. It required each country to:

– establish a competent national plant protection service whichcould survey its own territory to determine what pests werepresent, and that could inspect plants and plant products toensure absence of pests and issue phytosanitary certificates toattest to this fact;

– publish its phytosanitary regulations, including a list of thequarantine pests against which measures were taken;

– to collaborate with neighbouring countries to establish region-al plant protection organizations which could coordinate allphytosanitary activities.

A major element of the IPPC was the concept that a particularcountry does not have to act alone in defending its borders frominvading pests, but also can depend on exporting countries to take

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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 ofnational plant protection organizations so that importing countriescould have confidence in the thoroughness of the phytosanitaryinspection of the exporting country.

The IPPC also urged governments to create regional plant protec-tion organizations (RPPOs) to coordinate the quarantine activitieswithin specific geographic regions, where the environmental and cli-matic conditions and the pest spectrum would be similar for thecountries in the region. RPPOs have been established for all parts ofthe world, and their establishment has contributed massively to theexchange of information about potentially dangerous exotic pests,about their distribution and about methodology that could be appliedto prevent their spread. The RPPOs also allowed countries to acttogether to protect the region as a whole from alien pests; it is clearlya more effective strategy to prevent the initial introduction into aregion rather than trying to prevent spread from country to countryafter the pest has become established somewhere in the region.

The concept of quarantine pest was established by the IPPC andit can be defined as any pest (that is, any animal, plant or pathogenicagent injurious to plants or plant products; note that the EuropeanUnion uses “harmful organism” with the same meaning) against whichofficial measures are taken by a country to prevent its introduction tothat country; it should be of potential economic importance to thecountry, and it should be absent from the country or, if present,should be of limited distribution and subject to official national meas-ures to prevent spread. Thus a quarantine pest only has relevance inrelation to a particular country. The concept of quarantine nematodeis, of course, included with this definition, but the name is often usedalso for nematodes where the measures to prevent introduction havealready failed and now efforts are being made to eradicate, to limitspread or to reduce the damage caused by the nematode.

It should be noted that, from a global perspective, the principlesof the IPPC were not applied equally diligently throughout theworld. There were many countries, who, although signatories of theIPPC, did not fully trust either their trading partners or their neigh-bouring countries in the region to provide protection from exoticpests and they, therefore, maintained their traditional, phytosanitarypolicy of relying only on their own, thorough, border inspections.Such a policy tended to slow trade unnecessarily.

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After approximately 40 years of operation of the IPPC, duringwhich time the convention had been once revised and most countriesof the world had signed the convention, the World Trade Organization(WTO) came to the conclusion that the IPPC (as well as other inter-national agreements on human and animal health) was ineffective. TheWTO was of the opinion that countries were using the pretext of pro-tecting themselves from exotic pests of humans, animals and plants inorder to block undesirable imports into their territories, for example,imports that might be in direct competition with their own nationalindustries. In other words, they were using sanitary and phytosanitaryregulations as non-tariff barriers to trade. In 1994, the WTO drew upthe Agreement on Sanitary and Phytosanitary Measures (the SPSAgreement) which aimed to establish more rigorous criteria for devis-ing and implementing such measures.

The SPS Agreement recognised that countries have the sovereignright to take phytosanitary measures to protect plant health in theirterritory and that the country can decide on the level of protectionthat they consider appropriate. However, the measures should bebased on international standards, guidelines or recommendations, or,if not, they should be based on scientific principles. As well, perhapsmost importantly for the WTO, they should have a minimal effecton trade. The SPS Agreement allowed countries to challenge eachother’s phytosanitary regulations in a WTO court, with possible sanc-tions if severe measures are maintained unjustifiably.

The arrival of the SPS Agreement was a “wake-up call” for thoseof us involved in plant quarantine. It indicated to us that we hadbeen concentrating on maximising the efficacy of quarantine meas-ures and ignoring the fact that phytosanitary regulations were beingused by some countries to block unwanted trade. Plant quarantinecan be seen as the attempt to balance the two aims of preventing, asmuch as practical, the spread of plant pests while, at the same time,allowing international trade to proceed as fully as possible. We didnot have the balance right.

The other lesson learnt from the advent of the SPS Agreementwas that, in any confrontation between plant protection and worldtrade, it will be trade that will always win. Particularly as the WorldTrade Organization can impose punitive sanctions on any transgres-sion, whereas, the IPPC can only suggest an arbitration procedurebetween disputing countries.

The plant quarantine community moved quite rapidly in reac-

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tion to the SPS Agreement and set about revising the IPPC to bringit into line. The revision was completed in 1997. One of the require-ments in the SPS Agreement was that phytosanitary measuresshould be based on international (that is, global) standards, but, infact, no such global standards existed. Regional standards had previ-ously been produced by some of the regional plant protectionorganizations. In particular, EPPO (the regional organization for theEuropean and Mediterranean region) had produced quite a numberof such standards but these could not be considered as global stan-dards without having been approved and accepted by all countriesof the world. The revision of the IPPC envisaged a system to devel-op global standards; this involved a Secretariat of the IPPC workingwithin FAO with a coordinator responsible for a programme on thedevelopment of standards. The Commission on PhytosanitaryMeasures (CPM) supervises the creation and international approvalof these standards. All member countries of FAO are given theopportunity to study the standards during their preparation, to com-ment on them at this stage and to approve them on completion.

One of the most important international standards required bythe SPS Agreement was on pest risk analysis (PRA). This is neededin order to decide which pests should be quarantine pests for a par-ticular country, or part of a country, and which measures should betaken to prevent their introduction. A standard based on the princi-ples of PRA was, therefore, one of the first to be produced by theSecretariat of the IPPC. PRA, in a standardized format, has becomethe basis for modern phytosanitary measures. This is not to say thatrisk analysis had not previously been used in deciding on quarantinepests and in developing phytosanitary measures. In fact, the processwas generally performed in a non-structured way, either by theintuitive opinion of a particular expert or as a result of discussion“in smoke-filled rooms” between several experts on different aspectsof plant quarantine. Either way, the steps by which the final deci-sions were taken did not usually follow a systematic sequence, wereseldom accurately recorded and were unknown to other peoplewho were not involved.

Within the formalized PRA process developed by theSecretariat of the IPPC, there are three stages: 1. INITIATION, whichidentifies the pest that should be subjected to the process because itmight present a problem if introduced into the area being consid-ered; 2. RISK ASSESSMENT, in which an estimate of risk is obtained by

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combining the probability of introduction and the potential eco-nomic impact; it has not been possible to agree on any form ofquantitative estimate of risk and it is usually expressed in relativeterms. The first two stages require a considerable amount of biologi-cal data on the pest and its host plants and can be performed onlyby experts with the appropriate knowledge. For example, whenanalysing the risk from a particular nematode species, a nematologistwould be required to have available, and to take account of, infor-mation on the nematode’s geographic distribution, host range,host/parasite relations (including economic impact on specific hosts;possibly under different climatic/edaphic conditions), survival abili-ty during transport of the host plants and of other commodities, thepossibility of it becoming established in the area under considera-tion, and the efficacy of measures that could be used to ensure thatcommodities are nematode-free. In addition, information is neededon patterns of trade and on ecological conditions in the country oforigin and country at risk. 3. RISK MANAGEMENT, in which a decisionis taken as to whether the risk is sufficiently great to require mea-sures and, if so, which measures could, or should, be taken. This finalstage usually includes some political decision-making concerning therisk that a country could consider to be acceptable. All the steps inthis process must be recorded and may be re-examined later if newinformation becomes available. It is clear that this version of PRA ismuch more objective and transparent than the previous proceduredescribed above.

Other standards so far developed by the IPPC Secretariat(which are named International Standards for PhytosanitaryMeasures or ISPMs) include general standards on the principles ofplant quarantine, export certification, a phytosanitary import regula-tory system and a glossary of phytosanitary terms, as well as stan-dards on more specific subjects, such as, how to conduct surveil-lance for pests, how to establish and maintain pest-free areas andareas of low pest prevalence, the use of irradiation as a phytosani-tary measure, and the regulation of packaging wood in internationaltrade. Twentyfour ISPMs have been published (Table 1) and othersare in the pipeline. As the development of ISPMs continues, it isobvious that the subject matter will become more specific. Forexample, at present, there is a programme in progress for producingdiagnostic protocols each of which will focus on individual pests, oron groups of related pests, and will provide guidance on how to

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detect the pest in question in traded commodities, and then how toidentify the pest. All of these standards are developed with theactive participation of relevant experts worldwide. Diagnostic proto-cols 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 frombeing introduced to new areas and to avoid cargoes being unneces-sarily blocked, it is essential that suitable methodology should beused to detect quarantine pests during inspection at export orimport and that any organism found should be correctly identified.The more scientifically developed countries may not see the needfor such diagnostic protocols when expert taxonomists for all themajor pest groups are available to their phytosanitary inspectors, butit should be recognised that most countries of the world do nothave such a well developed support service. This programme shouldcontinue with the aim of providing, in the short term, diagnosticprotocols for those quarantine pests where there are recognised taxonomic difficulties (and, among the quarantine nematodes, thereis 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.intfor 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 agentsISPM 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.

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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 publishsuch lists (and there are still several major countries of the worldthat do not yet publish their quarantine lists), one can usually find anumber of quarantine nematodes. The species most commonlyfound probably reflect the views of nematological advisors to thequarantine authorities as to which are the most important species orthe most likely to be transported with international trade. The fol-lowing species are the ten most commonly represented in lists:Globodera rostochiensis, G. pallida, B. xylophilus, Aphelenchoidesbesseyi, Radopholus similis/citrophilus, D. dipsaci, D. destructor,Heterodera glycines, X. americanum and Nacobbus aberrans. Thesespecies are probably justified to be quarantine nematodes, but thejustification for other nematode species found on the quarantinelists of some countries is difficult to imagine. For example, someEuropean countries have the following list entries:

1) “Meloidogyne spp.”, 2) “Hirschmanniella spp. other than H. gracilis” and 3) “Longidorus diadecturus. In my personal opinion,

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these nematodes could only have been declared quarantine nema-todes as a result of a failure to apply PRA or a lack of detailed infor-mation, 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 havesome Meloidogyne spp. present; 2) This list entry is probably intendedto prevent the introduction of species that attack rice, but, in fact,there are numerous Hirschmanniella spp. living in aquatic environ-ments throughout the world, that have little or no effect on theaquatic plants on which they feed. There are several species inEurope, in addition to H. gracilis; 3) L. diadecturus is listed as aquarantine nematode because of its supposed ability to transmitpeach rosette mosaic virus (PRMV). But PRMV belongs to thegroup 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 demonstratetransmission is open to question.

Conclusions

This brief history of the development of plant quarantine demon-strates that the early attempts by individual governments acting aloneto prevent the spread of dangerous pests from one region of the worldto another was largely unsuccessful. This was mainly because theexpertise necessary to recognise potentially dangerous pests and todesign suitably effective measures to prevent their movement withouthindering trade were not widely available. The first exploitation ofinternational collaboration, through the International Plant ProtectionConvention improved the efficacy of quarantine measures but wasnot, 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 aprocess of development of international standards that will surely leadto a more objective, transparent and harmonized system of plantquarantine. However, now in 2007, we have not reached that goal, asthere are many countries which have not yet fully adopted the philos-ophy of international collaboration, and there are still examples ofquarantine legislation, for nematodes at least, that need to be reviewedin the light of current more enlightened thinking.

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There is a need for more input from nematologists into theinternationalization process so as to provide information regardingpotential quarantine nematodes on all aspects that could influenceplant quarantine decisions. Furthermore, although most of this history relates to nematodes (or other types of organisms) as plantpests, it should be noted that nematode species that are not para-sites of plants but are fungal feeding or saprophagous members ofthe soil ecosystem are becoming increasingly examined as potentialalien invasive species, that is, as organisms that could be introducedinto new areas and have the potential themselves or through theorganisms that they carry to disrupt the existing ecological balance.Such alien invasive species are often covered by the same types oflegislation and the same agencies as quarantine pests.

Much more information is needed about these nematodes, butthey have been very little studied as compared with the parasiticspecies; 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 thepinewood nematode, goes back to 1905, when Yano, Japan, reportedan unusual decline of pines from Nagasaki. For a long time thereafter,the cause of the disease was sought, but without success. Because ofthe large number of insects that were usually seen around and oninfected trees, it had always been assumed that the causal agent wouldprove to be among these insect species. However, in 1971, Kiyoharaand Tokushike found a nematode, of the genus Bursaphelenchus, ininfected trees. This nematode multiplied on fungal cultures, was inocu-lated into healthy trees and then re-isolated from the resulting wiltedtrees. The subsequent published reports were impressive: thisBursaphelenchus species could kill fully-grown trees within a fewmonths in the warmer areas of Japan, and could destroy completeforests 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 ofnematode extracted from the wood of diseased pines; it was namedBursaphelenchus lignicolus. Since 1975, the species has spread to thenorth 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 andreduced rainfall in the years 1978 and 1979, the losses were morethan 2 million m3 per year. From the beginning, B. lignicolus wasalways considered by Japanese scientists to be an exotic pest. Butwhere did it come from?

That this nematode could also cause damage in the USA

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became clear in 1979 when B. lignicolus was isolated in great num-bers from wood of a 39 year-old pine tree (Pinus nigra) in Missouriwhich had died after the colour of its needles changed to a reddishbrown colour (Dropkin and Foudin, 1979). In 1981, B. lignicolus wassynonymised by Nickle and colleagues with B. xylophilus, whichhad 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 beennamed Aphelenchoides xylophilus, the wood-inhabitingAphelenchoides, but was recognised by Nickle, in 1970, to belong inthe genus Bursaphelenchus. Its common name in the USA was the“pinewood nematode” (PWN). After its detection in Missouri, itwas found that B. xylophilus was widespread throughout the USAand Canada. It occurred there also on native species of coniferswhere, as a rule, it did not show the symptoms of pine wilt diseaseunless the trees were stressed e.g., by high temperature or lack ofwater. This fact that North America could be the homeland ofPWN was an illuminating piece of evidence. Dwinell (1993) laterreported the presence of B. xylophilus in Mexico.

The main vector of the PWN in Japan was shown to be the long-horned beetle, Monochamus alternatus, belonging to the familyCerambycidae. This beetle lays its eggs in dead or dying trees wherethe developing larvae then feed in the cambium layer. It was alreadyknown in Japan in the 19th century, and by the 1930s, it was said to bepresent in most areas of Japan, but was generally uncommon.However, with the spread of the pine wilt disease, and the resultingincrease of weakened trees that could act as breeding sites for beetles,the populations of Monochamus spp. increased significantly. In NorthAmerica, other Monochamus species transmit PWN, and the mainvector is M. carolinensis. There are also other, less efficient vectors inthe genus Monochamus. Possibly, all Monochamus species that breed inconifers can transmit the PWN, but the occasional transmissions byless efficient species of Monochamus or by some other wood or bark-breeding beetles are of little significance. In Europe, M. galloprovin-cialis and M. sutor are known to transmit the closely related nematodespecies B. mucronatus. Some speculate that these two insect speciesare “standing by” and waiting for the arrival of B. xylophilus. The fearbecame true, in 1999, in Portugal, where M. galloprovincialis transmitsB. 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

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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 spreadfurther and pine wilt disease has been reported from parts of theprovinces of Jiangsu, Anhui, Guangdong, Shandong, Zhejiang andHubei (Yang, 2003). In 1986, the spread of the PWN to Taiwan wasdiscovered and, in 1989, the nematode was reported to be presentin the Republic of Korea where it was detected first in P. thunbergiiand 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 intoFinland from the USA and Canada, and this was the impetus to estab-lish phytosanitary measures to prevent any possible spread intoEurope. Finland prohibited the importation of coniferous wood chipsfrom these sources, and the other Nordic countries soon followedsuit. EPPO (the European and Mediterranean Plant ProtectionOrganization) made a recommendation to its member countries, in1986, to refuse wood imports from infested countries. With itsDirective of 1989 (77/93 EEC), the European Community (latercalled the European Union or EU) recognized the potential danger ofB. xylophilus for European forests and imposed restrictions on importsinto Europe. PWN was placed on the quarantine list of the EU andalso of other European countries. Later, in 1991, a dispensation wasallowed by the Commission of the EU (92/13 EEC) for coniferouswood from North America provided that certain specified require-ments were fulfilled that would prevent introduction of B. xylophilus.

Helen Braasch: “The pinewood Nematode has been particularlyattractive 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 nematodeand by other nematode pests of agricultural or horticultural impor-tance were well known to us in Europe, but that a nematode couldcause 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 ofpalms in the Caribbean Islands. At this time, I was in charge of theQuarantine Laboratory in the Central Plant Protection Office of theformer East Germany (GDR) in Potsdam, close to Berlin. Beingalways on the lookout for new threats in plant quarantine, it wasquickly clear to me that PWN in Japan represented a major new

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quarantine problem. In 1983, I published, in Nachrichtenblatt für denPflanzenschutz in der DDR, the first European report on PWN, enti-tled: “The pine wood nematode, Bursaphelenchus xylophilus (Steinerund Buhrer, 1934) Nickle, 1970, from the point of view of plantquarantine” (in German).

I was concerned with PWN, in one way or another, for morethan 10 years without ever having seen it. Even obtaining the relevantliterature, for those of us “behind the Wall” often required it to beobtained by circuitous routes! When David McNamara, as AssistantDirector of EPPO visited our institute at this time (the GDR hadbecome a member of EPPO) I took the opportunity to ask him (qui-etly!) to support my participation in an EPPO Panel of Experts onPWN. What a request! For me, any participation in scientific meet-ings with the “capitalist abroad“ was forbidden. My boss at the time,the head of the Central Plant Protection Office, considered my inter-est in Bursaphelenchus to be an unnecessary interference with mywork as a quarantine nematologist. How often did I hear the expres-sion: “This is not the EPPO Panel on Bursaphelenchus!” The beliefsomehow persisted that the “Iron Curtain“ would keep the PWN out!But they did not reckon with my stubbornness. As I could not obtaina sample of the PWN, I concerned myself with its relatives. Thus, Idiscovered that B. mucronatus, the nearest relative of the PWN, wasnot only present in Germany but also frequently detected in pine tim-ber 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 avery long time within wood, together with the larval stages of theirinsect vectors. The sampling of the Russian sawn timber at our woodstorage sites was spectacular. We found boards which looked likesieves as a consequence of the Monochamus infestation, and the work-ers at the sites spoke of “swarms of flying beetles”. The beetles wouldbe able to obtain their maturation feeding in the surroundingBrandenburg pine forests and later would be able to lay their eggs inweakened trees. The dauer larvae of B. mucronatus, which are mainlycarried in the tracheae of the insects, would be transported to the treeswhere they could possibly find a new vector and perhaps mingle withnative populations. This could happen in a similar way, we believed,with the PWN which would then find the required environmentalconditions to take up residence in the dry pine forests of Brandenburgwhere summer temperatures are relatively high for Germany.

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My relationship with PWN improved with the collapse of theBerlin Wall and the unification of Germany. The first personal con-tact that I had thereafter with the “western world” was in theInstitute for Nematology and Vertebrate Research of the BBA(Federal Biological Research Centre for Agriculture and Forestry) inMünster, where Dieter Sturhan and Marlies Schauer-Blume had alsobeen concerning themselves with the Bursaphelenchus problem, andhad demonstrated the presence of B. fraudulentus in deciduoustrees. The title of their publication: “The occurrence of pinewoodnematodes (Bursaphelenchus spp.) in the Federal Republic ofGermany ?” (in German) (1989) led later to the misunderstanding incertain parts of the world that it was the pinewood nematode (B.xylophilus) itself that was present in Germany. The readers hadfailed to recognize the question mark in the title. I was laterrequired 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 meand I obtained a new position in the Kleinmachnow Branch of theFederal Biological Research Centre for Agriculture and Forestry.During my vacation, I made the first personal contacts withCanadian ‘Bursaphelenchists’, being received very amicably inVancouver by John Webster and Jack Sutherland. Now, I could alsoestablish my membership of the EPPO Panel of Experts on thePinewood Nematode, which was chaired by David McNamara.

The Department for Economic and Legal Affairs in PlantProtection of the BBA in Braunschweig, with an external branch inKleinmachnow, in which I was employed as a nematologist from1990, had the task, among others, of taking responsibility for fulfill-ing the requirements of Annexes I–V of EU Directive 77/93/EEC(Plant Quarantine Directive) and contributing to related internation-al working groups. The requirements concerning coniferous woodfrom North America needed particularly intensive action. Even ifthe occasional journeys to Brussels to participate in the EU StandingCommittee on Plant Health or in expert groups were inevitablystressful due to the need to leave home at 5 o’clock in the morningand to return at about 11 o’clock in the evening, I was always inter-ested in the statements from the representatives of other countrieson the time-consuming and expensive implementation of therequirements concerning PWN.

The Sword of Damocles was hanging over Europe: coniferous

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wood from infested countries was still being imported, althoughunder certain conditions. The phytosanitary measures considered nec-essary by the EU (especially heat treatment of wood to prevent intro-duction of B. xylophilus) were accepted only reluctantly by theexporting countries, because of the extra expenditure needed to beapplied to their wood exports. Surveillance of the cargoes importedinto Europe was limited to an insufficient number of samples, whilethe examination of documentation continued at a high rate. The dan-ger of introduction with packaging wood was not adequately recog-nized. Meanwhile, some experts in North America began to questionwhether the PWN could survive in Europe and, even if it did, wouldit cause damage anywhere there apart from the Mediterranean regionwhere the temperature was sufficiently high? There were even somesuggestions that the PWN might be already present in Europe andthat the import restrictions were, therefore, unnecessary!

One good thing came out of this situation: although the mainfunction of my department was to provide high quality scientificadvice, I was also placed in the position to be able to conductresearch on the PWN problem in order to provide scientific evalua-tion concerning this quarantine organism, and to present the resultsat international conferences. This privilege was not always free fromenvious glances from some of my colleagues, and also some obstaclesneeded to be overcome, or simply ignored. I surveyed the GermanStates for Bursaphelenchus species, studied their taxonomy and biolo-gy, compared the morphology and damage caused by B. xylophilusand B. mucronatus, conducted inter-species crossing experiments,researched the variability and climatic needs, the means of transmis-sion and spread, and collaborated with foreign nematologists. WithJohn Philis from Nicosia, I studied the Bursaphelenchus fauna ofCyprus; B. xylophilus was, luckily, not found there, despite patchesof dead pines. From a damaged pine in South Africa, an isolate ofBursaphelenchus was sent to me by A. Swart which, again fortunate-ly, proved not to be B. xylophilus, but B. leoni. Several internationalconferences between 1994 and 2004, with sections on pine wilt dis-ease, all contributed not only to the international exchange of experi-ence with the Bursaphelenchus problem, but also led to the founda-tion of a pan-European research collaboration on Bursaphelenchus.This collaboration reached its high-point at the end of the 20th centu-ry with the completion of the EU Project RISKBURS (1996-2000).During a visit to our partner in the research project in Vienna, I met

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Heinrich Schmutzenhofer, Secretary of the International Union ofForestry Research Organisations (IUFRO). In 1981, Dr.Schmutzenhofer had reported finding a Bursaphelenchus species asso-ciated with decline of fir trees in Austria; he made available to methe nematode material that he had collected at the time. During the‘Symposium on sustainability of pine forests in relation to pine wiltand decline’ in Tokyo, Japan (1998) I was able, for the first time, tohold discussions with Yasuharu Mamiya – an impressive, modest andyet radiating personality.

In the 1990s, it was recognized that a scientific response shouldbe given to the question of how dangerous would the PWN reallybe to Europe and to try to convince the doubters, especially inNorth America, of the need for the EU’s quarantine measures. TheEU, therefore, established an Expert Group from the member statesunder the leadership of Hugh Evans of the U.K. The results of theGroup’s deliberations were published in 1996 in the EPPO Bulletinas 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 mostimportant conclusions of this analysis were that the whole of thePRA area is suitable for colonisation by B. xylophilus, but that thedry Mediterranean and continental regions would be in particulardanger for the occurrence of pine wilt disease, that the occurrencein Europe would have important economic consequences, and thatthe greatest danger from wood imports would be when the nema-tode and its vector were both present at the same time. The finalconclusion was that shipments of coniferous wood from infestedareas required phytosanitary measures.

The EU-financed project RISKBURS, of which I was the projectleader, included biologists, forest scientists and molecular biologistsfrom Germany, Greece, Ireland, Italy and Austria. The projectallowed, for the first time in Europe, large-scale detection surveys tobe conducted to determine the Bursaphelenchus species present inEurope. Fortunately, B. xylophilus was not detected in the surveyedareas in Germany, Greece, Italy and Austria. Numerous pathogenici-ty tests conducted on young conifer plants with 15 Bursaphelenchusspecies suggested that B. mucronatus and B. sexdentati could alsohave a potential for pathogenicity, although, so far, no reliable con-firmation of these laboratory data have been observed on forest

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trees. May collaboration with Wolfgang Burgermeister (BBABraunschweig) and his colleagues, which had begun before the startof the project, on the molecular characterization of Bursaphelenchusspp., proved to be fruitful. The characterization of species that hadpreviously been identified morphologically, first used RAPD-PCR,later ITS-RFLP and finally sequencing. The methodology providedan essential contribution to the results of the project and then, later,to the confirmation of the first introduction of B. xylophilus intoPortugal; the methods are still used today. Techniques for molecularidentification of B. xylophilus and B. mucronatus were also elaborat-ed in Ireland, France and North America. The reference picturesbuilt up in Germany, with the aid of the extensive BBA collectionof Bursaphelenchus cultures (now continued by Thomas Schröder),permitted the differentiation of B. xylophilus from about 30 otherBursaphelenchus spp. by means of the ITS-RFLP technique.”

Manuel Mota: “In 1999, B. xylophilus was detected for the firsttime within the territory of the EU, more precisely in an area in theSetúbal Peninsula in Portugal. I am proud to have led a team ofresearchers from Portugal, which included Maria Antónia Bravo andEdmundo Sousa, from INIA. The discovery was made in May 1999while we were surveying cerambycid beetles and associated aphe-lenchid nematodes, in the area of Pegões, a town located in theSetúbal Peninsula, 30 km southeast of Lisbon. In collecting samplesof wood and insects, our intention was to establish which species ofnematode were present and with which species of tree and insectthey were associated. We had no thought that B. xylophilus mightbe present. To our great surprise (and alarm), one of the samplesyielded a tremendous number of specimens of a species ofBursaphelenchus. It was my M.Sc. student, Ana Catarina Penas whomade this observation during research work for her thesis, and shecalled me to confirm. We also asked Maria Antónia Bravo if she hadseen the same nematode in her lot of samples and she said ‘yes’. Thenematode appeared to us to be B. xylophilus, but we needed confir-mation as soon as possible. So, we contacted Helen Braasch andWolfgang Burgermeister in Germany who, more than anyone else,had the expertise to confirm quickly this initial diagnosis, usingmolecular techniques (ITS-RFLP).”

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Helen Braasch: “The greatest surprise of my nematological lifewas when I looked through my microscope, in spring 1999, at asample from a killed pine tree (Pinus pinaster) which had been sentto me by Manuel Mota from the University of Évora, Portugal, andsaw thousands of wriggling specimens of B. xylophilus. Thepinewood nematode in Europe! It was hard to believe.Morphologically, there was little doubt, but a molecular study byWolfgang 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 withthe 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 institu-tional authorities (University president, Research directors) about thisissue and immediately contacted the national plant protection authori-ty (DGPC), who would have to inform the EU Commission inBrussels about this. The initial intention of the EU, in September 1999,was to impose a general embargo on pinewood exports from Portugalto other countries of the EU. Fortunately, and following some intensepolitical lobbying, the quarantined area was restricted to the Setúbal

THE PINEWOOD NEMATODE: A PERSONAL VIEW 239

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.

Peninsula. On a short anecdotal note, I remember my friend JoséFrancisco Fernandes, owner of a large property inside the affected area,who in the past had joked and pulled my leg for studying these strangelittle animals under the microscope, with no apparent practical inter-est. Well, once his pine stand was subjected to strict and expensivequarantine measures, he suddenly became very interested in nematol-ogy, and wanted to learn everything about this new “enemy” whichcaused major economic damage to his financial situation!”

Helen Braasch: “I felt that I must go to Portugal as soon as pos-sible, and, shortly thereafter, I travelled there with WolfgangBurgermeister and Kai Metge. We were met at the airport byManuel Mota and a delegation of important people and very soon,during a working lunch, we exchanged our information. I insisted onseeing the infected tree, and after the meal we drove on the SetúbalPeninsula towards Évora to the affected forest plot. Of course, itwas not really an amusing situation but we laughed when Manuelcould not, at first, find the infected tree! After walking back andforth along the forest path, the tree was at last found; it had beensawn down and the stump was still standing; the tracks of beetlelarvae could be seen on it. We were shown a photograph of the treeas it appeared while still standing, but it did not look as though ithad died only in 1999. As we stood there in sad discussion aroundthe remains of the fallen tree, I heard the repeated expression fromthe representative of the Portuguese Ministry: “It’s a nightmare”!Samples taken from the site, from the stump and from nearby treesconfirmed the infestation. As well, in a second area lying close to atimber storage site, we made another find. A forest worker hadnoticed that the pines appeared to die unusually quickly.

The results of the sampling did not permit any doubt, and theysoon appeared in print (Mota, M. M.; Braasch, H.; Bravo, M. A.; Penas,A. C.; Burgermeister, W.; Metge, K.; Sousa, E., 1999, First record ofBursaphelenchus xylophilus in Portugal and in Europe. Nematology 1:727-734). Shortly afterwards, the vector was recognised to be M. gallo-provincialis which had previously been found only rarely in Portugal.The discovery of PWN in Portugal dramatically altered the Europeanview of the problem. The EU quarantine machinery rolled into actionand the Portuguese did their best to destroy any infected trees, delimitthe infested area, and prevent the spread of the pest to other areas ofPortugal and Europe. Although the spread of pine wilt disease has

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been prevented, it has proved impossible to eradicate the PWN infes-tation, despite the felling and destruction of all suspected trees in thedemarcated zone and the buffer zones.

The EU carried out inspection visits to evaluate the situation inPortugal. During the first inspection visit, we were pursued by thepaparazzi, but our delegation was not prepared to speak to thepress. The press photographers succeeded in taking pictures and, on16th September, 1999 an article appeared in the Portuguese pressentitled: ‘Inspectores Europeus em Silencio’ (‘The European Inspectorsremain silent’). It included a picture of our delegation, under whichmy name was given with the additional information that it was Iwho had confirmed the presence of the pest in Portugal.

I had hardly returned to Kleinmachnow before the telephonebegan to ring incessantly: the European press wanted to know some-thing about the PWN. The German Press Agency spread the newsabout the occurrence of B. xylophilus in Europe. The press reportsranged from more or less scientifically correct to rather distorted,and had headlines such as: “New Pest spreads Shock and Awe“, “TheAdvancing Worm”, “The Invasion of the Worms”, “Pine Stands inPortugal Dying of Thirst”, “Worm becomes a Global Threat”, “TinyWorm Destroys Complete Pine Forests”, “Scientist fromKleinmachnow Protects the World’s Coniferous Forests”– and theseare just a few of the many headlines.

According to EU Directive 2000/29/EC, which replaced theearlier Directive 77/93 EEC, the most important quarantine require-ments for imported conifer wood from infested countries were(depending on the commodity type): heat treatment (56°C in thecentre of the wood for at least 30 minutes), debarking, drying andfreedom from bore holes (galleries of cerambycid larvae and exitholes of beetles). Only the last three requirements were applied toconiferous packaging wood, which is frequently used to contain andsupport other commodities and which is often of inferior quality;this proved to be a serious mistake. There is considerable circum-stantial evidence that PWN was probably introduced to Portugalwith packaging wood from East Asia. The ports of Setúbal andLisbon are both near to the infested zone and there is a strong suspi-cion that infested wood passed through these ports. The impressionis certainly strengthened that importation of packaging wood playeda key role in the spread of PWN. For example, in China, saw mills,building sites and storage areas for packing wood are considered to

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be sources for new infestations. In recent years, in many Europeanand Asiatic countries, samples were taken from packaging woodcoming from infested areas, and PWN and living larvae of the vec-tor beetles were found. This is the greatest danger; both organismstogether in the wood!

In Brussels, the experts from the member states discussed howthe deplorable situation in Portugal should be combated. Thesemeetings are among the most interesting memories of my profes-sional career. It required intense concentration to ensure that theright thing was done, nothing should be overdone, nothing shouldbe neglected, and that the correct procedures should be completed.The decision of the EU of January 11th, 2000 (2000/58/EG) and itsmodified version of March 12th, 2001 (2001/218/EG), as well as lateradjustments, established the regulations whereby the transfer of thepest with coniferous wood and plants from the infested zone topest-free areas in Portugal and in other countries should be prevent-ed. At the same time, member states of the EU were obliged toconduct surveys in their countries to officially determine the distri-bution of PWN. The diagnostic protocol for B. xylophilus, whichhad been developed by EPPO, would facilitate identification. Manyof the non-EU countries in Europe also decided to examine theirpine stands. The results of our research from previous years indicat-ed that B. xylophilus had not, until the discovery in Portugal, beenfound in any European country.”

In order to prevent further introductions of the dangerous pestinto Europe with packing wood, the EU Commission decided, onMarch 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 orpressure impregnation and to be marked by the exporting countryto show the origin and treatment applied. Consignments from Chinahad to be accompanied by a phytosanitary certificate indicating theorigin of the packing wood and the identity of the executer of themeasures carried out. Furthermore, the member state receiving theconsignment must confirm by sampling that the required conditionshave been met.

But these requirements would not be sufficient. It has long beenclear that packaging wood is often re-used and, therefore, movesaround the world, and that Bursaphelenchus spp. survive such travel,as do many other pests. Because the origin of wood packaging mate-

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rial is often difficult to determine, globally applied measures are nec-essary in order to significantly reduce the risk of pest spread. FAOGuidelines for Regulating Wood Packaging Material in InternationalTrade (ISPM 15: FAO, 2003) are recognized as the basis for phytosan-itary measures applied by members of the World Trade Organization(WTO); non-contracting parties are also encouraged to observe thesestandards. These standards describe phytosanitary measures to reducethe risk of introduction and/or spread of quarantine pests associatedwith packaging material (including dunnage, crating, pallets, packingblocks, drums, cases etc.) made of coniferous and non-coniferous rawwood, in use in international trade.”

Helen Braasch: “After my retirement, I conducted a course inChina on the identification of Bursaphelenchus spp., together withWolfgang Burgermeister and Thomas Schröder of the BBABraunschweig; this took place in October, 2002 in Shanghai andNanjing, and was organised by Professor Maosong Lin of Nanjing.The course led to the development of useful collaboration with sev-eral Chinese scientists, especially with Jianfeng Gu of the TechnicalCentre, Ningbo Entry-Exit Inspection and Quarantine Bureau,Ningbo, Zhejiang, China. Almost all wooden packages importedthrough Ningbo harbour since 1997 have been sampled and inspect-ed. Bursaphelenchus xylophilus has been detected many times inlarge numbers in wood samples from different countries, and a con-siderable number of other Bursaphelenchus species, among themseveral undescribed species, were found. The results are alarming:The percentage of batches of packing wood containing any nema-todes averaged 21.3% (between 2000 and 2005), despite the claimson the accompanying phytosanitary certificates that the wood hadbeen 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 knownto occur, underlines the necessity of rigorous application of interna-tional agreements on the phytosanitary treatment of packing woodin international trade. Furthermore, the effectiveness of the quaran-tine measures agreed upon should be investigated and their applica-tion should be controlled more intensively.

In recent years, several new Bursaphelenchus species from EastAsia have been added to the already large number within this genus.Is it possible that we may find more species of Bursaphelenchus that

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are pathogenic to trees? An example of the difficulty of diseasedetection and diagnosis occurred in Vietnam (Lam Dong province)where symptoms similar to pine wilt disease, as well as dead Pinuskesiya trees were found in several locations, and were apparentlyassociated with a Bursaphelenchus species (EPPO Reporting Service2003). The disease was found in four locations in Lam Dongprovince in 36-48% of the trees. The species found in Vietnam ismorphologically distinct from B. xylophilus but shares the same vector, M. alternatus.”

Manuel Mota: “In 2001, my colleagues and I organized a scientificmeeting in Évora, Portugal, for the international community workingon the pinewood nematode in order to exchange views on recentresearch and to discuss control measures. About 50 researchers from14 countries attended the symposium. It was noted that pine speciesnative to North America and growing in the warmer regions areresistant to pine wilt disease, whereas two of the most commonpine species native to Europe, Pinus sylvestris and P. pinaster, arehighly susceptible to the disease and P. pinaster, in particular, occursin the hotter, southern region. Pine wilt disease is a threat to pineforests in southern and possibly eastern Europe, and the predictedclimatic changes, such as increasingly warm and unusual weatherconditions, may significantly influence the incidence of pine wiltdisease. The conclusions arrived at in Évora indicate that the finalresolution for controlling pine wilt should rely on eradication of thenematode or resistance-breeding strategies. Control measures shouldbe aimed at breaking the pine tree/pinewood nematode/pine sawyerdisease triangle. Spraying of insecticides, trunk injection, cutting anddestroying of trees presumed to be infected, restrictions in transport-ing wood, heat treatment and fumigation of timber are the controlmeasures most applied in countries with pine wilt disease. In spite ofthese various efforts, however, the total amount of pine timber lostto the disease is not decreasing conspicuously. This is also true forthe newly invaded region in Portugal. Therefore, strong quarantinemeasures and a particularly rigorous response at the early stage of anynew occurrence of the pinewood nematode are essential to hinderthe spread of this economically important disease in Europe.

A better understanding of the inter-relationships between thenematode, its vectors and the host trees is clearly a precondition forlimitation of damage by B. xylophilus. Additionally, an improvement

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in pest risk analysis systems would help to prevent further spreadand, hopefully, the EU project ‘Development of improved Pest RiskAnalysis techniques for quarantine pests, using pinewood nematode,Bursaphelenchus xylophilus, in Portugal as a model system’(PHRAME) will refine PRA techniques. Future symposia of theleading scientists working in this field will give us ideas for bettermanaging the problem in all its aspects.”

Helen Braasch: “An excellent monograph on PWN resultedfrom the conference in Évora in 2001, containing all the presenta-tions. I am confident that researchers working in this field will findthe booklet invaluable. But I hope also that they do not look tooclosely at the picture of Bursaphelenchus on the cover; it is not apicture of B. xylophilus! But maybe only someone with my continu-ing fascination with the taxonomy of the pinewood nematode andits relatives would notice.”

THE PINEWOOD NEMATODE: A PERSONAL VIEW 245

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, canbe detrimental or beneficial to humans. They parasitize plants andanimals, including many invertebrates, and as many invertebrates arepests, those nematodes that attack them are beneficial to humans.Our focus is on those nematodes that benefit us, especially thosethat 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-nema-tode associations were reported in the 17th to 19th centuries, but theuse of nematodes as biocontrol agents did not occur until the 20th century when Steinernema glaseri was applied in an augmenta-tive biological control program against larvae of the Japanese beetlethat had been introduced into the USA (Glaser & Farrell, 1935,Journal of the New York Entomological Society 43: 345–371). A specta-cular success was achieved when an insect-parasitic (entomogenous)tylenchid nematode species was used as a classical biocontrol agentagainst 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 forthe control of an invasive mole cricket. However, the number ofprojects using entomopathogenic or entomogenous nematodes asclassical biological control is limited.

Commercial interest in using nematodes as biological insecti-

246 HISTORY OF THE DEVELOPMENT OF NEMATODES AS BIOCONTROL AGENTS

cides surged in the 1980s. Private companies refined technologies formass production of nematodes based on the initial research by uni-versity and government researchers. But mass production is only onefacet of using nematodes as biocontrol agents. Both fundamental andapplied research programs have provided a wealth of knowledgethat has led to successful commercialization of these nematodes. Forexample, CSIRO scientists in Australia discovered the two differentlife cycles and morphologies of the tylenchid nematode used in clas-sical biological control against the Sirex woodwasp which eventuallyled to the first commercial nematode product. In the USA,researchers demonstrated that a mermithid nematode had consider-able promise against mosquitoes and was briefly commercialized inthe 1970s. Although this effort failed, because of production/stor-age/transport problems and the discovery of a potent bacteriumthat was more effective against mosquitoes than the nematode,much information about the mermithid’s biology, host range andsurvival was obtained. In the late 1970s and early 1980s, research inmass production technology with entomopathogenic nematodes andefficacy tests against various soil insect pests paved the way to suc-cessfully commercialize the nematodes. Today, a number of compa-nies are producing and marketing entomopathogenic nematodes inAsia, Europe, and North America, and there is interest in otherparts of the world to produce these nematodes commercially. In the1990s, a nematode species that parasitizes slugs was found in theUnited Kingdom and mass production and efficacy tests resulted inits commercial production. These accomplishments demonstratethat nematodes can be important biocontrol agents of insects andslugs. In fact, with the spread of the Sirex woodwasp into otherparts of the world, the tylenchid nematode continues to be used asa classical biological control agent.

Scientists continue to do novel research that furthers the use ofnematodes as biological control agents. For example, using predatorynematodes as biocontrol agents against plant parasitic nematodesand plant pathogenic fungi has generated interest among researchers.In this chapter, we identify the nematodes and the researchers byproviding a brief overview of events that led to the development ofnematodes as biocontrol agents. In presenting this historical perspec-tive, we could have discussed the significant findings chronologicallyas events occurred, but this would have resulted in a discontinuouspresentation of several nematode species and pest organisms. A more

HISTORY OF THE DEVELOPMENT OF NEMATODES AS BIOCONTROL AGENTS 247

readable approach, which we will use, is to do a treatise on thedevelopment of biological control of insects and some other organ-isms by a range of nematode species. Accordingly, we divide ourpresentation into four categories – entomopathogenic nematodes,entomogenous nematodes, malacogenous (malacopathogenic) nema-todes, and predatory nematodes. For more complete information ofthe various nematodes used as biocontrol agents, the reader isreferred 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 familiesSteinernematidae and Heterorhabditidae which are mutualisticallyassociated with bacteria in the genera Xenorhabdus andPhotorhabdus, respectively. Once the infective juvenile nematodesenter a susceptible host, the mutualistic bacteria are released result-ing 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. Thereare now over 36 species of Steinernema and 10 of Heterorhabditisdescribed from around the world. George O. Poinar Jr. wrote thefirst book on this topic, “Nematodes in Biological Control” in 1975.This was followed by “Entomopathogenic Nematodes in BiologicalControl” 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-UdoEhlers and David I. Shapiro-Ilan (eds) (2005).

Seminal discoveries that had significant impact on the commer-cial development of entomopathogenic nematodes are listed in Table1, and we describe below some of the historic details behind someof these discoveries. Rudolf Glaser was the first to establish a cultureof the entomopathogenic nematode, S. glaseri, and to conduct fieldtrials for the control of the introduced Japanese beetle in New Jersey.The remarkable discovery of the symbiotic relationship betweenSteinernema and the bacterium Achromobacter nematophilus wasmade by George O. Poinar Jr. and G. M. Thomas (1966, Parasitology56: 385). This bacterium was later renamed, Xenorhabdus nematophilus.Another nematode genus, Heterorhabditis, with biology similar to

248 HISTORY OF THE DEVELOPMENT OF NEMATODES AS BIOCONTROL AGENTS

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 trans-ferred to a new genus, Photorhabdus. It is now understood that allSteinernema species have mutualistic symbioses with species ofXenorhabdus and all Heterorhabditis species with Photorhabdusspecies (see Noel Boemare’s chapter in EntomopathogenicNematology, 2002).

Indeed, the discovery of symbiosis between entomopathogenicnematodes and bacteria was a major turning point in the develop-ment of the nematodes as commercial biological control agents.Exploiting the discovery of the symbiotic relationship between thenematodes and bacteria, Robin Bedding (1981, Nematologica27: 109–114) was the first to establish a successful mass-productionsystem which has come to be known as a solid culture due to hisinnovative use of polyether polyurethane sponge as a three-dimen-sional support structure allowing nematodes to move through thematrix and provide air exchange. He demonstrated that the nema-todes can be mass-produced on symbiotic bacteria by impregnatingthe sponge with an artificial diet. This led to the formation of thefirst commercial company, Biotech Australia, selling nematodes forcontrol of the black vine weevil in Australia and Europe. The firstcommercial production of entomopathogenic nematodes in liquidculture was established by a team of researchers led by MiltonFriedman at Biosys Inc., in Palo Alto, California. This was soon fol-lowed by MicroBio, a company based in Littlehampton, UK, whichestablished liquid production of Steinernema feltiae. Ralf-Udo Ehlers(1998, Biocontrol 43: 77–86) led the development of the first com-mercial scale production of heterorhabditids in liquid culture.

Formulation development for the application of entomopatho-genic nematodes has been slow. John Webster and Joan Bronskill(1968, Journal of Economic Entomology 61: 1370–1373) were the firstto use a water-holding polymer and a UV protectant mix with thenematodes for application. Robin Bedding (1988, World Patent No.WO 88/08668) developed a “clay sandwich” formulation in whichnematodes were placed in layers of clay to remove surface water.This formulation formed the basis for the commercial introductionof the first entomopathogenic nematode product against the blackvine weevil in Australia. Scientists at Biosys developed an alginateformulation in which sheets of calcium alginate spread over plastic

HISTORY OF THE DEVELOPMENT OF NEMATODES AS BIOCONTROL AGENTS 249

screens were used to trap nematodes. This formulation was intro-duced in the USA in the late 1980s. Continuous efforts to improvenematode formulations led Biosys to develop the first water dis-persible granular formulation in which the nematodes were encasedin 10–20 mm diameter granules consisting of a mixture of varioustypes of silica, clay, cellulose, lignin, and starches (Silver et al., 1995,World Patent No. WO 95/0577). This was the first formulation inwhich up to 7 months of room temperature shelf-life was achievedfor the commercially produced S. carpocapsae.

Quality control is an important activity in the commercial suc-cess of any product in the market. Rick Miller (1989, Journal ofNematology 21: 574) reported the development of the first qualitycontrol method to assess the virulence of commercially produced S. carpocapsae. This method, called the one-on-one Galleria mel-lonella bioassay, spurred the development of several other methodsto enhance nematode quality control.

As nematodes can be easily applied using conventional pesticideapplication equipment, the advances in nematode application tech-nology have been limited. P.S.P. Rao (1975, Indian Journal ofAgricultural Science 54: 275) was the first to show that S. carpocapsaeis compatible with certain insecticides and can be tank mixed.Research has occurred more recently on the effects of pumps, pres-sure differentials, contraction flow fields, agitation, and hydraulicnozzles on entomopathogenic nematodes. Although, nematodes arestill not used extensively against foliar pests, studies have been con-ducted on the use of spinning disks and the application of additivessuch as desiccation and UV protectants. Harry Kaya and C. Nelson(1985, Environmental Entomology 14: 572–574) suggested that thenematodes could be applied to the soil in alginate gels for increasedpersistence, and this concept was commercialized in the applicationof nematodes to tree trunks. Demonstrations that the nematodescan be applied through trickle, center-pivot and furrow irrigationsystems have enhanced the commercial utility of nematodes. It hasbeen demonstrated that the infected insect cadavers can serve asslow release systems for nematodes (Jansson & Lecrone, 1994,Florida Entomology 77: 281–284) and formulating nematode infectedcadavers for application has been attempted (Shapiro-Ilan et al.,2001, Journal of Invertebrate Pathology 78: 17–23).

Studies demonstrating the safety of entomopathogenic nema-todes to mammals and soil invertebrates were instrumental in

250 HISTORY OF THE DEVELOPMENT OF NEMATODES AS BIOCONTROL AGENTS

obtaining exemption of registration requirements for their commer-cial use in the USA, while studies demonstrating the susceptibilityof scores of insect pests to nematodes led to the introduction ofnematode products in various markets round the world. Predictabilityof control was first addressed in a seminal paper summarizing datafrom 82 field trials conducted on the use of nematodes against whitegrubs by Georgis, R. and Gaugler R. (1991, Journal of EconomicEntomology 84: 713–720). A major advance in the field of entomopa-thogenic nematology has been the recognition of dichotomy in thehost-finding behavior of entomopathogenic nematode species. Studies,particularly in Professor Gaugler’s laboratory, have shown that nem-atode species that use the ambushing type of foraging behavior arebetter adapted to finding and parasitizing hosts that are highlymobile and remain on the soil surface while nematode species thatuse 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 theappropriate nematode species with the target insect pests for mosteffective insect control.

Entomogenous nematodes

Entomogenous nematodes are true parasites that do not kill theirhosts quickly. The infective nematode enters a host and obtainsnutrients, affecting host fitness by reducing fecundity or causingsterility as well as reducing longevity, flight or causing other aber-rant behaviors. Nematodes in this group are diverse and include thefamilies Neotylenchidae [e.g., Beddingia (= Deladenus)] andMermithidae (e.g., Romanomermis). Unlike the neotylenchids, themermithids kill their hosts when they exit as 4th stage post-parasites.Although there are a large number of entomogenous nematodesincluding 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 nematodesin biocontrol is Beddingia siricidicola against the woodwasp, Sirexnoctilio (see Bedding & Iede, 2005, in Nematodes as BiocontrolAgents). The nematode, B. siricidicola, was initially discovered para-sitizing S. noctilio in New Zealand in 1962, and was later describedas a new species by Bedding (1968, Nematologica 14: 515–525).

HISTORY OF THE DEVELOPMENT OF NEMATODES AS BIOCONTROL AGENTS 251

Bedding (1967, Nature 214: 174–175) made the startling find that thisnematode had two, complex and separate life cycles. It has a free-living and a parasitic life cycle with two types of morphologicallydifferent adult female. The use of B. siricidicola as a biological con-trol agent against S. noctilio was greatly facilitated by the discoveryof the free-living life cycle. This nematode was mass producedmonoxenically on autoclaved, hydrated wheat in flasks that hadbeen inoculated with the mutualistic fungus.

Beddingia siricidicola was introduced experimentally into Tasmaniain 1970. It established, spread, and achieved high levels of parasitism. By1974, over 70% of the Sirex-infested trees contained nematodes and90% of the emerging S. noctilio adults where parasitized by them. Thefollowing 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 pineplantations. The nematode became established and was the major factorin suppressing S. noctilio populations.

In Brazil, B. siricidicola from Australia were released in 1989.Bedding and Iede (2005, in “Nematodes as Biocontrol Agents”) report-ed that in one area where Sirex infestations were high, the nematodewas released from 1990 to 1993 and resulted in levels of parasitismof 45% in 1991, 75% in 1992, and more than 90% in 1994. In 1995, itwas difficult to find Sirex-infested trees in this area. There can bevariation in nematode parasitism depending on the prevalence ofSirex infestation and the locality. Nematode parasitism varied from17% to 65% in four different areas of Sirex infestation, but para-sitism 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 biocontrolagent against S. noctilio, human intervention is required to monitorand assist the spread of the nematode. In Australia, there is aNational Sirex Coordination Committee, which developed aNational Sirex Strategy. Detailed standard operating procedures forrearing, storing, formulation, and quality control have been estab-lished. In new areas of infestation, worksheets covering variousaspects of Sirex control, including inoculation and distribution ofnematodes, are needed to ensure continued success.

Commercial developments for the entomogenous nematodeshave been reviewed by Platzer et al. (2005, in “Nematodes as

252 HISTORY OF THE DEVELOPMENT OF NEMATODES AS BIOCONTROL AGENTS

Biocontrol Agents”). In particular, nematodes belonging to the familyMermithidae have potential for the control of mosquito larvae. Onespecies, Romanomermis culicivorax (syn. Reesimermis nielseni), whichwas initially isolated from mosquito larvae in Lake Charles,Louisiana and Gainesville, Florida (Petersen, 1985, in Plant andInsect Nematodes. W.R. Nickle (ed.), Marcel Dekker Inc., New York.pp. 797–820), was commercially available in the USA for a shortperiod of time. At least two companies pursued the developmentand commercialization of this mermithid nematode. However, ship-ping 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 companywas able to develop an effective shipping container after a couple ofyears of research, but changed its program before test marketing thecontainer and dropped all biological control products. The discoveryand eventual registration of another biological control agent formosquitoes, Bacillus thuringiensis subsp. israelensis, in 1981, proba-bly added to the decision not to produce R. culicivorax as a biologi-cal control agent for mosquitoes. Overall, the use of either R. culi-civorax or R. iyengari as inundative biocontrol agents may not becost effective in developed countries, but may have greater potentialfor use as inoculative agents for long-term control.

Malacogenous (or Malacopathogenic) nematodes

Only one nematode species has been commercially developed forthe control of pest molluscs. This species, Phasmarhabditis hermaph-rodita, was first described as being associated with the slug Arionater. Although an artificial culture of P. hermaphrodita in ‘‘rottingflesh” was developed in the 1900s, the biocontrol potential of thisspecies was not discovered until the 1990s when Wilson et al.(1993, Biocontrol Science and Technology 3: 503–511) patented theuse of Phasmarhabditis nematodes as biological molluscicides, fol-lowing a 5-year research program supported by MicroBio Ltd (nowBecker Underwood). This company developed the first commercialproduct ‘NemaSlug’, in 1994, for sale in Europe for the control ofslug and snail pests. Much of the early research on the biology, cul-ture, host range, and biocontrol potential of P. hermaphrodita wasconducted by Wilson and his associates. They noted that the nema-

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todes produce an infective juvenile stage that enters the shell cavityof the slug where they multiply and cause the characteristic symp-toms in the form of a swollen mantle. Methods used for mass-pro-duction and formulation of P. hermaphrodita are similar to thosedeveloped for entomopathogenic nematodes (see Wilson & Grewal,2005, in Nematodes as Biocontrol Agents). Improvements in applica-tion methods and field efficacy of P. hermaphrodita have beendemonstrated against several slug species in diverse cropping sys-tems and horticultural settings (see Ester & Wilson, 2005, inNematodes as Biocontrol Agents).

Predatory nematodesNematophagyCobb (1917, Soil Science 3: 431–486) was the first to note predatoryactivity among nematodes. He reported that some mononchidnematodes ferociously feed on other small animals in the soil,including nematodes. Predatory nematodes belong to several ordersincluding Mononchida, Dorylaimida, Diplogasterida, Aphelenchida,Enoplida, and Rhabditida. Although mononchs have several limita-tions as inundative biocontrol agents, studies have demonstratedthat they are natural enemies of plant parasitic nematodes. Bilgramiand Brey (2005, in Nematodes as Biocontrol Agents) make a goodcase for commercial development of predatory nematodes for thecontrol of plant-parasitic nematodes.

MycetophagyNematodes in the genera Aphelenchus, Aphelenchoides, Filenchus,Iotonchium, and Tylenchus are some of those known to feed ondiverse species of fungi. Apart from their role in nutrient cycling,some species have the potential to reduce intensity and incidence ofroot diseases caused by fungi. Most studies have concentrated onAphelenchus avenae which is ubiquitous in temperate zones andfeeds on over 76 species of fungi. Mankau and Mankau (1962,Phytopathology 52: 741) reported on the ability of A. avenae toreproduce on phytopathogenic fungi, and Barker (1964, PlantDisease Reporter 48: 428–432) reported a reduction of Rhizoctoniasolani induced disease on plants due to feeding by A. avenae.Ishibashi et al. (2000, Japanese Journal of Nematology 30: 8–17) werethe first to report on the development of mass-production of

254 HISTORY OF THE DEVELOPMENT OF NEMATODES AS BIOCONTROL AGENTS

A. avenae for commercial application. Ishibashi and his colleagueshave conducted much of the recent research on field efficacy of A. avenae in controlling fungal pathogens of plants, and he makes aconvincing case for further evaluation and the potential develop-ment of A. avenae as a prophylactic biocontrol agent of root dis-eases of plants caused by soil borne fungi (see Ishibashi, 2005, inNematodes as Biocontrol Agents).

BacteriophagyBacteriophagy is common in rhabditid and diplogastrid nematodes inthe soil. However, there are reports of both a decrease and anincrease in bacterial biomass in the presence of bacteria-feedingnematodes. Grewal (1991, Annals of Applied Biology 118: 47–55) hasdemonstrated the potential of the bacteria-feeding nematode,Caenorhabditis elegans, to spread the antagonistic bacterium,Pseudomonas flourescens, for suppressing the bacterial blotch diseaseof mushrooms, caused by Pseudomonas tolaassii. This area definitelyrequires additional research.

Table 1. Some seminal discoveries that led to commercial develop-ment of entomopathogenic nematodes as biocontrol agents.

Discovery Year Reference

Discovery of Steinernema 1923 Steiner, G., Zentralblatt für Bakteriologie,Parasitenkunde,Infektionskrankheiten undHygiene Abt. 1 orig., 59: 14.

Establishment of 1931 Glaser, R.W., Science 73: 614.Steinernema in culture

Susceptibility of the 1932 Glaser, R.W., New Jersey Departmentfirst pest species of Agriculture. Circ. 211.

First field application 1935 Glaser, R.W., Farrell, C.C., against a pest Journal of New York Entomological.

Society 43: 345–371.

Discovery of a 1966 Poinar, G.O., Jr., Thomas, G.M.,symbiotic bacterium Parasitology 56: 385.

First prototype 1968 Webster, J.M., Bronskill, J.F., Journalformulation of Economic Entomology 61: 1370–1373.

HISTORY OF THE DEVELOPMENT OF NEMATODES AS BIOCONTROL AGENTS 255

Desiccation, survival 1973 Simmons, W.R., Poinar, G.O. Jr.,of Steinernema 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 1975 Bedding, R.A., Akhurst, R.J.,Galleria-bait technique Nematologica 21: 109–110.

Pesticide compatibility of 1975 Rao, P.S.P., Steinernema Indian Journal of Agricultural Sciences.

54: 275.

Establishment of the 1981 Bedding, R.A., Nematologica 27: 109–114.first commercialmass-production system(solid culture)

First demonstration of 1981 Bedding, R.A., Miller, L.A.,effective control of Annals of Appied. Biology 99: 211–216.black vine weevil withnematodes

Encapsulation 1985 Kaya, H. K., Nelsen, C.E.,of nematodes in calcium Environmental Entomology 14: 572–574.alginate

Development of liquid 1986 Pace, G.W., Grote, W., Pitt, D.E., culture of nematodes Pitt, J.M., World Patent

No. WO 86/01074.

Development of 1988 Bedding, R. A., World Patentclay-based formulations No. WO 88/08668.

Development of the 1989 Miller, R., Journal of Nematology 21: 574.one-on-one qualitycontrol bioassay

Establishment of the 1990 Friedman, M.J.,first commercial liquid In: Gaugler R., Kaya, H.K., (eds.),mass-production for Entomopathogenic Nematodes

Steinernema 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.

256 HISTORY OF THE DEVELOPMENT OF NEMATODES AS BIOCONTROL AGENTS

Development of 1995 Silver, S.C., Dunlop, D.B., Grove, I.D.,water dispersible granules World Patent No. WO 95/0577.

Establishment of the 1998 Ehlers, R.-U., Lunau, S.first commercial liquid Krasomil-Osterfeld, K., Osterfeld, K.H.,mass-production Biocontrol 43: 77–86.for Heterorhabditis

Synergism with nicotinoid 1998 Koppenhöfer, A.M., Kaya, H.K.,insecticides Journal of Economic Entomology

91: 618–623.

Development of the 1999 Grewal, P.S., Converse, V., Georgis, R.,sand-well bioassay Journal of Invertebrate Pathology

73: 40–44.

First assessment of the 2000 Gaugler, R., Grewal, P., Kaya, H.K.,quality of commercially Smith-Fiola, D., Biological Controlproduced nematodes 17: 100–109.

HISTORY OF THE DEVELOPMENT OF NEMATODES AS BIOCONTROL AGENTS 257

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 asso-ciated information in nematology relies to a considerable extent onjournals, newsletters and conferences as essential components of aninterlinking infrastructure. In the last 50 years, there have been sev-eral important changes and developments to facilitate informationexchange, the most important of which is the advent of the internetand the associated repository of freely available information,amongst which molecular data are arguably the most significant. Inthis short overview, we aim to trace the development of the differ-ent aspects of the infrastructure of the European Society ofNematologists (ESN) and the Society of Nematologists (SON), andmake some educated guesses about future changes.

Societies

The first international conference for nematology (called the‘International Nematology Symposium and Training Course’) washeld at Rothamsted Experimental Station (now RothamstedResearch) in Harpenden, UK in 1951. Twelve subject areas were cov-ered, each being examined in relation to Heterodera (=Globodera)

258 DYNAMICS OF NEMATOLOGICAL INFRASTRUCTURE

rostochiensis, Ditylenchus dipsaci, Aphelenchoides spp., andPratylenchus spp. The 46 participants came from different Europeancountries and also from countries such as Egypt, Haiti, Indonesia,Uganda; USA nematology was represented by Gerald Thorne. Themeeting was notable for including a one-day excursion to Cambridge.This inclusion of a tour day, in which the spouses and family ofnematologists also participate has become an enjoyable, standard fea-ture of almost all conferences organised by the ESN.

However, it was not until 1953, at the International Congress ofZoology in Copenhagen, that the idea of a European nematologysociety was envisaged. Two years later at the ‘InternationalSymposium on Plant Nematodes and the Disease they Cause’ inWageningen, Prosper Bovien from Denmark was a prime mover inestablishing the Society of European Nematologists, as it was initial-ly 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 the4th International Symposium for Nematology. During the 9th Europeansymposium, in Warsaw in 1967, the general meeting decided tochange the name of the society from the Society of EuropeanNematologists to the European Society of Nematologists. In 1958,the US nematologists in the American Phytopathological Society dis-cussed the formation of a separate society. The first officers of theSON were elected in 1961, with Merlin Allen as the President. Thefirst meeting was held the following year at Oregon State University.Unlike the ESN, which holds meetings biennially (Table 1), the SONmeetings are held annually (Table 2). The initial meetings of theSON, in the 1960s and 1970s, were held predominately on collegecampuses, whereas for the last 15 years the majority of the meetingshave been held in luxury hotels. There is a greater tendency amongstNorth American nematologists, compared with nematologists fromelsewhere, to incorporate the conference into a family vacation.Many of the ESN meetings have included accommodation inUniversity halls of residence, which in recent years have been builtmore specifically to attract conferences and provide en suite facilities,a great rarity in the early years. The frequent encounters in thoseyears with distinguished scientists plodding down the hall to a com-mon bath/shower facility was a great equaliser, although not alwaysappreciated by some senior nematologists!

DYNAMICS OF NEMATOLOGICAL INFRASTRUCTURE 259

Table 1. List of the International Symposia of the European Society of Nematologists.

1st Symposium Harpenden, England, UK 19512nd Symposium Copenhagen, Denmark 1953

(part of the InternationalCongress of Zoology)

3rd Symposium Wageningen, The Netherlands 1955(part of the InternationalSymposium on PlantNematodes and the Diseases they Cause)

4th Symposium Hamburg, Germany 1957(the first meeting after the official formation of theSociety of European Nematologists)

5th Symposium Uppsala, Sweden 19596th Symposium Gent, Belgium 19617th Symposium Auchincruive, Scotland, UK 19638th Symposium Antibes, France 19659th Symposium Warsaw, Poland 1967

(the Society’s name was changed to theEuropean Society of Nematologists)

10th Symposium Pescara, Italy 197011th Symposium Reading, England, UK 197212th Symposium Granada, Spain 197413th Symposium Dublin, Ireland 197614th Symposium Munich, Germany 1978

(as the Nematology section of the3rd International Congressof Plant Pathology)

15th Symposium Bari, Italy 198016th Symposium St Andrews, Scotland, UK 198217th Symposium Guelph, Canada 1984

(as part of the 1st International Congressof Nematology)

18th Symposium Antibes-Juan-les-Pins, France 198619th Symposium Uppsala, Sweden 198820th Symposium Veldhoven, The Netherlands 1990

(as part of the 2nd International Congressof Nematology)

260 DYNAMICS OF NEMATOLOGICAL INFRASTRUCTURE

21st Symposium Albufeira, Portugal 199222nd Symposium Gent, Belgium 199423rd Symposium Guadeloupe, Antilles 1996

(as part of the 3rd International Congressof Nematology)

24th Symposium Dundee, Scotland, UK 199825th Symposium Herzliya, Israel 200026th Symposium Tenerife, Canary Islands 2002

(as part of the 4th International Congressof Nematology)

27th Symposium Rome, Italy 200428th Symposium Blagoevgrad, Bulgaria 2006

Table 2. List of the Meetings of the Society of Nematologists.

1st Meeting Corvallis, Oregon 19622nd Meeting Amherst, Massachusetts 19633rd Meeting Boulder, Colorado 19644th Meeting Urbana, Illinois 19655th Meeting Daytona Beach, Florida 19666th Meeting Washington, D.C. 19677th Meeting Columbus, Ohio 19688th Meeting San Francisco, California 19699th Meeting Washington, D.C. 197010th Meeting Ottawa, Canada 197111th Meeting Raleigh, North Carolina 197212th Meeting Minneapolis, Minnesota 197313th Meeting Riverside, California 197414th Meeting Houston, Texas 197515th Meeting Daytona Beach, Florida 197616th Meeting East Lansing, Michigan 197717th Meeting Hot Springs, Arkansas 197818th Meeting Salt Lake City, Utah 197919th Meeting New Orleans, Louisiana 198020th Meeting Seattle, Washington 198121st Meeting Knoxville, Tennessee 198222nd Meeting Ames, Iowa 1983

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23rd Meeting Guelph, Canada 1984(as part of the 1st International Congressof Nematology)

24th Meeting Atlantic City, New Jersey 198525th Meeting Orlando, Florida 198626th Meeting Honolulu, Hawaii 198727th Meeting Raleigh, North Carolina 198828th Meeting Davis, California 198929th Meeting Veldhoven, The Netherlands 1990

(as part of the 2nd International Congressof Nematology)

30th Meeting Baltimore, Massachusetts 199131st Meeting Vancouver, Canada 199232nd Meeting Nashville, Tennessee 199333rd Meeting San Antonio, Texas 199434th Meeting Little Rock, Arkansas 199535th Meeting Guadeloupe, Antilles 1996

(as part of the 3rd International Congressof Nematology)

36th Meeting Tucson, Arizona 199737th Meeting St Louis, Missouri 199838th Meeting Monteray, California 199939th Meeting Quebec City, Canada 200040th Meeting Salt Lake City, Utah 200141st Meeting Tenerife, Canary Islands 2002

(as part of the 4th International Congress of Nematology)

42nd Meeting Ithaca, New York 200343rd Meeting Estes Park, Colorado 200444th Meeting Fort Lauderdale, Florida 200545th Meeting Kauai, Hawaii 2006

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The SON council is more structured than the equivalent inESN. As well as an executive board of the SON, with elected andnominated members, there are numerous committees that makerecommendations on various aspects from awards to specialist top-ics and policy. The President of the Society is elected by theSociety’s members. Traditionally, Presidents and executive boardmembers have been from North America, although Roland Perry(Fig. 1) became the first person from Europe to serve on the execu-tive board (2000–2003) and it is possible that a future Presidentmay be from the wider membership outside North America. TheESN President is the person in whose country the next ESN meet-ing will take place. Thus, participants at the annual general meetingvote on the venue for the next meeting and the President is fromthe country of the successful bid. The board of ESN comprises peo-ple nominated by the membership and recently has included anematologist from the USA; currently this is James Starr (Fig. 1).

An essential component of all scientific meetings is the availabilityof an appropriate place where liquid refreshment facilitates exchangeof information and discussion of research ideas. This has always been achallenge on US college campuses but changes, especially the provision

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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 theSociety of Nematologists.

of microbreweries, have been beneficial. Such changes are not just con-fined to campuses: in 1979, the SON meeting was held in Salt LakeCity and it was an extreme challenge to find any suitable establishmentbut, by contrast, when the Society met there again, in 2001, there weretwo 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 formedpart of the 3rd International Congress of Plant Pathology, was mem-orable for the welcome reception in the famous Hofbräu Haus! Thismeeting included, for the first time, posters and a paper on biologi-cal control. Looking through the programmes over the years, it isevident that the emphasis in the early years on papers about chemi-cal control gradually changed, and by the 15th symposium in Bari, in1980, there was a session on alternative control methods. This trendhas 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 interactionsamong individuals and groups from Europe and North America andthe advent of relatively cheap flights, especially within Europe, gen-erated closer links among nematologists world wide. Several individ-uals perceived a need for an official forum to link the nematologysocieties more closely. This was partially realised when the threelargest societies, the ESN, the SON and the Organisation forNematologists of Tropical America (ONTA; organized in PuertoRico in 1967 and first meeting in 1968), joined in the organization ofthe 1st International Congress of Nematology in Guelph, Canada in1984. The growing awareness of the adverse effects of nematicideswas reflected in a colloquium, at the Congress, on ‘Pesticides ingroundwater’. At the Congress dinner, one enthusiastic after-dinnerspeaker, whose native language was not English, caused someamusement when he exhorted the audience to publicise the scienceof nematology by telling them to “go out and expose yourselves”!During this Congress the demand for an integrated forum resultedin a proposal for an International Nematological Society.Subsequently, the second International Congress of Nematology, atVeldhoven, The Netherlands in 1990, established a pattern of hold-ing these meetings every six years.

The 21st ESN Symposium in Albufeira, Portugal in 1992, afterthe “Iron Curtain’ had been lifted, was notable for the plenaryaddress from Eino Krall on ‘A personal perspective on nematology in

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Eastern Europe”. The gradual incorporation of molecular and bio-chemical studies into mainstream nematology is evident from theprogramme and was further developed in the 22nd ESN Symposiumin Gent, Belgium, 1994. Belgium is justly famous for its extensiverange of excellent beers and the beer and cheese evening, with 11 different types of beer, certainly stimulated some animated dis-cussions, some of which were scientific. A similar effect wasachieved at the 24th Symposium, in Dundee in 1998, with whisky asthe essential catalyst. In between these two meetings, the thirdInternational Congress of Nematology was held in luxurious sur-roundings in Guadeloupe, French West Indies, in 1996. A hurricaneand an earthquake caused some problems during the first couple ofdays, with the welcome reception being cancelled and several delegates unable to travel. The hardy individuals who endured theinitial 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 someother nematology societies. In 1995, The Russian Society ofNematologists was able to hold its 1st English Language InternationalSymposium in St. Petersburg with 30 invited delegates from theWest. This was very successful and subsequent conferences havebeen held biennially with delegates also from outside Russia. Thesemeetings 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 Federationof Nematology Societies (IFNS) as a global communications forumwith the stated aim ‘to foster communication among nematologistsworld-wide’ (www.ifns.org). IFNS comprises 14 individual societieswith a total of about 2,500 members (Table 3). There was a long ges-tation period (the seed of the idea was sown at the 1st InternationalCongress of Nematology in Guelph, Canada in 1984) before the birthof the IFNS because of differences in the decision making processesof the various societies. The establishment, in 1996, and success ofthe Federation were due primarily to the hard work, tact and persist-ence of Kenneth Barker (Fig. 2), of North Carolina State University,who became the first President of the IFNS. An enjoyable and scien-tifically excellent 4th International Congress of Nematology was heldin Tenerife, Canary Islands in 2002 and the 5th is to be held in

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Brisbane, Australia in 2008. This will be the first opportunity for thenematology societies to meet in Australia, and it should provide anideal forum for enhancing links between nematologists fromAustralasia and other parts of the world.

Table 3. The 14 nematology societies which are affiliated to theInternational Federation of Nematology Societies.

Afro-Asian Society of NematologistsAustralasian Association of NematologistsBrazilian Nematological SocietyChinese Society of Plant NematologistsEgyptian Society of Agricultural NematologyEuropean Society of NematologistsItalian Society of NematologistsJapanese Nematological SocietyNematological Society of IndiaNematological Society of Southern AfricaOrganization of Nematologists of Tropical AmericaPakistan Society of NematologistsRussian Society of NematologistsSociety of Nematologists

The International Congresses of Nematology have been verysuccessful, despite some initial growing pains, caused mainly byindividual societies insisting on adhering to their own traditional formats. This was especially evident in the banquets of the first twocongresses where the after-dinner presentations were inordinatelylong and tedious. Sense has prevailed and the banquets are now anenjoyable part of the meetings. Undoubtedly, the InternationalCongresses will continue to be important adjuncts to the conferencesof individual societies.

Newsletters

Society newsletters have traditionally been used to convey to themembership minutes of executive meetings, announcements offuture conferences and general items of interest to members. TheESN newsletter (initially called Nematology News) was first sent to

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forty colleagues as a few typed A4 sheets stapled together.Subsequently, in the first issue of Nematologica, NematologicalNews was incorporated as an appendix of the journal. Later it wasseparated again, and in the 1990s it developed into a much moreextensive and professionally produced document, including sum-maries of Ph.D., theses, articles commenting on specific aspects ofnematology and news from various centres of nematology. The lat-ter items were often a catalogue of overseas trips made by travel-mad nematologists and degenerated, in some places, to one-upman-ship contests! The ESN newsletter is now much slimmer and morefocused than in previous years, with interesting contributions suchas laboratory profiles. The SON newsletter is somewhat similar incontent to the ESN newsletter but is used more frequently as aforum for discussion of specific topics; for example, correct termi-nology of races, pathotypes and strains etc., journal publication poli-cy and scientific ethics. Both newsletters are now available as elec-tronic versions and sent by e-mail.

Although, newsletters still have a role in alerting the member-ship to meetings of interest, their future existence as hard copyofferings is in doubt. The advent of the internet and the Societies’websites now fill much of the previous role of newsletters.

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Fig. 2. Kenneth Barker, first President ofthe International Federation ofNematology Societies.

Fig. 3. Michel Luc, founding editor ofRevue de Nématologie and a current mem-ber of the editorial board of Nematology.

Journals

The primary method of information exchange of any scientific disci-pline is through the printed word via scientific journals. This is stilltrue, although, as we discuss below, the use of electronic transfer ofinformation may supersede the traditional journal format. For plantnematology, the importance of information transfer with developingcountries still justifies the traditional methods.

It is always slightly frustrating that nematology as a discipline isdivided into different subdisciplines, such as plant nematology, animalnematology (usually as a component of parasitology), Caenorhabditis ele-gans groups, free-living nematology and, increasingly, entomopathogenicnematology. Each sub-discipline has its preferred conferences and thefragmentation is also reflected in the journals, as each group has its ownpreferred journals for publishing. In universities, nematology is rarelytaught as a separate discipline, but the sub-disciplines are often includedas components of other courses. In the USA, plant nematology is usuallytaught as part of a plant pathology degree course, whereas in the UK andsome other parts of Europe, plant nematology was included as a topic inzoology and biology degree courses, often as a “pure science” discipline.In the past, this was reflected in a more field-based approach to nemato-logy research in the USA, whereas the European nematologists had a farmore laboratory-based, pure science research approach.

In the past, the fragmentation of nematology had three majoreffects. First, research developments in one area, such as animal nema-tology, were often not utilised in others, such as plant nematology. Inthe past, a paper given by a plant nematologist at a parasitology meet-ing would result in the speaker being deafened by the noise of peopleexiting the lecture theatre. Now, the use of common molecular tech-niques to examine a wide range of host-parasite interactions, forexample, means that the research on plant parasitic nematodes hasmore 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 tokeep abreast of the literature. As the electronic era progressed, moreeffective access to a wide range of other journals was ensured. Now,although work on molecular biology of plant parasitic nematodes maybe published in molecular journals, it is a simple task to access theinformation quickly on-line. The third effect is that the journals pub-lishing most of the plant nematology papers have a low impact factor.

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In the present era, when impact factors are considered (by administra-tors and grant review panels!) to be of paramount importance as indi-cators of research output quality, such journals may not be able toattract the top quality papers, especially those with molecular biologycontent. However, they are still an important part of the nematologyinfrastructure and it is interesting to examine the evolution of thejournals and to consider their possible future.

The two main international journals for papers on plant nema-tology are currently Nematology and the Journal of Nematology, withNematropica, the Russian Journal of Nematology (first appeared in1993) and Nematologia Mediterranea (1973) providing a forum thatincludes papers on more regional aspects. Nematology is owned andpublished by Brill, The Netherlands, and there are no page chargesfor publication but a high subscription fee. By contrast, Journal ofNematology (JON) is owned by the Society of Nematologists andimposes page charges but is sent to all members of the society as apart of the modest membership fee. In the 1970s and 1980s, manyEuropean universities and research establishments were reluctant tofinance page charges if a paper could be published at no cost else-where. Consequently, there were few papers from European authorsin JON. The situation has changed slightly as research scientists haveto obtain grants to justify their continued existence and grantmoney can, in some cases, provide European nematologists theopportunity to fund publication costs. In the future, journals,whether wholly on-line or hard copy and on-line, may movetowards the JON format, with the authors bearing the publicationcosts and the journals provided free of charge.

JON first appeared in 1969 with Seymour D. Van Gundy as itsfirst editor. The first issue of Nematology was published in 1999, butthe history of this journal goes back to the 1950s when the journalNematologica (also published by Brill) first appeared, in 1956, withJ.H. Schuurmans Stekhoven as Editor-in-Chief. Nematologica wasestablished as a journal to cover the field of nematology in general,except for papers on medical and veterinary subjects. It attractedpapers on plant nematology that previously would have been submit-ted to the Journal of Helminthology, a journal that still exists butwhich is now more mainstream parasitology. In 1978, Michel Luc(Fig. 3) started a second journal, published in France by the Office dela Recherche Scientifique et Technique d’Outre-Mer (ORSTOM),called Revue de Nématologie, with an A4 format (compared to the

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smaller Nematologica). Revue de Nématologie quickly became a verysuccessful journal but its title counted against it. At one promotioninterview in the UK, the candidate was asked why he published in aFrench national journal; it took some persuasion to convince the ques-tioner that Revue de Nématologie was a well respected internationaltitle. Partly because of this type of perception and also to ensure thatthe journal was included in Current Contents, the title was changed, in1992, to Fundamental and Applied Nematology, published byGauthier-Villars – ORSTOM and, subsequently, Elsevier. When Brillacquired the title, Fundamental and Applied Nematology was amalga-mated with Nematologica to form the present journal, Nematology.

From 1987 until 2001, the SON published also the Annals ofApplied Nematology, which was a single issue per year produced as asupplement to the Journal of Nematology. Annals of AppliedNematology served as an outlet for strictly applied aspects of plantnematology. Publication ceased because of insufficient submissionsto support both SON publications. Just prior to this action, theJON changed to the larger A4 page format and discontinued its tra-ditional orange coloured cover. These changes caused great concernto some members of the society!

In terms of impact factors, neither Nematology nor JON ratesvery highly, rarely breaking the 1.0 barrier, but this is not a reflectionon the quality of the science. In part, it is due to the small number ofplant nematologists but it also is a consequence of the need for scien-tists to publish in high impact journals, and many of the papers onmolecular nematology, for example, are published elsewhere.However, on the plus side, the citation life of papers in the plantnematology journals is long. On-line publishing is likely to be thescenario of the future, although there will continue to be a demandfor hard copies of the journals for several more years. However, withlibraries increasingly cutting back on journal subscriptions and theescalating cost of publishing, production of hard copies of nematol-ogy journals may, in the long term, not be economically viable.

Websites

In the 1990s, both ESN (www.esn-online.org) and SON (www.nema-tologists.org) set up websites. These are gradually taking over the roleof newsletters and they also provide registration facilities for confer-

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ences and access to relevant sources of information. Manuscript sub-mission for JON is now through the SON website. Such sites willbecome more important in the future. With the demise of the Annalsof Applied Nematology and the concomitant need for a place to pub-lish large data sets, such as results of germplasm screening and nema-tode distribution, websites are likely to become the medium ofchoice. This mirrors the access to genetic information, such as DNAand protein sequence data, that is freely available on the web.

Electronic communications, whether to replace journals or foraccess to data sets, may take some years to realise fully their poten-tial. In part, this is because the Societies have to consider the needsand resources of all interested parties and, in part, because of theundoubted reluctance of some nematologists to embrace theadvances 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 inthe above sections, electronic communication will be central to thefuture infrastructure of the Societies but conferences will continueto be essential. One aspect we have not mentioned: the fact that allfacets of communication essential to science depend on the timeand effort of volunteers. Moving to a predominantly electronicforum will not ease this burden. The present generation of nematol-ogists should be grateful to all those who have ensured the healthydevelopment of the ESN and SON and the journals over the past 50years. The future will still depend on the goodwill and effort of thenext generation of nematologists to keep the Societies and theirassociated activities alive and active. At the end of the day, we allget fun out of nematology!

Acknowledgements

We are grateful to Maurice Moens, Merelbeke, Belgium for makingavailable the text of his address to the ESN meeting in Israel on thehistory of the ESN.

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18.(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 hope-lessly risky business, and is more likely to produce laughter at the2057 meetings of the nematology societies than acknowledgementof a (by then very old) nematologist’s gift of prophecy. I do havesome meager credentials, however, as I made a few predictions forthe following ten years at the 1993 APS/SON meeting in Nashville.Those predictions have come at least partially true, although Imust concede that they were not difficult to foresee. Ten yearsand fifty years are vastly different lengths of time. When I wasborn, the Korean War had just erupted. There was no interstatehighway system in the U.S. at that time, and Studebakers, DeSotos,and Packards were still being manufactured, B.G. Chitwood hadjust published a paper that placed root-knot nematode taxonomyonto a sound footing, soybean cyst nematode was not yet knownin the U.S., and trichodorids were not recognized as significantplant pathogens. Advances did not come at such a rapid rate asthey do now. The pace of all aspects of life was much slower andmore relaxed 50 years ago. Do we willingly speed up our pace aswe grow older or do we do it because we must? I tend to believethat our sense of life’s pace is set early in our lives, and so I sus-pect that the younger contributors to this compilation may do abetter job of hitting the technical high spots as they gaze into thecrystal ball. I could beat the original Mario Brothers, but I don’teven try the stuff my sons play. The advantage to nematologistswho span eras is that they may see the whole picture a little more

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NEMATOLOGY:DREAMS AND VISIONS OF THE FUTURE

clearly, and can engage in sweeping generalities without being heldto specifics! These generalities should provide lots of amusementfor mid-century colleagues.

The science of nematology is viable primarily because of theimportance of crop-parasitic nematodes in agriculture. Without thiseconomic 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 societiesthat place great value on the application of knowledge to crop pro-duction and protection. Therefore, the future of nematology is tiedclosely to the future of agriculture, and as we cannot do away withagriculture, 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 climatewith sufficient rainfall, but at no time since the medieval Little IceAge has humanity faced a challenge like the next 50 years willpose. If current scientific consensus is correct, runaway productionof carbon dioxide will profoundly alter world agriculture by makingsome areas hotter and drier, and others hotter but wetter.Agricultural lands just a little above sea level may be inundated asthe Antarctic and Greenland ice caps melt. I suspect the nematologylessons that are being learned now in dryland agriculture in Africa,Asia, and the American Southwest will be put to good use over amuch larger area of the world. On the other hand, some water-lovingcrops will shift northward. The wetter and warmer Canadian Plainswill become immensely important producers of soybeans that willstill be in Maturity Groups 0-II, but will be infested withHeterodera glycines. However, arable land and aquifer capacity arelikely to be diminished worldwide within 50 years, reducing foodsurpluses and requiring the utmost scientific effort and ingenuity toimprove and protect crops.

Nematode management in 50 years will be much more sophisti-cated and environmentally friendly than it is today, largely becausethe trend toward truly committed environmentalism is building indeveloped societies. My guess is that we will be decidedly “green” in50 years. Students will shudder when really old nematologists reflectwistfully on the days when they injected chlorinated and brominatedhydrocarbons into soil. Crop protection will center mostly on themanipulation of plant and pest genomes. However, if there is anythingwe should have learned already, it is that nematodes often can adapt

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and select themselves faster than we can change cultivars. Economicnematology will be tied even more closely to cultivar developmentand testing in 50 years than it is today. I suspect that the sources fornew germplasm and cultivars will have swung back to university andexperiment station laboratories. Growers will have become extreme-ly sophisticated in their approaches to growing plants, partly becausecrop prices will be higher due to the contraction of arable land.Producers will turn to the very best, most reliable, and least biasedscience they can obtain to optimize their yields, and this will beforthcoming from the universities. There will be minor though significant work still being done on the implementation of biologicalcontrol and the transfer of soil suppressiveness, but soil stasis willstill be a difficult nut to crack. However, the maturing interest in andlove of environmental harmony will lead to nematode synecologybeing studied to a far greater degree than it is today.

Fifty years from now, universities will remain the centers fortraining nematologists, but the distribution of strong programs willbe much different from what it is today. We can expect to seeworld-class institutions well-distributed around the world.Instruction in nematology will, of course, still be available, but in aform completely different from what we now have. Our ability tostore and process huge quantities of data will continue to growtogether with concomitant leaps in the sophistication of computergenerated graphics. This means that students in 2057 will learnalmost totally by immersion in virtual reality scenarios, where theycan be a nematode living its life – whether it be penetrating rootepidermis, sucking on a fungal hypha, scooping up bacteria or beingattacked and destroyed by another nematode. Students will be ableto try out different stylets for feeding or wiggle through soils of dif-ferent textures. For that matter, they will also be able to be a plant,insect or vertebrate reacting to invasion of a parasitic nematode. Ifthis vision of instruction by virtual reality proves to be correct itwill result in instruction and research being fused – acting out dif-ferent scenarios will result in the generation of hordes of testablehypotheses 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 theirinstruction on their home institutions; rather, there will be a worldcurriculum, taught primarily through simulation, to get the bestknowledge from every institution. For instance, taxonomy instruc-

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tion may come from Europe, organic amendment knowledge fromIndia, potato nematode instruction from Peru, cropping systemsfrom the U.S., ecology principles from Poland, dryland farming fromAfrica and New Mexico. Going a step further, virtual reality-basedinstruction will enable experts from all continents to cooperate inthe development of courses on an intimate scale not previouslyachieved. Today, we can link together for video conferencing, butthe technology is clumsy and prone to breakdowns – or at least willbe considered clumsy and fragile 50 years from now!

When I was a cute little newborn more than 50 years ago, myparents had just managed to obtain a private telephone line toreplace the party line that they had been sharing with three otherhouseholds. The most glorious event I can remember from my pre-teen years was the 1962 launch of the Telstar 1 satellite, riding amighty Delta-Thor rocket into space. It seems almost paltry now totalk of advances in communications. Rather, we are barrelling head-long (not just advancing) into a future where the pace of changewill be breathtaking; the changes wrought in that future will bemuch greater than what has occurred since 1950. I plan to be at the2057 SON meeting (maybe all meetings will be virtual by then!) toreview the predictions that my colleagues and I have made, and todrink a toast to the next 50 years after that.

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(ii) VISION OF NEMATOLOGY IN CANADAIN 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 forthe next 50 years? Well, as the weather man would say, it’s going tobe a mixture of sun and clouds with some possibility of rain orsnow. Which basically means that “Everything is possible.” It isalmost impossible to predict the political choices that will be madeat national and international levels. However, one can only hopethat a high priority is given to research and that significantly greaterfinancial resources will be injected into research by industrializedcountries (10% or more of GDP per country) to pursue both funda-mental and applied research in nematology.

Over thousands of years of human activity on our planetmankind has been able to adapt to numerous changes along his/herevolution. Now, what we are experiencing is something quiteunique; a phenomenon most probably occurring for the first time inour planet’s long history. The planet earth is trying to adapt to envi-ronmental changes caus ed by centuries of human activity. Fromwhat we have seen in the last decade, it’s having a hard time coping.Yes, global warming is a huge challenge, with enormous conse-quences across the planet. We can hear it almost every day on thenews report. Somewhere, someplace, the earth is shivering, sneezingand desperately trying to adapt to global warming. What does thisphenomenon have to do with nematology? Well, I believe that inthe next few decades, or even sooner, this global change will have atremendous impact on pests, and diseases in agriculture and forestry.

Temperate agricultural production areas will see their short listof pests and diseases expanding significantly due to milder tempera-

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tures during the winter and more heat during the summer. Forexample, in Canada, the soybean cyst nematode, Heteroderaglycines, and the root-knot nematode, Meloidogyne incognita, willmost probably become common residents of agricultural soilstogether with many new root and foliar fungal diseases. After a con-tinuous and steady decline over the next 10-20 years, the worldwidenumber of nematologists will expand again to reach new peaks asthe necessity for both basic and applied research in agriculture,forestry, and medicine will be identified as top priorities by univer-sities, industry and governmental institutions.

Field extension nematology will advance to meet the challengesby using more accurate tools for monitoring damaging nematodepopulations. From a soil sample, both scientists and extension work-ers will be able to make an assessment of the number of pathogenicnematodes using quantitative molecular kits. Traditional morpholog-ical identification will still be required to keep track of new speciesin a given territory. Using internet and interactive web sites, agrono-mists will have access to information on damage thresholds and alsopredictive models for managing major pathogenic species.

New regulations and rules between industrialized countries onglobal market issues will be implemented in order to maintain andassure the survival of agriculture in all producing countries. Thedemand for healthier, quality foods will be booming and chemicalpesticide applications reduced as Governments legislate restrictionsin their use. The outcome will require increased research on alterna-tive control methods, including biological controls, and this will gen-erate new tools. The list of entomopathogenic nematodes availablefor insect control will grow, and the in-vitro multiplication of severalspecies of insect parasitic mermithids will be achieved and madeavailable commercially. Steinernematid and heterorhabditid nema-todes, 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 activeingredients which will be applied as environmental friendly com-pounds with increased specificity against harmful pests. Other basicresearch on these bacteria will be pursued to successfully introducethe bacterial genes into plants to provide high resistance to bothfoliar and root insect feeders.

The availability of fresh water is already a major issue in numer-ous countries. Irrigation of large production areas will be reduced

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and in some areas prohibited in order to maintain sufficientamounts of water for human consumption. Research on droughtresistant crops will be included in breeding programs, together withmultiple resistance to plant parasitic nematodes and diseases, andthis will be the major tool for maintaining high yield production inindustrial and, more importantly, in third-world and developingcountries. I believe GMO’s will be extensively used, providing allmajor crops such as wheat, rice, sorghum, millet, etc., with multipleresistance to plant pathogens. Basic research on model organisms,such as Caenorhabditis elegans, will be expanded as numerous reve-lations in intra- and extra-cellular communications and gene expres-sion will have contributed to the development of new ways ofdetecting 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 researchteams. This ametabolic state of life could essentially permit life topersist indefinitely until environmental conditions are hospitable.The increasing energy costs for producing food for humanity willsignificantly reduce the livestock industry and stimulate the creationof alternative protein sources. New sources, such as nematode-basedtofu, will be investigated by the food industry. Basic new mathemat-ic models based on the study of the development and growth of C. elegans will explain the secret to the formation of life on ourplanet and, ultimately, will provide all the information needed torecreate life under laboratory conditions from basic molecules.

Many of my colleagues may consider this wish list to be some-what out of proportion … and they are probably right. But whenyou think about it, life is actually all a matter of choices. One of ourpriorities as human beings is to take full responsibility for our actionsas creators of change. Every day, we create our own lives.Imagination and dreaming are also part of our divine capabilities forcreating a “today”, a “tomorrow” and a “future”. On this same level ofthinking, we must imagine (John Lennon, 1971) and integrate intoour own lives, now more than ever, unconditional love, tolerance andhappiness for ourselves and for others in order to help make it hap-pen 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 StreetWest Lafayette, Indiana, USA

As one who began her nematology career prior to academic use ofcomputers or the discovery of the double helix, I assume thattoday’s young nematologists will experience comparable paradigmchanging discoveries, inventions and ways of doing research duringtheir careers. Rather than speculate about general changes in nema-tological research over the next 50 years, I prefer to reflect on thefuture impact on nematode systematics research of changes inthinking and technology that are already underway.

Probably the most crucial of the coming innovations that willaffect nematode systematics 50 years out cannot even be envisagedat the present time. For example, although wide-spread use of com-puters was probably predictable, as well as the unravelling of thenature of DNA, who could have predicted the discoveries of restric-tion endonucleases and DNA cloning, automated DNA sequencing,large array and high throughput molecular analysis, or genetic trans-formation for pest resistance in plants. We did not, perhaps couldnot, envision the extent and rapidity of technical improvements tocomputer hardware and the vast array of software developed to uti-lize such enormous quantities of new molecular data. Polymerasechain reaction (PCR) and the companion invention of the thermo-cycler alone were career-changing for me because these toolsenabled me to obtain DNA from a single nematode specimen foruse in molecular systematics and phylogeny.

Molecular sequencing technologies will continue to improve.Just as we evolved from the period of large sequencing gels, labori-

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ously poured, loaded and analyzed with light boxes in our laborato-ries to automated sequencing and computer generated output, nano-sequencing is coming at breakneck speed. Because they can be pro-duced so rapidly the generation of nano-sequences of increasinglength will change the face of systematics and phylogenetic analysis.Large array molecular analyses of all kinds, generated to explain howorganisms behave and function, are proliferating; and even large com-panies find crucial needs for people skilled in in silico research.Increasingly, graduate students are generating over a few weeks moredata than can be analyzed in years. This will lead to increasinglymore complex software to make sense of polybites of new data andwhole new fields of research in bioinformatics. Many nematologygraduate students will find that their entire research project revolvesaround data mining, not in libraries of books, but of computer datastored in electronic data repositories. They may never enter a lab orsee a whole nematode under a microscope even though they are try-ing to understand living organisms.

I can foresee continued heated discussion for the next decade ormore about DNA barcoding and its place in systematics. The cur-rent discussions about DNA barcoding are an opening salvo in whatI believe will be an ongoing and prolonged discussion about thekinds of data that will be utilized in future taxonomic and systemat-ic research, i.e., the outcome of these important discussions willdetermine what nematode systematists of the future will be doing.

A DNA barcode at the present time is defined as a specific sub-set of the DNA of an organism believed by barcode proponents todelimit species boundaries, facilitate rapid identification (by com-parison of barcode data among all known species of interest), andprovide sufficient data for phylogenetic analysis. A principal andearly proponent of DNA barcodes has been the entomologist PaulHebert, University of Guelph, Guelph, Canada. He is joined bymany colleagues, especially people who have major interests in bio-diversity, ecosystem inventories and conservation, e.g., DanielJanzen, University of Pennsylvania, Philadelphia, USA, and AllenHerre, Smithsonian Institution, Panama City, Panama (both evolu-tionary biologists). Dr. Hebert has designated about 600 base pairs(bp) of the DNA of mitochondrial cytochrome oxidase I (COI) asthe barcode of choice, and many concur. Often researchers predictthat pocket gadgets will be available that will enable any researcherto input a tiny sample of the specimen into the gadget and, based

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on barcode data, quickly learn the identity of the specimen and itsrelationship to other taxa. Although I laughed with a recent class ofmine at this notion, a few weeks later a student brought me a paperthat suggested ongoing development of such a gadget by an entre-preneurial company. An important nematology spokesman for DNAbarcodes has been Mark Blaxter, although his examples haveemphasized barcode sequences of ribosomal DNA (rDNA) forspecies previously described. Others have suggested that barcodesfrom several genes will be needed for identification and analysis.Although barcode proponents often mention the value of “tradition-al taxonomy”, they likewise point out that experts in taxonomy aredisappearing from university and other laboratories, and that rapidadvances in cost-effective technology will likely force society toabandon traditional taxonomy for more economic approaches.

Opponents of DNA barcoding provide vigorous argumentsabout the perils of an emphasis on DNA barcoding at the cost of atruly integrative taxonomy that includes traditional elements as wellas new molecular sequence data. Among these spokesmen are ento-mologists Quentin Wheeler, Natural History Museum, London, UK,and Kipling Will, University of California, Berkeley. They argueforcefully that the notion that DNA barcodes can replace traditionaltaxonomy and systematics is a very bad idea. They are skeptical thata 600 bp piece of the mitochondrial COI gene can suffice to sortout all of the life forms that may be encountered (and this skepti-cism is shared even by some writers who basically like the idea ofDNA barcodes to circumvent traditional systematic procedures). Anumber of writers express concerns that the “hype” over the DNAbarcode approach will appeal to research grant panels and boardswho distribute funds but who have little knowledge of systematics;and that such shifts in funding will result in even fewer practitionersof integrative taxonomy who use many different kinds of characters.

This debate will continue and I am unable to predict the out-come. Currently, we are not able to sequence entire organismsquickly and efficiently in our own laboratories, and therefore sys-tematists must use morphological, behavioral and other data, inaddition to whatever molecular data we are able to acquire. Whennano-sequencing techniques make it possible to easily and economi-cally obtain the entire sequence for every specimen, and new hard-ware and software enable appropriate comparisons and phylogeneticanalyses of these sequence data, the barcode controversies about

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which piece of DNA to use may abate. However, the controversiesregarding the need for an “integrative taxonomy” will likely persist.Will nematologists find a need to collect other kinds of data abouttheir specimens and have the funds to do so? Will DNA sequencestell the whole story regarding species similarities, differences andphylogenetic relationships? Will a remnant of nematologists remainwho take their students on collecting trips and marvel over thestructural beauty of specimens they find in soil or stream? Willnematologists still have microscopes in their laboratories? Today’syoungest 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 Resourcesand Applied Life Sciences, A-1190, Wien, Austria

I was scared and disturbed by a penetrating knocking on the door ofmy office. “Mr Grundler, are you ready? Your lecture will start soonand as it is for your farewell ceremony you should be on time!” Mysecretary – what good luck to have her around! I wonder whatwould have happened to me without her during the past 20 years. Iglanced at my watch – two o´clock, 4th of October 2025 – and con-tinued to go through my manuscript. It comprised an overview ofthe last 40 years of Plant Nematology and an outlook on the future.

Since the late 1980s, plant nematology has received a strongboost triggered by the rapid developments in ecology, molecularbiology and computer sciences. Nowadays, complete maps andsequenced genomes are available for many nematodes and for allimportant crop hosts, and routine nematode identification is almostcompletely based on sequence analysis. We have just established ourmost recent purchase in the lab: a fully automated “life analyzer”that identifies organisms and provides information on the mostimportant biological parameters through the integrative analysis ofnucleic acid sequences, transcription activities and metabolic prod-ucts. A single nematode, a small sample of microbes or a piece ofplant, and the name of the organism or organisms are presentedtogether with all available information.

Many problems in plant nematology that have kept generationsof scientists busy, seem to be solved today: we know a lot about theinteractions between plant and nematodes. For example the func-tion of plant resistance genes are now well understood. It was veryexciting to learn about the function of nematode compounds trig-gering feeding cell development in root-knot and cyst nematodes.Now they are identified and the cascades of events in plants leadingto the development of feeding cells are well explained.

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We also know a lot about the nematodes. For most of thedescribed nematode species their relation with their host plants canbe clarified and their economic and ecological impact identified.

The knowledge gained also has been transferred into controlmeasures. Transgenic approaches have been developed to engineerresistance in crop plants by the transfer of resistance gene compo-nents in a wide range of host plants. In other cases the signal cascadeleading to the formation of feeding cells can be locally interruptedthus preventing the development of nematodes. In other cases,defence pathways are triggered whenever nematodes start to invadeor feed on the particular plant.

Nematicides are still available but their development has headedin a completely new direction. Some of them are harmless syntheticcompounds that can be sprayed on plants. At sites of nematodeinfection, however, they are processed to effective nematicides bythe action of plant or nematode compounds. An important role isplayed by microbial nematicides. After decades of research a numberof highly active microbial nematicides are available on the market.

But to be honest, there are still a lot of problems caused bynematodes and other pathogens. Although the ideological altercationbetween conventional and organic farming is now history and thereal integration of culture methods and control methods has broughtconsiderable improvement to the situation, our little worms are stillalmost omnipresent. The fact is that in a number of cases artificialor natural resistance has been overcome by new pathotypes, whileon the other hand the establishment of new plant species as cropsfor food and industrial uses has put nematode species on the mapthat were “no names” previously.

A problem that has become more and more important is thealmost unpredictable occurrence of extreme weather events espe-cially in moderate climates. While heat and drought has broughtabout a continuous and significant expansion of deserts and theSahel, in our former moderate climatic zones the weather nowadaysoscillates between extreme frosts or warmth in the winter and rapidchanges from heavy rainfall to long drought periods in the summer.Unfortunately, our crops seem to be much less well adapted tothese challenges than nematodes or pathogens which often findgood conditions in the weakened plants and thus aggravate thealready serious damage. It will be a challenge for all disciplines toimprove plant production under these conditions.

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I settled back in my chair and deliberated again on the future asit would pass the half century. The number of described nematodespecies has increased especially over recent years when taxonomicprocedures have become more and more automated. Nematodeswere confirmed to live in nearly all environments and to be the“absolute exploration experts”. Some of them are excellent biologi-cal models in genetics and cell biology and these have been studiedin great depth. However, the great wealth of variation in physiologi-cal and behavioural adaptations is still not fully realized and by farnot fully used. I suggest that nematodes will be more and more usedas a source of knowledge and a resource for industrial purposes. Forexample, high value proteins and other biologically active com-pounds will be produced by fermentation of specific nematodes fornutritional, pharmaceutical or technical application. On the otherhand they will be employed e.g., to improve degraded soils or byreleasing specific species combined with microbes. Many moreapplications will be found. Just recently…

The telephone rings. Tapping around in the dark I try to findthe receiver. “Mr Grundler, this is your wake up call, it is seveno´clock!” A look at the illuminated alarm clock confirms what thefriendly voice said. I hesitate when reading the date on the display:12th of October 2006. I try to remember. I have been somewhereelse. What happened? Maybe a dream, but, unfortunately, I cannotremember 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 futureof 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 wasunable to accurately portray the basic changes through which thetwentieth century would go. It is also unclear to me what substan-tial advances will take place in nematology during the next fiftyyears. Nevertheless, in view of the knowledge that this new disci-pline has already reached we can expect a promising future.

As a discipline and a society we should remember what hasalready been accomplished in the field of nematology in a relativelyshort time. Important resources are already available from the internetand from genome-based technologies, and are likely to increase.With globalization, nematology will face many challenges whileminimizing the introduction of new nematode pests (“non-beneficialnematodes”). At the present time there are several diseases causedby nematodes that, although not completely under control, havebeen successfully managed. For some diseases, such as pine wilt disease, caused by Bursaphelenchus xylophilus, the best disease management is prevention such as minimizing introductions, forestmanagement, tree species selection, etc. Nematologists will continueto participate in inter-disciplinary approaches to overcome the complexity of such diseases.

Very recently, the secretary of FAO called for a new “green rev-olution” in the face of the growing human population and the needto obtain more food and fiber. It has been calculated that an addi-tional 1 billion tons of cereals will be needed to feed the world. Thisincrease in crop production carries a concomitant need to protect

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plants, be they field crops, horticultural crops, ornamentals or forestspecies. Nematodes, as major pests and pathogens, will certainlycontinue to play an important role in limiting the production ofthese 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 certaingroups of nematodes, such as entomopathogenic nematodes (EPNs),more relevant. However, the public, as a whole, still needs to learnthe importance and potential of nematodes as beneficial organisms(e.g., Caenorhabditis elegans and EPNs).

The major developments in science and technology often drawfrom curiosity-driven research, and over time these developmentshave had a great impact on national interests. As a consequence ofrecognizing economic benefits, nematologists will face new paradigms,such as to perform research mainly for the economic interest and ben-efit of a region or country. I wonder if this will lead to inequalitybetween those who have access to research and those who do not.

Nematology could split into several sub-disciplines and thuslead to tough competition between them, which could ultimatelylead to the disregard of some areas (e.g., classical taxonomy). In sucha scenario, the future of this discipline should stimulate a better andmore determined discussion among nematologists, and perhaps abetter definition of itself. From the beginning of my career in sci-ence I have always had the following quote in mind – Science cameinto its own when it managed to refuse the subjective and embrace theobjective. Looking ahead into the next 50 years my dream is thatnematology will always keep this in mind, and embrace the objec-tive (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 com-munity. Since the inception of the European Society ofNematologists in 1956, and up to the 1980s, plant pathologists,nematologists included, have largely achieved their purpose ofincreasing and protecting strategic world food supplies and commer-cial crops, including forestry. Much of the achievement was drivenby staff from Europe and North America, where governments hadinvested heavily to i) increase strategic, national production and com-mercial returns from agriculture and forestry, and ii) circumvent aperceived world food shortage as a result of global populationincrease. In these two continents in more recent times there has beena progressive and increasingly rapid decline in numbers of these staff.

To speculate on the future of nematology over the next 50 yearsthe 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 emergenceof international supermarket chains have each contributed to majorchanges in agricultural and forestry production to service popula-tions in these regions. With high disposable incomes, basic food costsare increasingly becoming a smaller proportion of the individual’sbudget. In these regions an increasing quantity of basic food productsare imported due to lower production costs available in otherregions and accompanying low international transport costs, whilstdemand for alternative foods also increases. This results from higher

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production costs for home grown foods, and inexpensive interna-tional travel that exposes individuals to alternative foods.

Substantial current emphasis in nematology in these regions isdirected towards technology transfer and education, ecology andmolecular 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 ofimportance except in plant quarantine and to provide an opportunityfor technology transfer and education, i.e., training of overseas post-graduate students that financially contribute to the national income.

Technology transfer and education is short term, as trained for-eign students returning to their home regions will supply this func-tion locally. As a result of the non-recognition of nematology, fund-ing for nematode ecology will rapidly decline as will granting formuch molecular biology that is already considered “taxonomic tin-kering”. Genetically manipulated crops have spectacularly failed togain public acceptance, and this also has contributed to fundingdecline. International supermarket conglomerates that control globalfood production can, almost overnight, shift crop production fromone country to another, and frequently between continents. In thefuture, global supermarkets will be the source of plantpathology/nematology funding, but such funding will be small,short term, highly focused on an end product result, and much of itprovided to “local” staff. Plant quarantine services provide govern-ments with a rationale to control global trade through prevention ofthe introduction of non-indigenous harmful pests and pathogens.Thus, plant pathologists with some nematology training, will beemployed as nematode taxonomists in plant quarantine services.Consequently, there will be many fewer nematologists than current-ly 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 toserve the populations in these regions. Also, there is an increasingdemand by populations in Regions “A” for food and forestry productsproduced in Regions “B”. Nematologists are already working in theseregions and will continue to be required to provide strategic input

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to increase agricultural and forestry production. As well, globalsupermarkets will employ local nematologists for specific researchwhen required. Plant quarantine personnel, including nematologists,are already working in these regions as part of the global endeavourto prevent the introduction of non-indigenous pests and pathogens.It appears unlikely that there will be any significant change in thenumber of nematologists employed in these regions during the next50 years. In some countries, e.g., Brazil, there is likely to be a reduc-tion in numbers whereas in others, such as China, there may be amodest increase.

Regions “C” – Others

Few nematologists are employed in these regions, mainly as a resultof local national economic situations. This situation may change ifglobal supermarkets shift food production to these regions. Anyincrease in the number of nematologists that might be employed inthese regions during the next 50 years will be relatively small.

The alternative scenarios

Globalization, particularly as it affects agricultural and forestry pro-duction, is dependant on low income nations being used to providefood/services to high income nations and inexpensive transport is akey factor. With existing oil supplies steadily declining, combinedwith increasing demand from developing nations such as China andIndia, it can be expected that international transport costs will sub-stantially increase during the next 50 years. When the importationcosts become equal to, or greater, than local production costs it canbe anticipated that home grown production will replace imports. Insuch a scenario there will be renewed interest in “regenerating”national plant pathology, including nematology, to help in increasingstrategic national production and commercial returns from agricul-ture and forestry in Regions “A”

Global climate change may provide incentives and opportunitiesfor “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

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Regions “B” and “C” This change of production centers wouldrequire the assistance from plant pathologists, including nematolo-gists, who would be employed in Regions “A”.

With one, or other, or both of these scenarios the number of ne-matologists that might be employed in Regions “A” will be relativelysmall, and quite unlike the number of nematologists employed previously.

Conclusion

It would be gratifying, having spent 32 years as a nematologist, to bepositive about the future prospects for nematology. Sadly, withextensive global travel I have reached the painful conclusion thatglobally the science of nematology, as with plant pathology in gener-al, is in increasingly rapid decline. There appears to be little chanceof recovery, and by 2056, the 100th anniversary of the EuropeanSociety of Nematologists, there will be many fewer nematologiststhan at present. It is not inconceivable that even within the next 25years rather than waiting for 50 years, the European Society ofNematologists 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 yearsfrom now. I hope my vision is not unduly skewed by my ownresearch career having been focused on a single crop (cotton) andonly two species of nematode. Fortunately, I’ll not be around toaccept the blame for factual errors. Please ascribe occasional irrever-ence 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 innematode systematics, and functional genomics had provided a goodframework for investigating and understanding at least in part, thebasis of plant parasitism by nematodes, host plant resistance, andhost specificity. Molecular methods had long ago identified numer-ous candidate suppressive agents within soil and provided regulatoryagencies with powerful tools for identifying taxa of regulatoryimportance. The world (finally) had embraced transgenes in cropplants. Nematode resistance transgenes from fungi, not plants, hadbeen successfully inserted into plant genomes with promoters fromviruses, and these constructs (like their insect resistance transgenepredecessors) made some plants but not others resistant to somenematodes, but not to others.

Consequently, transgenes had found their place, the novelty wasover, and nematologists were again intensely exploring fundamental(in some cases new and in some cases long-neglected) componentsof nematode biology – pheromones, hormones, the species concept,plant host finding (!), and the underlying biochemistry, morphology,toxicology, and physiology of parasitism of nematodes by antago-nists, and ditto for parasitism of plants and insects by nematodes.

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Intensive studies had extended nematode ecology beyond com-parative nematode community structure, to the exploration andmodeling 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 bydiurnal thermal waves through soil. A burgeoning new field expand-ed the Bernardian rule to predict trophic and species diversity in dis-turbed and undisturbed soil-plant systems. (Apologies to ErnieBernard, as it must have been his intriguing symposium at the Societyof Nematologists meeting in Kauai that triggered this reflection)

In applied nematology, a major breakthrough had created manynew jobs, increasing teaching and research positions in nematologyby several-fold (reminiscent of the demonstration of the huge yieldresponses to soil fumigation more than a century earlier), but whenI started to read in detail about the breakthrough in a recent journalarticle, I awoke.

In a (relatively) crazy dream, I saw new waves of nematologistssequentially clearing the way for new dogma by freeing themselvesof the previous wave’s, starting with our own. One such waveviewed the plant damage models of today as antiquated oversimpli-fications suitable for management predictions only in Mediterraneanenvironments, but provided no immediate substitute. Another wavediscarded the concept of host race, returning it to plant pathology,placing plant host preference and virulence within a large set ofgenetic plasticity dimensions.

I dreamed of a Journal of Nematology with expanded freedomof expression and no page charges (now, that was REALLY crazy,but probably inevitable, following the conversion to 100% on-linepublication). As a new requirement, all field plot manuscripts hadto include a small diagram illustrating the experimental design (Thisseems like an excellent idea to me!).

In dreams of research funding (dreams about the long-termfuture, that is, not the short term!), I saw partial globalization ofresources for agricultural research, and the consequent refocusing ofcostly, technologically elite methodology at centers of nematologicalexcellence in tropical, subtropical, and Mediterranean latitudes(where nematological problems historically have been arguably themost important and certainly the most complex, and yet the leastwell funded). I saw stationery letterhead (in three colors of ink,which suggests to me, lots of money) for the European Research

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Institute for Mediterranean Nematology (at Bari), and similarly col-orful letterhead for the Global Center for Fundamental and AppliedNematological Research (at Delhi), and the International TropicalNematology Research Center (at Turrialba). I also saw evidence ofrecognition, by research granting institutions, that plant nematodesare 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 majorresearch grants were being (without precedent) routinely awardedfor the study of non-“entomopathogenic” insect parasites, and thebiology of marine, freshwater, and free-living soil nematodes.

In a dream about the worldwide nematological community, Isaw nematologists continuing to focus on specific groups of nema-todes, for example, on entomopathogenic nematodes, on plantnematodes important to a specific agricultural commodity, on para-sites of domestic animals, on nematodes as bio-indicators, etc.,because nematologists continued to be funded for the most part tostudy specific groups. However, as might be projected from today’ssituation, I saw geographically isolated nematologists outpacingother scientists by utilizing the internet as part of a global scientificcommunity. This was characterized by refreshingly vigorous interac-tion among scientists studying the same group in widely separatedlocalities. I also saw frequent collaborations among geographicallyseparated scientists for the purpose of comparing ecologicallydiverse nematode taxa. These developments and universal conver-sion to on-line journals had led, essentially, to the establishment of aWorld Society of Nematology with its own on-line journals, replac-ing the International Federation of Nematology Societies, which hadoutlived its usefulness. Was this really all a dream? A nightmare? Orperhaps, just happy ruminations!

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(viii) MY DREAM OF THE FUTURE OF NEMATOLOGY AND CHEMICALCOMMUNICATION RESEARCH – 50 YEARS FROM NOW

EKATERINA RIGA

Washington State University, Prosser, Washington, USA

For several years nematologists have been working on nematodebehavior, orientation and the mechanism of nematode orientation toa range of chemical and non-chemical cues. Cues that evoke nema-tode responses are potential candidates for integration into nema-tode control strategies for commercial use in crop protection.Disruption of nematode orientation in response to species-specificpheromones, sex specific pheromones or host chemical cues couldhave environmental, economic and biological importance because itcould lead to the discovery of new tools to manage plant parasiticnematodes. Although behavioral assays to study pheromones andhost chemical cues for several plant parasitic nematode species havebeen devised, the specific chemical structure of nematodepheromones and host chemical cues have not yet been identified. Inthe future, semiochemical cues will be identified that will disrupt orsaturate nematode reception especially when the nematode issearching for a host plant or for a mate.

Sex and species-specific pheromones will be used in the field asnematode control agents. For amphimictic nematodes, sexpheromones will be applied in the field to saturate the soil environ-ment, thus the male nematode sensory organs will become saturatedand unable to respond to the signals from their females. Therefore,mating will either be inhibited or delayed to the extent that maleswill die due to starvation. Since hermaphroditic nematodes do notuse sex pheromones to communicate with each other, we could saturate their environment with compounds that are antagonistic to

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their species-specific pheromone, thus preventing their membersfrom communicating with each other.

Plant host cues will be identified for both amphimictic and her-maphroditic nematodes and used to design antagonistic compoundsthat mask the host cues and lead to nematode disorientation. Forexample, plant seeds or seedlings could be coated with nematodeantagonistic compounds for protection against plant parasitic nema-tode invasion during the plant’s establishing period. In addition,plant host antagonistic compounds will be applied in the field, thesame way as sex pheromones, to saturate the soil environment andto disorientate the nematodes. Compounds mimicking plant hostroot exudates will be used to trigger egg hatching prior to planting,thus the juveniles would hatch and in the absence of plant hosts diefrom starvation. Alternatively, antagonistic compounds to plant hostroot exudates will be designed to block egg hatching during plant-ing, thus protecting the young roots from juvenile invasion andfeeding. We will be able to genetically modify non-host plants torelease host exudates in yet another nematode control strategy.

Host plants will be modified to release compounds that attractpredators of plant parasitic nematodes to the host root zone. Thus,plant parasitic nematodes will be eliminated due to the presence ofpredatory nematodes (e.g., mononchids), collembolans, predatorymites and other soil invertebrates.

Trap crops will be genetically modified to release exudates with nematode pheromone properties, thus attracting nematodes tonon-host roots. For example, genetically modified non-host plants ortrap plants could be planted prior to host plants or as intercrops tomislead nematodes.

I predict that 50 years from now we will be using these semio-chemicals e.g., sex pheromones, species-specific pheromones, andhost root exudates, to control plant parasitic nematodes by alteringtheir behavior. The semiochemicals will have a narrow target rangeas they will be species-specific and nematode developmental stage-specific. As well, in the future, I believe that effective nema-tode control based on semiochemical isolation and identificationwill 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 redplanet. We don’t even know how much space radiation they will beexposed to or what its effects may be.

The Brookhaven National Laboratory used its particle accelera-tor to attempt to simulate the radiation exposure of a round trip toMars. The results showed an expected exposure of approximately130,000 millirem – more than 370 times the annual average dose forAmericans (350 millirem). It gets worse. According to theUniversity of California Davis’ environmental health and safety web-site, the human LD50 (dose lethal enough to kill 50 per cent of thepeople exposed) is about 500,000 millirem. So a round trip to Marscould expose the voyagers to more than one-quarter LD50, whichmeans that one person in eight could die from radiation poisoningwhile the rest would probably be very sick. This does not even takeinto account unpredictable solar flares which could increase thedose many times.

The Brookhaven results may or may not be true. Little is knownabout the effects of long exposure to space radiation either aboardthe International Space Station (ISS) – where there has been contin-ual habitation for a few years – or for long-duration spaceflightssuch as the planned Mars mission. While manned spacecraft havebeen equipped with excellent radiation detection devices, they don’tmeasure the biological damage caused by space radiation.

Caenorhabditis elegans is a good model system for collecting

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data on the biological effects of radiation in space. These data couldlead to the development of radiation countermeasures enablinglong-duration manned spaceflight and continual habitation aboardthe ISS. C. elegans has many advantages. A prime one is its smallsize (adults about 1mm long) and this enables C. elegans experi-ments to be very compact. This is important because it is expensiveto launch weight into orbit, and spacecraft and the ISS, are verycramped. In addition, C. elegans has a short generation time. Itstwo-week lifespan enables a single hermaphrodite to produce about300 offspring, each with less than 1,000 somatic cells, which areeasy to maintain. These advantages allow short, flexible and costeffective experimental procedures. The nematodes can be culturedin small bags so that all an astronaut has to do is inject liquid foodinto the bags every couple of weeks. Another advantage is that C.elegans is the simplest multi-cellular organism with a completelyknown genomic DNA sequence. Like humans, C. elegans has about20,000 genes, about 4,500 of which are orthologous to human genes.These orthologs include a large set of DNA damage repair genes andthus C. elegans is an excellent model for predicting potential biolog-ical damage to humans in the space environment. Our laboratoryhas Canadian Space Agency funding to evaluate the use of C. elegansDNA-array chips, containing bits of every gene, for their efficacy inthe rapid analysis of space radiation induced deficiencies.

Caenorhabditis elegans has already travelled in space on severalmissions. 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), whichcrashed in Texas; and the 2004 First International C. elegans experi-ment (ICE-first) (Zhao et al. 2005. Gravitation Space Biology Bulletin18: 11–16) on the Delta mission. Amazingly, living nematodes wererecovered from the Columbia disaster. They survived an impact2,295 times the force of Earth’s gravity. “This is a very excitingresult,” said Catharine Conley of NASA, “It’s the first demonstrationthat animals can survive a re-entry event similar to what would beexperienced inside a meteorite. It shows directly that even complexsmall creatures originating on one planet could survive landing onanother without the protection of a spacecraft.”

The two JPL experiments and ICE-first used the eT1 mutagentesting system (Rosenbluth et al., 1983. Mutation Research 110:

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39–48; Johnsen & Baillie. 1991. Genetics 129: 735–752). eT1 is a balancerthat can capture mutations in the genome of C. elegans for laboratoryanalysis after a completed spaceflight.

While the first nematodes-in-space experiments were for short-time (about one generation) exposure to space radiation, ICE-first lasted a little longer – 11 days in space. These short experimentsyielded only a few mutations, not much above the number of spon-taneous mutations we would expect on Earth. This is probablybecause satellites in low Earth orbit (LEO), including the ISS, areprotected by Earth’s magnetosphere. Trips to the Moon or Mars willlast a lot longer and will not be protected by the magnetosphere.

So far, all the C. elegans space flights have been to LEO. In thefuture we would like to send nematodes on a very long unmannedmission outside the Earth’s protective magnetosphere. We would usean automated feeding system and take advantage of fluorescent proteins such as green fluorescent protein (GFP) and a fluorescencedetector. The system would be designed so that new mutationswould alter the nematode’s fluorescence in a detectable manner. Thiswould give much better data then the LEO missions on the biologicaleffects of long term exposure to fluctuating dosages of different typesof radiation in deep space. The data could lead to spacecraft designsthat will protect humans as we move out through space exploringand colonizing our solar system. Nematodes led us in the humangenome sequence project now they may lead us into space.

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