Practical Plant Virology: Protocols and Exercises

450 Pages·1998·8.199 MB·English

by  Dr. Jeanne Dijkstra, Dr. Cees P. de Jager (auth.)

SPRINGER
LAB MANUAL
Springer
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Jeanne Dijkstra Cees P. de Jager
Practical Plant Virology
Protocols and Exercises
With 77 Figures, Including Two Color Plates
Springer
DR. JEANNE DIJKSTRA
DR. eEES P. DE JAGER
Department of Virology
Wageningen Agricultural University
Binnenhaven 11
Wageningen
The Netherlands
ISBN 978-3-642-48981-5 ISBN 978-3-642-72030-7 (eBook)
DOI 10.1007/978-3-642-72030-7
Die Deutsche Bibliothek - CIP-Einheitsaufnahme
Practical plant virology: protocols and exercises I Jeanne Dijkstra ; Cees P. de Jager. - Berlin; Heidelberg;
New York: Springer, 1998 (Springer lab manual)
ISBN 978-3-642-48981-5
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Preface
The idea for this book arose from what we perceived as the need for an
up-to-date guide to class exercises in plant virology. We were encouraged
to proceed after receiving 29 positive responses (out of 30 replies to our
enquiries) from colleagues worldwide.
To the best of our knowledge, no such publications have appeared since
D. Noordam's book containing practical exercises (Noordam 1973) and
the latest (1988) edition of the American Phytopathological Society's
Laboratory Exercises in Plant Pathology, in which 4 out of its 31 chapters
discuss plant viruses.
Our original plan was to aim this publication at students and teachers of
plant virology, plant pathology, plant breeding and microbiology. How
ever, both colleagues and our publisher suggested widening the scope of
the book by making it useful also for research workers and laboratory
technicians. Therefore, we decided to prepare a laboratory manual of
interest to all groups.
We have tried to cover all relevant branches of plant virology, including
the molecular aspects, in as far as they pertain to the detection and basic
characterisation of plant viruses. We have not included protocols for the
molecular biology of plant viruses (sequencing, construction of recombi
nants, transgenic plants, etc.), as they are presented adequately in many
other recent publications.
The protocols in this book are described in a manner which should be
understandable to those with a basic knowledge of biology and chemistry.
Although most of the methods and techniques used in plant virology have
already been published in scientific papers, the latter seldom give suf
ficient practical details to enable an inexperienced student, or other new
comers, in the field of plant virology to carry them out successfully.
We are aware that new methods are continuously being developed and
existing techniques modified. However, we considered it inappropriate to
include recently developed protocols with which sufficient experience has
not yet been obtained. Therefore, we have restricted ourselves to incor-
VI Preface
porating only those methods and techniques that have been adequately
tested and found to be successful by us, and/or colleagues in our depart
ment and other centres of plant virological research. We realise that not all
techniques will be universally applicable and that modifications may be
required to suit the specific experimental conditions or a particular strain
of virus. However, we hope that sufficient background information has
been given to make such alterations as may be necessary.
The 89 protocols and subprotocols are arranged in 14 different parts.
Each of the protocols begins with a general introduction to the topic
and a list of required materials (other than those found as standard
laboratory equipment). In the latter instance, wherever a manufacturer
is mentioned, it does not imply endorsement and any comparable product
can be used. Thereafter, a step-by-step procedure is given, in some
instances preceded by preparations, and/or followed by interpretation
of data, if applicable. Also included are tips and troubleshooting sugges
tions for facilitating the experiments and overcoming likely problems. At
the end of a protocol or an introduction, a reference list is given that con
tains references mentioned in the text and other relevant publications.
Whenever appropriate, detailed recipes and formulations of ingredients
are appended at the end of the book.
Parts I-VII are concerned with biological characteristics of viruses,
whereas Parts VIII-XIII deal with intrinsic viral properties. Part XIV
serves as a guide to diagnosis and identification for which the necessary
tools are provided in previous parts. Some parts contain class exercises;
in addition, many of the protocols in other parts are suitable for student
experiments.
Virus names in this book are basically those provided by the sixth
Report of the International Committee on Taxonomy of Viruses (1995)
with addition of the generic designation.
We are grateful to the late Dr. J.W. Seinhorst for his major contribution
to Protocol 14 on nematodes and we wish to thank particularly Dr. E.R.
Tiongco of the Philippine Rice Research Institute (PhilRice), Maligaya
(Philippines) for providing the protocol drafts on transmission by leaf
hoppers (Protocol 17).
We owe a special debt of thanks for valuable advice and substantial
contribution to Dr. J.A. Khan of the Plant Virus Laboratory, NBRI, Luck
now, India (Protocol 18) and to H. Lohuis (Protocols 36,41 and Part XIII),
Dr. D. Peters (Protocols IS, 16, 19,35) and Dr. J.W.M. van Lent (Part XII),
all of our department.
For critical comments and helpful suggestions, we thank H. Bloksma
and M. Kikkert of our department, and Dr. J.E. Wellink of Department
Preface VII
of Molecular Biology ofWageningen Agricultural University (Protocol 2);
J.1. Lindner of Research Institute of Plant Protection (IPO), Wageningen
(Protocol 12); J. Vink of IPO (SubprotocoI28.10); Dr. 1. Bos, formerly of
IPO (Part VI); Dr. F. Quak, formerly of IPO, and Dr. A. van Zaayen of
Inspection Service for Floriculture and Arboriculture, Roelofarendsveen
(Part VII); Dr. E.M.J. Jaspars of Department of Biochemistry, University
of Leiden (Subprotocol 28.1); Dr. B.J.M. Verduin of our department
(Parts X;XIII); and Dr. U. Jayasinghe of Northern Philippines Root Crop
Research and Training Center, Benguet State University, La Trinidad
(Philippines) (Subprotocol 40.3).
Last, but not least, we would like to thank the following respondents
to our exploratory letter who paved the way for this publication by their
positive reactions: Drs. A. Ahoonmanesh, Isfahan (Iran); O.W. Barnett,
Raleigh, North Carolina (USA); R.N. Campbell, Davis, California (USA);
Y.-X. Chen, Nanjing (Peoples' Republic of China); J.A. Dodds, Riverside,
California (USA); Y. Ehara, Sendai (Japan); G. Erdiller, Ankara (Turkey);
F. Garda-Arenal, Madrid (Spain); A.J. Gibbs, Canberra (Australia);
R.O. Hampton, Corvallis, Oregon (USA); B.D. Harrison, Dundee
(UK); K. Izadpanah, Shiraz (Iran); E.M.J. Jaspars, Leiden (Netherlands);
R. Koenig, Braunschweig (Germany); A.F. Lana, Morogoro (Tanzania);
W.J. Lucas, Davis, California (USA); K. Maramorosch, New Brunswick,
New Jersey (USA); K. Miczynski, Krakow (Poland); G.L Mink, Prosser,
Washington (USA); Y. Okada, Utsunomiya (Japan); LV. Pop, Bucharest
(Rumania); E. Sander, Tiibingen (Germany); S. Sarkar, Stuttgart
(Germany); C. Stussi-Garaud, Strasbourg (France); R.H. Symons, Glen
Osmond (Australia); M.A. Taiwo, Lagos (Nigeria); K. Tomaru, Tokyo
(Japan); M.B. von Wechmar, Rondebosch (South Africa); Z-J. Zhou,
Fuzhou (People's Republic of China). We trust that this book, which has
been specifically designed as a laboratory manual, will be of use to all
who work with plant viruses.
JEANNE DIJKSTRA AND CEES P. DE JAGER
VIII Preface
References
Noordam D (1973) Identification of plant viruses. Methods and experiments.
Centre for Agricultural Publishing and Documentation (Pudoc), Wageningen,
p 207
Baudoin ABAM, Hooper GR, Mathre DE, Carroll RB (eds) (1988) Laboratory
exercises in plant pathology: an instructional kit. The American Phytopatholo
gical Society, St. Paul, Minnesota, p 196 (Teacher's Manual), p (300) (Student
Exercises)
Contents
Part I Virus Inoculation ......................... 1
Introduction I Virus Inoculation 3
Protocol 1 Mechanical Inoculation of Plants 5
Protocol 2 Inoculation of Protoplasts Isolated from Leaves 14
2.1 Inoculation of Cowpea Protoplasts with
Cowpea Mosaic Comovirus in PEG ...... 15
2.2 Inoculation of Barley Protoplasts
with Brome Mosaic Bromovirus-RNA by
Electroporation ................... 21
Protocol 3 Differential Temperature Treatment
of Infected Plants ....................... 29
Exercise 1 Infectivity of Tobacco Mosaic Tobamovirus 32
Part II Symptomatology ......................... 35
Introduction II Symptomatology ....................... 37
Protocol 4 Description of External Symptoms .......... 42
Protocol 5 Determination of Internal Symptoms ........ 48
5.1 Staining with Trypan Blue ............ 50
5.2 Staining with Phloxine-Methylene Blue 51
5.3 Staining with Methyl Green-Pyronin 52
5.4 Staining with Azure A ............... 53
5.5 Staining with Orange-Green (O-G) ...... 54
Exercise 2 Systemic Acquired Resistance (SAR) ......... 57
X Contents
Exercise 3 Demonstration of Phloem Necrosis in Plants
Affected by Potato Leafroll ................ 60
Exercise 4 Inclusions in Hair Cells of Leaves of Tobacco
Infected with Tobacco Mosaic Tobamovirus .... 63
Part III Infectivity Assays ........................ 65
Protocol 6 Infectivity Assay on Local-Lesion Hosts ....... 67
6.1 Infectivity Assay on Detached Leaves .... 68
6.2 Infectivity Assay on Nondetached Leaves 74
Protocol 7 Infectivity Assay on Hosts Producing
Starch Lesions ......................... 78
Protocol 8 Infectivity Assay on Hosts Producing Systemic
Symptoms Only ........................ 82
Exercise 5 Effect of Temperature on the Susceptibility
of Plants ............................. 86
Exercise 6 Effect of Abrasives on the Infection .......... 88
Exercise 7 Effect of Inhibitors on the Infection ......... 90
Exercise 8 Effect of Light on the Susceptibility of Plants ... 92
Exercise 9 Effect of Time of the Day on the Susceptibility
of Plants ............................. 94
Part IV Infectivity of Virus in Crude Sap ............... 97
Introduction IV Infectivity of Virus in Crude Sap ........... 99
Protocol 9 Determination of the Dilution End-Point ...... 100
Protocol 10 Determination of the Thermal Inactivation Point 102
Protocol 11 Determination of the Longevity In Vitro ...... 105
Exercise 10 Determination of the Dilution End-Point ...... 108
Exercise 11 Determination of the Thermal Inactivation Point 110
Exercise 12 Determination of the Longevity In Vitro ...... 111