NEW TECHNOLOGIES TO IMPROVE PHYTODIAGNOSIS

Ziel

GENERAL DESCRIPTION OF THE ACTION

1. GENERAL BACKGROUND

Introduction: One third of the world's agricultural plant production is destroyed by pests and diseases. Early detection of the presence of pathogens in crops, and more particularly in material intended for propagation or shipment to other countries would make it possible to eliminate infected plants and seeds and thus decrease the otherwise inevitable losses. Further development and extension of new technologies for the early detection and identification of plant pathogens is therefore of the utmost economic importance.

Early detection of plant diseases not only allows proper forecasting and preventive control measures to be taken, but also prevents the necessary use of pesticides. Thus this helps to protect the environment and decrease farm costs and the level of pesticide residues in food crops.

The selection of healthy plant propagation material by means of appropriate pathogen detection techniques is a cheap preventive disease control measure and the best guarantee for a successful future crop.

Early detection of pathogens in selected lines of crop plants in breeding programmes saves valuable time in the search for improved plants particularly those resistant to diseases.

Rapid detection and unequivocal identification of diseases in plant material submitted to quarantine conditions helps to prevent spread of diseases in unaffected ecological regions.

More sensitive pathogen detection methods facilitate the study of the biology and epidemiology of numerous plant diseases with unknown etiology.

In a European Community "without borders", with an open market for plant propagation material and crops, sanitary certifications of propagation material will be compulsory and of utmost importance to avoid disease spread leading to severe losses of crops. Collaboration between private and public national programmes for eliminating diseases and finding alternatives for crop protection is required. Certification can only be established with standardized methods to be applied and approved by each Member State. Modern immunochemical and nucleic acid-based techniques methods can best be standardized and are sensitive enough to allow early detection and identification of plant pathogens.

Achievements of the past COST Action 88: "Methods of Early Detection and Identification of Plant Diseases" [1987 - 1993].

The Action was particularly successful in:

1) The characterization of plant disease detection methods as well as standards.

2) The exchange of results, reagents and standards between different laboratories through the organization of more than thirty international workshops and meetings.

3) Management of centralized ring-tests to establish standards and evaluate diagnostic reagents.

4) The establishment of a European Database and Reference Collection within the "Deutsche Sammlung fr Mikroorganismen und Zellkulturen" (DSM) in Braunschweig.

5) Transfer of technology into industrial and agricultural applications.

In addition several co-ordinated EU-research proposals have been initiated by the COST-88 Network. The network was enlarged to include East European countries with the financial help from the PECO programme.

Test Methods
Advantages
Disadvantages

Biological Testing

simple; insensitive to mutational changes; sensitive;
laborious; non-differentiating; time consuming; greenhouse or field space necessary; unreliable

Antibody-based Technologies

quick; easy sample preparation; can be automatized; quantitative; cheap; reliable;
sensitive to mutational changes of the antigens; quality of antisera of high impact; serological variability of antigens need to be studied

Nucleic acid-based Technologies

extremely sensitive;
expensive chemicals; laborious sample preparation; experienced personnel necessary; still needs R&D

(a) Current state-of-the-art in the field: Methods for the detection of plant pathogens are based on the following: 1. Biological testing; 2. Antibody-based testing; 3. Nucleic acid-based testing. Each of these different technical categories has its explicit advantages and disadvantages as summarized in Table 1.

Biological testing is still required when the pathogen responsible for a particular disease is not yet identified. New greenhouse or in vitro bioassays might replace some laborious greenhouse or field tests. However, it is hoped that molecular or immunochemical technologies will replace them in the near future.

Antibody-based methods are commonly used in large-scale testing programmes especially since the introduction of ELISA (Enzyme-Linked Immunosorbent Assay). With the now widespread monoclonal antibody technique the problems of low specificity and sensitivity have been partially overcome. Nevertheless only a few satisfactory tests have been developed for bacterial or fungal pathogens. The new technology to improve and modify existing monoclonal antibodies (MAB) by means of genetic engineering appears a very promising tool to redesign and optimize already existing antibodies for particular applications.

With respect to the pathogens where serological methods have failed so far or are inappropriate, nucleic acid-based techniques have shown great potential to reach the same importance as ELISA. In particular PCR-technology (Polymerase Chain Reaction), which by itself already has been applied successfully as a detection method, or whose variants like RAPD or PCR-RFLP will allow a rapid development of suitable specific tests. In summary nucleic acid (NA)-based techniques will almost certainly be a very valuable technique for bacterial and fungal pathogens as well as for plant pathogens of higher orders like nematodes, insects and so forth.

(b) Why in the context of COST?: Modern agriculture and agroeconomy have superseded geographical and climatic borders and thus allowed an almost unlimited spread of plant products together with their pathogens. A COST Action is the best forum to build a network among European plant

pathologists to harmonize and standardize testing methods for important plant pathogens. This is important to build confidence in testing among states. It would also strengthen the impact and acceptance of the annexes and guidelines of Directive 77/93/EEC. We must protect our European borders from the ingress of damaging pathogens and thereby ultimately improve the competitiveness of European agriculture in the world market.

(c) Relation to other international scientific programmes: An important aspect of this Action will be the co-operation with other COST Actions in the field of biotechnology, e.g. In Vitro Culture, Mycorrhiza or COST 813 "Diseases and Disorders in Forest Nurseries" and several projects within the AlR-programme. The outcome of the proposed Action will be extremely beneficial for the EPPO (European Plant Protection Organization).

2. OBJECTIVES OF THE ACTION

This COST Action has two main objectives:

1) The primary aim of this Action is to aid in a standardization of new technologies for phytodiagnosis and their practical application, as well as the quick transfer of new technologies from laboratory level to practice.

2) A secondary aim is to promote standardization and harmonization of phytodiagnosis by extension of existing databases and collections of references and standards as well as the organization of ring-tests to define the latter.

This Action will supply the different governmental institutions of participating countries with expert information about references, diagnostic standards and the most appropriate test procedures for plant pathogens. The collaboration of different expert laboratories throughout the whole of Europe, including East European Countries, will ensure a uniform test procedure, update the list of important pathogens for Europe, provide a broad spectrum of pathogens to be included into the reference collection and, after a ring-test, assure test-quality.

3. SCIENTIFIC CONTENT

The new proposed Action is an immediate and logical successor to the successful COST Action-88 "Methods of Early Detection and Identification of Plant Pathogens". It will take advantage of the network of scientists that was established by COST-88. Considering the results that were achieved by COST-88 and the emergence of new technologies, the new Action will address the following topics:

1) Antibody-based Technology

With respect to tests based on antibodies we intend to investigate:

- Improvement of test sensitivity and specificity by the application of genetically engineered antibodies for detection, including the integration of non-structural proteins as possible targets.

- Modifications of already existing MABs by means of genetic engineering to cope with non-optimal performance.

- Exploitation of immunotolerance mechanisms towards production of specific antibodies.

- Application of Biosensor technology to diagnosis and antibody design.

2) Nucleic Acid-based Technology

Nucleic acid-based test techniques are predominantly based on PCR-technology. Developments of techniques like RAPDS (Random Amplified Polymorphic DNA), RFLP (Restriction Fragment Length Polymorphism) and sequencing of rRNA-operon will produce rapid improvement in test techniques for pathogens. The development of a pathogen-independent extraction method to obtain nucleic acids for use in PCR is still of central importance.

Finally the combination of antibody- and nucleic acid-based techniques such as immuno-capture-ELlSA, DNA-ELISA and Biosensor-based assay principles need to be evaluated and tested with a broad spectrum of pathogens and host plants in order to explore the value for practical applications.

3) Diagnostic Forum

The new antibody- and nucleic acid-based techniques will be evaluated for use in practical routine testing. In addition feed-back from practical testing to the research level will enhance the value of these techniques. This will lead to standardization and harmonization of methods and diagnostics and thus strengthen the impact and acceptance of the annexes and guidelines of Directive 77/93/EEC.

4) Databases and Collections

Existing COST-88 databases will be expanded to include all pathogens and attempts will be made to link them to other databases like the international virus database. We will also compile a database for laboratory protocols defined by the COST Action. The Reference Collection will contain the pathogens that have been worked on during the Action.

4. TIMETABLE

The time required to pursue the scientific projects will be four years with workshops as required and annual management meetings to evaluate the progress. A brief proposal for the first two years is as follows:

Year 1: Establishment of Working Groups

Organize workshops on: Evaluation of Immunotolerance
Techniques used to identify pathogen-specific probes
Tospovirus Ring-test

Year 2: Organize workshops on: Fungal Detection [Botrytis]
Database [extension to other pathogens]
Sample Preparation for PCR Bacterial Detection [Erwinia]
Viral Detection [Nepoviruses]

International experts will be invited to participate in these workshops. Results from the different activities will be made public in yearly reports and preferably by publications in refereed journals.

5. ORGANIZATION AND MANAGEMENT

In contrast to the previous Action, Working Groups (WG) will be organized mainly according to techniques and not according to pathogens. The following groups will be established from the beginning:

1) WG Antibody-based Technology
2) WG Nucleic Acid-based Technology
3) WG Diagnostic Forum
4) WG Databases and Collections

The organizational structure is outlined in Fig. 1. Each of the WGs will have a co-ordinator that should also be a member of the Management Committee. Sub-WGs may be initiated for each WG according to specific needs, especially WG 3 where many different pathogens will be included during the course of the Action. These SWGs will be in operation only for a limited period of time to validate the test methods and the reagents.

ECONOMIC DIMENSION OF THE ACTION

It is estimated that the Action will involve 80 senior scientists, 120 junior scientists and 100 technicians. This will amount to a total of personnel costs of ECU 12 100 000 per year.

Current status
The MoU of this Category B Action has now been signed by 17 countries. The Action is organised as shown in Fig. 1 into 4 Working Groups, under which time-limited sub-Working Groups can be established as needed.
The Management Committee is chaired by G. Adam (D) and C. Manceau (F).
The WG Antibody-based Technology (co-ordinated by L. Torrance, UK) is primarily concerned with :
improvement of the sensitivity and specificity of serological tests by application of gentechnologically produced antigens and antibodies;
modification of already existing monoclonal antibodies by gentechnology to optimise their performance;
exploitation of immuntolerance-mechanisms towards production of more specific antibodies;
application of biosensor-technology for diagnosis and antibody-design.

The WG Nucleic Acid-based Technology (co-ordinated by C. Manceau, and F. and J. Spak, CZ) is primarily concerned with methods based on the polymerase chain reaction (PCR) :
the development and application of techniques such as RAPDS (Random Amplified Polymorphic DNA), RFLP (Restriction Fragment Length Polymorphism), and AFLP (Amplified Fragment Length Polymorphism) and sequencing of rRNA-operons will lead towards a rapid improvement of test methods for all pathogens of plants. It will also be useful for the development of group- or genus-specific tests in order to reduce the necessary number of tests;
the development of pathogen-independent extraction methods for preparation of PCR-suitable nucleic acid is still a central topic of interest;
in 1996 it was decided to establish a sub-WG for the application of NA-based techniques to identify invertebrate plant pests and their vector status.

The WG Diagnostic Forum (co-ordinated by P. Boonekamp, NL, J. van Vaerenberg, B, and M. Dewey, UK) is primarily concerned with :
the transfer of new laboratory techniques for phytodiagnosis into practical application, including the development of generally applicable protocols;
practical testing of methods and diagnostics developed by the two previous WGs on a larger scale;
performance of ring-tests for important European plant pathogens in order to explore the variability of the pathogens and diagnostic tools.

The WG Databases and Collections (co-ordinated by S. Winter, and D. and R. van der Vlugt, NL) is primarily concerned with :
keeping a reference collection of isolates that have been compared in ring-tests and can serve as well-defined and characterised positive controls and as a source to develop and screen new diagnostic tools;
making available a database containing information about existence and addresses for diagnostic tools and reference pathogens. This will be distributed and made accessible through the World Wide Web (WWW).

Programm/Programme

IC-COST - European cooperation in the field of scientific and technical research (COST), 1971-

Thema/Themen

4 - Agriculture and Biotechnology

Aufforderung zur Vorschlagseinreichung

Data not available

Finanzierungsplan

Data not available

Koordinator

CEC

EU-Beitrag

Keine Daten

Adresse

Rue de la Loi, 200
1049 Brussels
Belgien

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