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THE DEVELOPMENT AND APPLICATION OF NUCLEIC ACID PROBE TECHNOLOGY FOR RAPID AND RELIABLE DETECTION AND IDENTIFICATION OF PLANT QUARANTINE BACTERIA

Objective

The successful implementation of the common plant health policy in the European Community is largely dependent upon the availability of reliable test procedures for high risk statutory organisms. Essentially standardized methods for laboratory tests which are harmonized in all Member States are a prerequisite.

Recent advances in molecular biology have generated powerful fingerprint methods for rapid, specific and sensitive detection and identification of plant pathogens with obvious advantages over the common conventional methods, in particular serological tests.

The project aims to introduce these technologies in methods for reliable test-monitoring of the bacteria under the EC Plant Health Directives.
The successful implementation of the common plant health policy is the European Community is largely dependent on the availability of reliable test procedures for high risk statutory organisms. It is necessary to have standardized methods which are harmonized in all Member States. Recent advances in molecular biology have generated powerful fingerprint methods for rapid, specific and sensitive detection and identification of plant pathogens with obvious advantages over the common conventional methods, in particular serological tests. Work is being done to improve the detection, diagnosis and identification of bacteria which are quarantine or high risk statutory organisms by the development and exploitation of nucleic acid probe techniques.

Probes are now available for 5 quarantine bacteria. Protocols were prepared for deoxyribonucleic acid (DNA) hybridization tests for 2 organisms, ring tests made with bacterial cell suspensions and hybridization conditions, labelling and detection systems evaluated. Protocols have been evaluated for colony hybridization tests and direct blotting and ring tests made with bacterial cell suspensions and contaminated plant material. Primers were prepared for polymerase chain reaction (PCR) amplification tests, reagents distributed and the first round of tests made for sensitivity.
The programme consists of (1) testing of probes in dot blot hybridization, (2) development and quantification of target DNA amplification by the Polymerase Chain Reaction (PCR), (3) identification and genetic characterization in Restriciton Fragment Length Polymorphism (RFLP) analysis and (4) rapid detection of single cells with fluorescent RNA oligonucleotide probes.

The work consists of the preparation of the test protocols and experiments to evaluate the methods, which are performed in three consecutive stages :

1) testing of cell suspensions of the target bacteria, related and non-related pathogenic and saprophytic bacteria, in particular those naturally present in association with the plant material,

2) reconstruction tests with artificially contaminated plant extracts,

3) ringtests using samples plant material with different levels of natural contamination.

Coordinator

RIJKSSTATION VOOR PLANTENZIEKTEN
Address
Burgemeesters Van Gansberghelaan 96
9820 Merelbeke
Belgium

Participants (10)

BENAKI PHYTOPATHOLOGICAL INSTITUTE
Greece
Address
Stephanou Delta Street 8
14561 Kifissia (Athens)
Biologische Bundesanstalt für Land- und Forstwirtschaft
Germany
Address
Messeweg 11-12
38104 Braunschweig
DANISH INSTITUTE OF PLANT AND SOIL SCIENCE - RESEARCH CENTRE FOR PLANT PROTECTION
Denmark
Address
Lottenborgvej 2
2800 Lyngby
DIRECCION GENERAL DE SANIDAD DE LA PRODUCCION AGRARIA
Spain
Address
Paseo Infanta Isabel 1
28000 Madrid
Fédération Régionale des Groupements de Protection des Cultures des Pays de la Loire
France
Address
10 Rue Le Notre
49044 Angers
INSTITUTO NACIONAL DE INVESTIGACAO AGRARIA - CENTRO NACIONAL DE PROTECCAO DA PRODUCAO AGRICOLA
Portugal
Address
Quinta Do Marques
2780 Oeiras
MAFF Central Science Laboratory
United Kingdom
Address
Hatching Green
AL5 2BD Harpenden
PLANTENZIEKTEKUNDIGE DIENST
Netherlands
Address
Geertjesweg 15
6700 HC Wageningen
State Laboratory
Ireland
Address
Abbotstown - Castleknock
15 Dublin
UNIVERSITY OF BOLOGNA
Italy
Address
Via Zamboni 33
40126 Bologna