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VIRUS DETECTOR
Improved diagnostic tools for the certification of strawberry propagation material

Viruses constitute a significant threat to the strawberry industry, causing severe economic losses. These viruses include the causal agents of quarantine diseases of major economic importance, such as crinkle, mottle, vein banding, and mild yellow edge disease. Rapid and simple methods for the detection of the major aphid-borne strawberry viruses are unavailable, due to problems with the purification of these viruses from plant material. Control of the quarantine viruses is difficult and relies completely on excluding viruses by meristem culture and rigorous certification programmes to maintain clean planting stock. Most aphid-borne strawberry viruses can only be detected by using time-consuming biological indexing procedures introduced over 40 years ago, such as grafting to sensitive indicator plants. Methods based on the detection of viral nucleic acid would offer an alternative to the expensive and time-consuming procedures currently used.

Objectives
This project proposes to develop cost-effective, standardised methods for the rapid, reliable and sensitive detection of the major viruses and to implement these protocols in screening procedures. These methods could be used by the certification and quarantine services within the European Union and in Central and Eastern European countries. The detection methods will be adapted to enable large-scale screening of propagation material, including the screening of in vitro material. They will be used by organisations involved in the production and classification of certified elite propagation material.
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Progress to Date
The following work packages were completed:
Work Package 1: Characterising strawberry viruses and determining their genetic sequences
Work Package 2: Investigating the variability of strains of the various viruses
Work Package 3: Optimising and comparing AmpliDet RNA-based detection procedures and extraction methods for the detection of different strawberry viruses
Work Package 4: Developing a procedure for the detection of all four viruses in one reaction
Work Package 5: Conducting field evaluations of commercial strawberry cultivars with the developed AmpliDet RNA multiplex variants
Work Package 6: Communicating the objectives and results of the project to all interested European organisations and institutions involved in the strawberry industry.

Results
1) The project was able to isolate partly purified strawberry crinkle virus (SCV) suspensions and to isolate the genomic RNA from the particles
2) cDNA was synthesised to the RNA and a cDNA library was compiled
3) The project isolated strawberry mottle virus (SMoV) and associated dsRNA from infected N. occidentalis and used the dsRNA to synthesise cDNA
4) Single sensitive Nucleic Acide Sequence Based Amplification (NASBA) systems were developed for SCV, SMoV, strawberry vein banding virus (SVBV) and strawberry mild yellow edge potexvirus (SMYEPV)
5) A multiplex reverse transcriptase polymerase chain reaction (RT-PCR) method for the simultaneous detection of all four viruses, in combination with a plant RNA-specific internal control, has been developed. It can be used as an indicator of the quality of the plant extract and reverse transcription in general.
6) To evaluate the new methods for detecting aphid-borne strawberry viruses, chosen samples of infected strawberry plants and Fragaria sp. indicators originating from various countries were tested using RT-PCR and AmpliDet RNA techniques.
7) Successful field evaluations on commercial strawberry cultivars were performed using the AmpliDet RNA mono and duplex variants.
8) A workshop entitled 'Detection of strawberry viruses - recent advances in molecular diagnostics' was held in September 2002.

Classified in FRUIT, CROP PESTS AND DISEASES, GENOMICS

Scientist responsible for the project

Dr CORNELIS D. SCHOEN
Box 16
6700 AA Wageningen
Netherlands (The) - NL

Phone: +31 317 476156
Fax: +31 317 418094
E-mail: c.d.schoen@wur.nl

References

Project ID QLRT-1999-01553
Organisation Plant Research International B.V.
Area 5.1.1
Start date 01 February 2000
Duration (months) 36
Total cost 1 073 071 €
Total EC contribution   724 996 €
Status Completed

The partners

  • Research and Breeding Institute of Pomology Holovousy LTD, Czech Republic (The) - CZ
    karesova@vsuo.cz
  • Institute of Plant Molecular Biology, Academy of Sciences of the Czech Republic, Czech Republic (The) - CZ
    spak@umbr.cas.cz
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