Abstract:
A Greek SME involved in production and trade of nursery plant material, has developed a diagnosis technique using Reverse Transcription - Polymerase Chain Reaction (RT-PCR) technology to identify simultaneously all known Closter viruses, vitiviruses and fovea viruses infecting grapevine. It can be used for routine testing of grapevine propagation material of viruses involved in the aetiology of grapevine leaf roll and rugose wood complex. Partners for all kind of agreements are sought.

Description:
A Greek company has developed diagnostic tests, directed to the detection of Vitivirus, Fovea virus, and Closterovirus species, in grapevine. In particular the test comprises oligonucleotide primers and optimised reverse transcription (RT) polymerase chain reaction (PCR) methods with high sensitivity, specificity and polyvalence that allow reliable, rapid and simultaneous generic detection of members belonging to said genera. An improved simple sample preparation suitable for large-scale applications was also developed and a large number of samples can be tested very easily within a short time.

The test starts with a simple method that uses a rapid template preparation protocol, by spotting of plant sap extract on a nylon membrane, and a thermal treatment processing before RT-PCR. The spotted extract is used in a first-stage RT-PCR, at the end of which an aliquot of the resulting mixture is carried over into a second-stage nested PCR. The amplification products of the first- and second-stage polymerase chain reactions may be analysed to identify the presence or absence of the first and second targeted nucleotide sequences.

Innovative Aspects:

• Multiplex generic detection of viruses (simultaneous detection of at least 12 viruses infecting grapevine using one assay)
• Reliable detection using simple sample preparation and alleviating the need for laborious RNA extraction

Main Advantages:
This method can be useful in large-scale detection applications, because of the following advantages:

1. Increased detection polyvalence, and multiplex generic detection of viruses that reduced time and cost per assay
2. Increased specificity and sensitivity, compared to other generic PCR methods known in the art for grapevine virus detection, by using a nested PCR format.

The increased sensitivity also allowed the use of a simple and rapid template preparation protocol, by spotting of plant sap extract on a nylon membrane, and a thermal treatment processing before RT-PCR

These improvements permit constant PCR amplification from petiole and cortical scraping preparations of infected grapevines, and enable multiplex detection of viruses belonging to three genera. This method compared to other generic PCR methods for grapevine virus detection known in the art has increased specificity and sensitivity, and compared to other PCR methods known in the art for specific single grapevine virus detection, has higher polyvalence and similar sensitivity and specificity.

Description of the specialization of the organization:
VITRO HELLAS S.A. is a private company established in 1986, consisting of a nursery, greenhouses tissue culture laboratory and a laboratory where both serological and molecular methods are performed to check the sanitary condition of plant material. The company is involved in both production and trade of plant propagation material. The production potential of the laboratory is higher than one million acclimatized plants per year. The main products are:

Laboratory:

Rootstocks: GF 677, MAXMA 14, 29C, R110, 140Ru, 1103P.
Fruit trees: Kiwi (cv Hayward and male clone ?Tomuri?)

Rusberrys (cvs Sunberry, Thornless, Evergreen, Tayberry, Thornfree)
Potatos: minitubers cv. Spunta, cv. Jaerla, cv. Kennebec
Aromatic plants: Oregano, menthol, Melissa sp.

Nursery:

Peaches, nectarine, apple, apricot, plum, cultivars, grafted on rootstocks produced in the laboratory
Olive (Greek cultivars)

The permanent staff of the company is consisted of Nine agronomists (1 MBA and 3 MSc., two of which are Ph.D. candidates), nine technicians, specializing on tissue culture methods of large scale production, accountant, accounting clerk, secretary and ten workers specializing in greenhouse acclimatization procedures and nursery work. Additional staff is seasonally engaged to account for increases in production.

Additional Information:
National Documentation Centre
Mr George Apostolopoulos
+30 10 7273920
+30 10 7246824
E-mail: gapo@ekt.gr