Call Topic B : PSTVd

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Call topic title

Epidemiology and diagnosis of Potato spindle tuber viroid and other pospiviroids of plant health concern in relation to Solanaceae.

Background

As a consequence of findings of Potato spindle tuber viroid (PSTVd) in ornamental plants of Solanum jasminoides and Brugmansia spp. in several Member States of the EU in 2006 the Commission Decision 2007/410/EC provides for emergency measures to prevent the further spread of PSTVd with these plants. Recent publications report both on other pospiviroids in S. jasminoides and infections by PSTVd and other pospiviroids in tomato and in other ornamental plant species e. g. Cestrum sp., Petunia sp., Solanum rantonettii, Streptosolen jamesonii, and Verbena sp. Currently, there is no knowledge about occurrence of these or other pospiviroids in wild plant species of Solanaceae and more ornamental host species are being expected. In relation to risk assessment for the main solanaceous crops, potato and tomato, it is essential to know the status of PSTVd and other pospiviroids of potential relevance for these important crops. This requires good diagnostic tools, surveying and understanding of some basic biology and pathways for introduction and spread.

Research need

The project application should address two main aspects.

Epidemiology:

Contributions to a realistic judgement of the potential risk of viroid transmission from ornamental or wild host plants to potato and tomato (and possibly sweet pepper) crops. This includes knowledge on:

host range of PSTVd and the pospiviroids Citrus exocortis viroid (CEVd), Chrysanthemum stunt viroid (CSVd), Columnea latent viroid (CLVd), Tomato apical stunt viroid (TASVd) and Tomato chlorotic dwarf viroid (TCDVd) including an indication of the infection rates in the host plants
different transmission pathways by which these pospiviroids might spread from ornamental/wild plants to solanaceous crops; aspects to be studied are mechanical transmission during cultural practices, aphid transmission (e.g. hetero-encapsidation with other aphid-borne viruses), transmission with other insects (e.g. during pollination), the role of environmental conditions such as temperature
the role of viroid-host interactions in the epidemiology of the pospiviroids

Diagnosis:

For detection and identification of pospiviroids a variety of methods have been published, including return-PAGE, nucleic acid hybridization, reverse transcription-PCR, real-time PCR. Each of these methods has advantages and disadvantages in respect of sensitivity, specificity, reliability, suitability for different host plants or host tissues, speed and labour requirements. Until now there only exists an EPPO protocol on the diagnosis of PSTVd in potato leaves.

The project should address the following:

Approved protocols for the detection in other host plants (e. g. tomatoes, solanaceous ornamentals) and other tissues (e. g. seeds) are required for both PSTVd and other pospiviroids.
For surveillance purposes, a set of reliable and robust methods, applicable under different laboratory conditions, should be compared. Definite identification should at least be based on sequence analysis of the total viroid genome.
Validation of methods by a ring-testing should be done by at least the diagnostic laboratories in the Consortium. However, a larger number of laboratories would be desirable, and final protocols might also be provided to national plant protection organisation's diagnostic laboratories to trial or implement the methods. (N.B. A test on PSTVd in potato leaves should not be included, except for comparison, as this was carried out recently.)
Finally, there is a need for reliable detection and identification of pospiviroids in tomato seeds. Specifically, a diagnostic protocol for ungerminated seeds is required comprising RNA extraction method(s) from ungerminated seeds and RNA-based detection/identification method(s).

Expected benefits

The project will complement the official survey according to article 3(1) of Decision 2007/410/EC carried out by the EU Member States.
Transmission experiments carried out under 'worst case' conditions will indicate whether there are potential pathways for viroids spreading from ornamental or wild plants to potato, pepper and tomato crops. Results will give a first indication whether such events will result in stable establishment in these crops. The relevance and probability of the different transmission pathways will be assessed.
Diagnostic methods will be improved and will provide a basis for future diagnostic protocols to be proposed to the EPPO. A set of ring-tested methods for detection and identification of the specified viroids in different host plants will be available for use by the Plant Health authorities responsible for surveillance and control of regulated/quarantine plant pests.
The results will provide important elements for future pest risk analysis of pospiviroids affecting solanaceous plants other than PSTVd. The project will underpin future policy decisions (e.g. in the EC Standing Committee on Plant Health) in relation to improving phytosanitary regulations aimed at preventing of the spread of pospiviroids in important solanaceous crops. Plant Health Services of the Member States, who are in charge of surveying and testing crops for the presence or continued absence of viroids, will benefit from the results that will build a foundation for harmonising diagnostic methods for these pospiviroids within the EU.

Organisations interested in participating in a research consortium

Name: Mojca Virscek Marn Organisation: Agricultural Institute of Slovenia

Expertise: Dr. Mojca Virscek Marn is the coordinator of survey of PSTVd in Slovenia and is responsible for detection and identification of PSTVd. For this purpose a two step RT-PCR test is used according to the EPPO Diagnostic Protocol. A real time PCR protocol ha also been introduced. In Slovenia the survey started by the end of 2006, after PSTVd infection was confirmed on a pot plant of Brugmansia cordata, imported from Netherlands. Until now 109 samples were taken and analysed. Infection was confirmed in only one sample of Brugmansia, 41 samples of S. jasminoides and 2 samples of S. rantonnetii. Most of the PCR products were sequenced. Sequence analyses confirmed the presence of PSTVd.

Apart from the coordination of PSTVd Mojca Virscek Marn is the coordinator of sharka survey in Slovenia and responsible for detection of viruses on fruit crops, field crops and grapevine. She is also involved in basic and applied research on isolation and characterisation of fruit, grapevine and field crop viruses and viroids mainly using test plant inoculation, ELISA and molecular biology techniques, study of plant virus – vector interactions, development of laboratory protocols for detection and identification of viruses and viroids on fruit, grapevine and field crop viruses and viroids, influence of virus or vioroid infection of host plant, study of virus and viroid distribution in Slovenia. Her bibliography is abvailable on
http://izum.izum.si/bibliografije/Y20080317152453-03853.html
and http://sicris.izum.si/search/rsr.aspx?lang=eng&id=4932&opt=1

At the Agricultural institute of Slovenia we are interested in collection and testing of cultivated, wild and ornamental plants (potential hosts of PSTVd), harmonization and optimization of detection methods (RNA extraction, RT-PCR and quantitative RT-PCR detection), mechanical transmission studies and studies of the effect of environmental conditions on viroid expression. We have gathered some experience of growing Brugmansia sp. and S. jasminoides in growth chambers and started some inoculation tests and would like to continue the research.

Name: Mariyana Gozmanova Organisation: University of Plovdiv, Bulgaria

Expertise: Plant Molecular Virology group at the Dept. of Plant Physiology and Molecular Biology, University of Plovdiv, Bulgaria has as a main area of research the study of molecular interactions between PSTVd and host factors mediating viroid movement and replication in host and parasitic plants. A role of post-transcriptional gene silencing (PTGS) in viroid pathogenicity is currently under investigation.
The group is aware of the higher significance of viroid survey in Bulgaria related to epidemiology and management of this plant pest.
The team believes that unraveling of new potential viroid hosts among Bulgarian plant cultivars and the identification of susceptible and resistant to viroid infection Bulgarian host genotypes will contribute to better understanding of specific bilateral interactions.
More information can be found at the web page: www.plantgene.org

Name: Valérie Molinéro-Demilly Organisation: LNPV

Expertise: Implementation and optimisation of PSTVd ans Pospoviroïdes detection by RT-PCR and molecular hybridization. Development of methodology for ring test management.French PSTVd survey on ornamental plants.

Name: Elvira Schecklies Organisation: pab productions

Expertise: COMPANY PROFILE

Pab productions produces polyclonal antibodies in rabbits and chicken. Using the yielded sera and yolks – mainly affinity purified - immunological test systems, e.g. ELISA, are developed according to the requirements of market and customers.
Addtionally pab productions has developed an innovative product line based on renewable resources (commercially available):
- microtiterplates from polylactic acid (PLA) with advantages compared to polystyrene:
- low backgrond and variation
- standard curve range: 5ng- 10 µg
- optimized surface even for hydrophilic molecules

- innovative immunostick-cuvette-system for ELISA (adhesive and covalent binding) from polyhydroxybutyrate (PHB)

- adjuvant for immunstimulation from PHB
- no necrosis
- direct binding to the antigen

Contact: pab productions
Elvira Schecklies
Bgm.Herzog Str. 5;
D – 85241 Hebertshausen
Phone: +49 (0) 8131-20709
Fax: +49 (0) 8131-20709
e-mail: pab.productions@web.de
www site: www.pabproductions.de

Advantages of pab-immunosticks compared to microplate ELISA:

- in contrary to microtiter plates, no sample extraction is necessary
- no need or waste of organic solvents
- users with only spectral photometers or even no equipment can carry out the tests
- locally tests by just using precipitation conjugates
- untrained stuff or amateurs can use the stick system without problems
- ideal for users with low turnover of samples

The main advantage is, that you there is no need to seperate the analyte from the rest of the sample. Just pulps – only if the sample is dry – are made and the stick can directly be transferred. This is especially useful, when the analyt can not be seperated, e.g. because of adhesivity to other components of the sample.
The coated antibodies on the surface of the stick will catch the antigen selectively. The further ELISA can be carried out as usual.
For example, the the case of

- antibiotics in milk, eggs or baby food
- mycotoxins in cereals
- pesticides
- detection of bacteria, funghi and parasites in stool, food, water,

the stick can be directly transferred in the sample. After just washing with water, the bound analyte can be detected using e.g. an enzym-antibody-conjugate. Final step is the substrate reaction.
A test like this can be distributed by pharmacies, so eyeryone can control a possible contamination at home.
Tests, locally carried out in the environment, can quickly detect contaminated areas or water.
This wide range of controls has an additional advantage for your tests for laboratory use: positive samples should be checked using your microtiterplate ELISA to control the result.

Name: test@nonexistentdomain12341.com Organisation: test@nonexistentdomain12341.com

Expertise: test@nonexistentdomain12341.com