Call Topic A: Erwinia amylovora

A call text for this topic is provided below. Please see the Applicants Guide for national eligibility, available national funding, and application processes

<< Back

Call topic title

Development and validation of innovative diagnostic tools for the detection of fire blight (Erwinia amylovora)

Background

Erwinia amylovora, causal agent of fire blight, is a quarantine bacterium in Europe. Rapid and on-site diagnosis is critical for optimal implementation of phytosanitary control measures.

Different diagnostic screening tests for a rapid diagnosis of E. amylovora are commercially available and new methods have recently been published. However, these have not yet been validated for phytosanitary applications and require ring-testing for validation. Validation tests should be performed including those protocols which were recently revised by the European Plant Protection Organization (EPPO/OEPP), thus supporting the preparation of the new protocol for the Food and Agriculture Organization of the United Nations (FAO/IPPC). Usually these tests are designed for application in laboratory settings and they may not be adequate for on-site diagnostics. Thus, innovative detection methods should be developed specifically suitable for on-site diagnosis. New diagnostic tests (e.g., commercial PCR methods, immunological tests) have been developed since the last European validation trials. The application of these tests for laboratory and on-site use requires validation in ring-trials. Innovative tests suited for on-site applications need to be developed in case the existing tools are insufficient. Recent advances with other diseases using immunological and on-site PCR tools could be used as models for developing similar tools for fire blight.

Determination of asymptomatic infection is newly mandated by EU regulations, but currently no appropriate protocols are available. Sampling for asymptomatic infections is particularly difficult, since testing of a high number of randomised samples is necessary and the isolation of latent bacteria from woody tissues is challenging. Sampling protocols and tools are currently lacking and should be developed in this project.

The long-term control of fire blight requires eradication of inoculum reservoirs. Methods to source-track E. amylovora considering sampling protocols and molecular identification to the strain level should be developed.

Research need

The following work is required:

• Development of sampling protocols and tools for asymptomatic infections
• Development of methods to source-track E. amylovora considering sampling protocols and molecular identification to the strain level.
• Ring-testing trials to validate new test methods for application for:
  1. detection of E. amylovora in asymptomatic plants on-site or in laboratories
  2. detection of E. amylovora in symptomatic plants on-site or in laboratories

Expected benefits

The following beneficiaries are anticipated:

1. Quarantine laboratories of several countries facing fire blight epidemics, (and countries which may face fire blight occurrence in the near future) since:

1. newly available methods are reviewed with respect to their specificity, sensitivity and performance. Innovative methods are developed as needed for on-site applications.
2. the use of validated methods improves the quality of the laboratory
3. the validation of methods in ring-tests is a major requirement for use in accredited laboratories
4. the availability of validated screening test would allow larger number of samples in the detection of E. amylovora in asymptomatic plants
5. these methods are quicker and therefore labour costs are reduced (Note: this implies that the methods should be developed to be rapid as well as reliable and inexpensive)

2. Research laboratories, since the availability of a reliable screening method would allow accurate epidemiological studies to improve fire blight control measures

3. Plant protection service, since results are quickly available and decisions on phytosanitary measures can be reached earlier (especially in situations of imminent danger, e.g. in protected zones with a sudden disease outbreak)

4. Fruit growers and nurseries, since the availability of a reliable screening method would allow an economically feasible testing of plants intended for planting and would facilitate the development of certification schemes.

Organisations interested in participating in a research consortium

Name: paulin Organisation: inra

Expertise: Our team has been working on fire blight for long. Although our expertise is not strain characterisation or detection, we could possibly help in attempts at detection in symptomless plants?

Name: Poliakoff Françoise Organisation: LNPV, FRANCE

Expertise: Implementation and optimisation of Erwinia amylovora detection on asymptomatic samples by isolation, PCR, IF, biological tests. Develpment of methodology for ring-testing organisation.

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)










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.






- 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