COST-EFFECTIVE HAND-HELD DEVICE FOR RAPID IN-FIELD DETECTION OF FLAVENSCENCE DOREE PHYTOPLASMA IN GRAPEVINES

Final Report Summary - VITISENS (COST-EFFECTIVE HAND-HELD DEVICE FOR RAPID IN-FIELD DETECTION OF FLAVENSCENCE DOREE PHYTOPLASMA IN GRAPEVINES)

Executive Summary:

The wine sector is a fundamental pillar of European identity and one of Europe’s foremost global lead markets. However, small and medium companies in this vital European lead sector are currently facing an epidemic threat caused by the Flavescence dorée phytoplasma (FDp). This unicellular organism that grows in the sap of grapevines is responsible for a destructive and dangerous grapevine disease that severely damages plant productivity and has an important economic impact. Already widespread in Italy and France, where it is catalogued as catastrophic, it is present in Slovenia and Portugal and also occurred in Spain, Switzerland, Austria and Serbia. FD incidence is rising rapidly showing a clear epidemic pattern that threatens to engulf European winegrowers and vine nursery SMEs despite mandatory national and EU control measures. One major reason behind the spread is the fact that visual inspection remains routine practice to determine the presence of phytoplasma, despite being clearly insufficient for detecting latent infections. Additionally, symptoms of the disease can be easily misled with other diseases or physiological alterations. Since visual inspections are insufficient for Flavescence dorée diagnosis, a reliable in-field detection of FDp as early as possible in the season is needed to avoid disease epidemics.

VITISENS project aims to provide vineyard and nursery managers a low cost, portable, rapid, reliable, in-field detection system of FD in grapevines which will prevent contamination by Flavescence dorée in countries where the pest is not yet established, will stop its spread in countries where the distribution is still limited and will help to efficiently fight against the disease in countries already affected. The proposed portable hand held device aims to determine the presence of the FD in grapevines by means of an innovative integration of the extraction, amplification and detection steps required for the recognition of specific nucleic acid sequences.

With a cost below €1000 per platform and €10 per test, specialized vine nurseries will be able to assess the condition of vines, root stocks, vine grafts and other planting material before trading benefit from selling value-added certified healthy planting material and from saving in laboratory costs, while wine growers will gain more precise, rapid and effective field interventions: a faster eradication of the actual infected vines and surrounding areas will prevent contamination of the whole vineyard and will avoid eradication of healthy vines improving production. The VITISENS system will be manufactured and distributed by consortium technical SMEs who will also benefit from opening new markets and income sources by means of a new portfolio product.

Vitisens is the result of a European Project funded by the European Commission for 28 months and a total investment of €1,096,879, under the “Research for the Benefit of the SMEs” at the Seventh Frammework Programme (FP7). The consortium provided the complementary business capabilities, commercial networks and research expertise to guarantee the technology a quick route to the market and all members are fully committed to ensuring the success of the project.

The project was divided into research, demonstration, dissemination, exploitation and management activities by means of Work packages. After 28 months of research VITISENS has achieved most of the objectives foreseen, sampling material needed and protocol based on seasonality effect and a sample treatment protocol optimized with a DNA extraction method using new laboratories methods (LAMP) adapted for in field conditions.(VITISENS Diagnostic kit). Moreover the portable handheld device and special instrumentation for detection have been designed and constructed (VITISENS Reader and disposable plastic cartridge).

Project Context and Objectives:

Of the 2.25 million hectares of grapevines (Vitis vinifera) grown globally, Europe, with a 45% of world’s vineyard area, remains the main producer and exporter of wine. Italy, France and Spain are the main producers accounting for a third of the total world production of 66 million Mt (FAOSTAT 2007). The European Union is also world leader in the market for vine nursery products, with an annual output of 360 million cuttings (EP, 2009). Small and medium companies in this vital European lead sector are currently facing an epidemic threat caused by the Flavescence dorée (FD) phytoplasma. This unicellular organism that lives and grows in the sap of grapevines is responsible for the most destructive and dangerous phytoplasma disease of grapevines and severely damages plant productivity (Boudon-Padieu, 2007). The occurrence of the Flavescence dorée disease has been catalogued as catastrophic in France and Italy (Boudon-Padier, 2002).

The phytoplasma relies on an insect, the leafhopper Scaphoideus titanus, to act as a spread vector, transporting the phytoplasma as it moves between vines to feed (Christensen, 2005). Because of this, pest occurrence depends on the simultaneous presence of both phytoplasma and vector. If appropriate control measures are not applied, the number of infected vines may increase steadily 10-fold every year, reaching 80–100% within a few years, which underlies its catastrophic potential. To this risk is added human trade activity to establish and expand new vineyards with rootstocks, vine grafts and other planting material which may be latently contaminated.

This situation indicates that in spite of the mandatory national and European regulations for control, namely a) Eradication of infected plants, b) Insecticide treatments against the insect vector and c) Use of healthy planting material, the disease is spreading actively representing a serious threat for European winegrowers and vine nursery SMEs (Boudon-Padieu, 2003), as once FD is established in the vineyard, the means to reduce losses caused by the phytoplasma are extremely limited.

VITISENS proposes a portable handheld device answering to the need of wine producer and vine plant nursery SMEs to perform rapid in-field tests to determine the presence of the Flavescence dorée phytoplasma in grapevines by means of an innovative integration of the extraction, amplification and detection steps required for the detection of specific nucleic acid sequences, with an estimated price per device of €1,000 and a cost per test of €10.

The VITISENS system consists of two parts:

 A plastic cartridge (B) where the sample will be injected and which includes the syringes with the required reagents, the reservoir where the amplification and detection will take place and a waste reservoir. This is a single closed disposable system which prevents the problem of sample contamination.
 A handheld device (A) which contains a simple heating system and a fluorescence reading equipment. The amplification reservoir is cube shaped so that excitation and emission come through two different sides, with the heating system placed at the third side of the cube.

Scientific and Technological objectives of VITISENS project

Scientific objectives: To fill the gaps in the current state-of-the-art in FD detection methods while improving their specificity and sensitivity through the combination of molecular methods and fluorescence detection technologies, as well as to innovate far beyond the current laboratory applications of the Loop Mediated Nucleic Acid Amplification technology (LAMP) through its optimization and application for the early detection of the Flavescence dorée phytoplasma.

Technological objectives: To develop a homogeneous, simple, rapid, accurate, hand-held and cost-effective diagnostic tool for detecting the Flavescence dorée phytoplasma based on the LAMP technology, with a cost below €1,000 per unit and €10 per test.

These main objectives are achieved by integrating, combining and building upon the scientific and technological know-how and experience of the project partners. The following measurable specific scientific and technical sub-objectives will need to be fulfilled:

Scientific sub-objectives (defined to support the technological development):

1. To fix details regarding technical, biological and design specifications of the VITISENS system in order to adapt it as much as possible to end-user requirements, such as ease of operation, measurement time, cost per test and cost of the device, etc.
2. To select the most appropriate plant material (e.g. leaves, berries, root tissue), quantity and season for sampling as well as sample processing method (e.g. roller, crystal beads) to ensure maximum test sensitivity (by optimizing the efficiency of the DNA extraction) and minimize cross-reactivity risks as well as to establish a sample treatment protocol including the testing frequency required for preventing the spread of the disease.
3. To select the ideal silica matrix (e.g. silica beads or silica membranes) to be integrated in the disposable cartridge for immobilising and purifying the sample DNA with maximum efficiency.
4. To define the extraction buffer in which the sample will be injected into the disposable cartridge to allow an optimum DNA adsorption on the silica matrix and its volume. To define also the rinsing buffer, the elution buffer to be used for desorbing the DNA from the silica matrix (if required) and their volumes and ensure that none of the buffer components inhibit the enzymes used for the amplification reaction.
5. To select the target sequences for the specific amplification of FD phytoplasma DNA in order to avoid cross reactivity with other pathogens or sample components and to design the primers that will start the amplification of such target sequences.
6. To optimize for selectivity and end point sensitivity the critical parameters of the LAMP assay protocol such as magnesium, betaine, primer, nucleotides (dNTP) and enzyme (BST I) concentrations.

Technological sub-objectives:

1. To deliver a simple sample treatment kit adapted to field conditions for sample processing (e.g. roller, crystal beads) with a price below €1. The sample obtained must be filtered and ready to be injected in the disposable plastic cartridge by means of syringe 1 ensuring maximum test sensitivity (by optimizing the efficiency of the DNA extraction) and minimal cross-reactivity risks
2. To design and construct a low cost handheld reader suitable for use in field conditions (e.g. water-proof and protected against dust) which includes a simple heating system and a highly sensitive detection system based on fluorescence able to reach the 65ºC required for LAMP amplification in less than 1min. and to display the results in 20 min. It must also take into account the end-user requirements such as price, ease of use and robustness. Power autonomy must be of at least 12 hours with a Li-ion rechargeable battery
3. To develop a software package that will allow the exchange and management of the data stored in the portable reader (e.g. through USB or Bluetooth interface) and a firmware that will allow the system control with an intuitive user-interface through the display / touch screen.
4. To design and construct a disposable cartridge which integrates the silica matrix, the waste reservoir and the sample cell where the amplification reaction and fluorescence measurement will take place when introduced in the handheld reader as well as the tubing / microfluidic system for the injection of the sample (after being processed with the method developed by NIB under WP2), the buffers (defined by BIOENG in WP3) and the LAMP reagents (defined and developed by FERA under WP4). The design must take into account end-user requirements, and will ensure improvement on current available techniques in terms of speed, accuracy, sensitivity, specificity, price and risk of contamination
5. To perform exhaustive tests both in the laboratory (with samples of known pathogen concentration) and in the field (with samples of unknown pathogen concentration) to ensure optimum system functionality, to prove that the VITISENS system can compete with current detection methods and to demonstrate that VITISENS device fulfils the needs of the end-user SMEs.

Dissemination, training and exploitation objectives as well as management objectives are also highly relevant to this project. To that end, complete extra objectives have been foreseen:

1. To carry out training activities in order to facilitate the take-up of the project results by the consortium.
2. To disseminate the results and the foreground resulting from the project to the members of the consortium and beyond to a wider audience to maximize the project impact.
3. To protect the Intellectual Property Rights and to promote the exploitation of the foreground generated during the project to the greatest possible advantage for the SMEs.
4. To optimise the use of resources and to ensure that all aspects of the EC requirements for communication and reporting are met.

Project Results:

The project results per WP can be summarized as follows:

In the scope of WP1 a market study was carried out and the end-user requirements identified. In line with these requirements, the system specifications were defined. The market survey was performed by means of a questionnaire prepared by the SMEs with the support of the RTDs. The questionnaire was translated and distributed by each of the SMEs in the consortium to gather information within their own countries among their customers or any other end-user who might be interested in the new diagnostic tool. The main points highlighted are the following:

- 56% of the end-users questionned are based in a region where FD is present
- France and Italy (80% and 60% respectively) are the most affected countries and as a consequence most part of the answers come from those countries (from the total of 48, 23 come from France and 12 from Italy).
- Despite the low level of answers, the similar distribution between end users actually performing tests (44%) and the end users who don’t perform tests (56%) is interesting for the analysis of the questionnaire. When analizing these results per country it can also be observed that the most affected countries are also more commited on performing tests.
- It is quite difficult to recover information related to the amount of tests performed a year, as it depends on the country and on the region. As FD fight is managed by governmental authorities, they are the best ones to give global information regarding price of FD analysis (e.g. 5000 FD analysis have been performed in France in 2010)
- Regarding the time needed to obtain the results after having sent their samples to a dedicated laboratory it was observed that most of times it takes at least 2 weeks and due to the impact of FD disease this time period it is not quick enough
- On average, a FD tests cost 30 euros, but most of the times this is funded by public organisms. VITISENS device could be proposed up to 15 euros
- Regarding the interest on a new detection tool, 65% of the end users stated that it would be necessary to have a quick, cost effective and reliable field diagnostic test for Flavescence dorée. This is especially important for the affected countries. Moreover, the end-users in Germany, where the FD is not an issue at least for the moment, showed their interest in having another detection tool for the other important phytoplasma of greater concern for the vineyards, Bois Noir.
- Another important point was “where the end users want to use the new diagnostic tool” in order to evaluate some characteristics relatives to the portability of the device. Half of the questioned end users answered directly “in the field” when it’s only for quick symptom confirmation, and the other part in the laboratory when involved in a prospection campaign.
- The most important features of the system are: Accuracy, Easyness to use, Quickness, and robustness.

The information gathered was also used to define the technical specifications of the VITISENS system. This allowed a better adaptation of the system to the requirements and demands of the implied industrial sector, ensuring they are fully met, taking always into account the technical feasibility and the experience of project partners.

WP2 was focused on the definition of the sample collection and processing protocols for Flavescence doreé phytoplasma (FDp) detection and design the protocols for sample processing suitable for performing in field.

The sampling procedure is one of the most crucial steps in the process of pathogen detection and identification. It is advisable that the tissue is taken from at least three different shoots of the plant. In the case of testing leaf veins 1 g of material is needed. This is approximately two veins from 5 leaves. When testing berries, at least two, but if possible more, berries should be tested for a reliable detection of FDp DNA in a given berry cluster. Regarding the seasonal influence on phytoplasma titre, it was found out that FDp titre was higher in August tissues compared to tissues sampled in June, except in the cases of the remission of the infection. FDp was detected in all tissues on the symptomatic shoots, which confirms the system but uneven distribution of FD in grapevine.

On the other hand, two most suitable processing methods for grapevine leaf veins and berries were established. The first was the manual homogenisation with tubes filled with beads and sand. This approach is easy and can be done in-field, without any electrical equipment, and is applicable for homogenisation of leaf veins, berries and flowers. Ultra-Turrax Tube Drive (UTTD, IKA) assisted homogenisation was the second selected approach and can be used in small on-site laboratories. The device is small and affordable and results in more uniform homogenisation compared to manual shaking.

For the case of latent infection detection, it was found out that early in the season, before the appearance of symptoms on the leaves, the grapevine flowers were a good source of tissue showing the infection state of the plant. The titre of the phytoplasmas in the flowers was high enough for reliable detection also with on-site approach.

WP3 was focused on the development of a DNA extraction method. Different extraction buffers and silica based-DNA extraction methods suitable to be incorporated into the VITISENS handheld device for allowing LAMP amplification were tested. Flavescence dorée phytoplasma could be detected by PCR in “standard” DNA extracted and purified samples, however, when anion exchange column and silica beads were used to optimize DNA extraction for in-field conditions as an alternative to the “standard” method, no DNA was obtained with any buffer. Thus, in search of an alternative method for a suitable FD detection in field-conditions an additional task was included, using crude extracts directly to PCR. Plant crude extracts (leaves and fruits) seems to require a delicate compromise between getting the highest possible presence of FD DNA while keeping low the level of contaminants. The extraction buffer to be used with crude extract has been selected and optimized at the end of this work package together with the LAMP primers developed in WP4. The results showed that the FD detection is possible in infected material from crude grape in different dilutions. Moreover, it was possible to detect plant gen using COX as positive control of the assay.

The main objective of WP4 was to develop and optimize a LAMP amplification method for the specific detection of Flavescence doreé (FD) phyotplasma DNA from infected grapevine samples, suitable for incorporation into the handheld reader. A range of genes commonly used in the detection and characterisation of phytoplasmas were selected as targets for the development of LAMP assays. Four genes were identified where DNA sequences were available for a range of phytoplasma species, allowing the application of primer design criteria to develop specific assays for FD. Between one and three assays were designed to each gene to allow for variance in performance characteristics. Initial evaluation of each assay was performed to determine the specificity against a range of different phytoplasma species, and the speed of amplification. Following this evaluation five assays were found to be specific to 16SrV phytoplasmas and the quickest three assays (with rapid amplification times of less than 21 minutes) were selected for further validation. These assays were assessed against a wide range of non-target species including; other phytoplasma species, bacterial and fungal agents commonly causing grapevine diseases, a range of grapevine endophytes, field infected FD plant samples, healthy and infected alternate FD host plant species, a wide range of healthy grapevine varieties and two species of infected and healthy insect vector. Analytical sensitivity was evaluated using serial dilutions of FD DNA, and diagnostic sensitivity by dual testing of samples with a range of FD concentrations by LAMP and the laboratory based gold-standard method of real-time PCR. Additionally optimisation of the assays to maximise performance in terms of speed of amplification was undertaken by assessment of a range of amplification temperatures. Based on the results of all of these appraisals an assay designed to the 23S rRNA was selected as having the best overall performance characteristics and meeting the work package objective. This assay did not show any cross reaction with any of the specificity samples, including the phytoplasma Bois Noir (16Sr XII) which commonly causes disease in grapevines and is indistinguishable based upon symptoms. This also revealed the LAMP assay has a very high sensitivity. The selected assay was validated to EPPO standards. Additional work was undertaken to assess the viability of lyophilisation of LAMP reagents, with testing performed using three assays, however lyophilisation caused the complete failure of the LAMP reaction, most likely due to lyophilisation destroying the functionality of the enzyme, therefore LAMP primers must be used in liquid form.

WP5 was focused on the development of the two main modules of the portable VITISENS reader, fluorescence detection unit, temperature control module and detection instrumentation. The optical system consisted on a solid state light source (LED) for excitation of fluorescence on the sample, the optical set-up needed to focus the excitation light onto the sample and collect the fluorescence emitted from the excited sample and the fluorescence detector. All components were carefully selected from commercially available components and evaluated to ensure maximum signal, low noise and maximum sensitivity. The temperature control system includes a power load resistance to be used as a thermal source in contact with one of the faces of sample cell, a precision temperature sensor is on the opposite face of the sample cell, a security device to cut off the power on the load resistance in case of over-temperature detection and a digital control system (e.g. PID control strategy) to adjust the temperature of the heater to the set-point. When developing the detection instrumentation first an electronic schematic design was developed including the following elements: power management and Li-IO Battery charge system, optical system, thermal system, communications system, user interface to configure the system and to display the results, non volatile memory and Time stamp systems and a microcontroller unit which will control the whole system. When the electronic schematic design was finished, all components were assembled in a printed circuit board (PCB). The hardware was then tested and all the functional blocks were adjusted and optimized. The next step was to program the microcontroller to create an operative firmware. The firmware has been exhaustively tested to ensure the correct operation of the device and all of their functions, to guarantee a bug free firmware.

The disposable cartridge and portable reader were designed and constructed under WP6 integrating and adapting all the technologies developed in previous work packages. The design of cartridge and reader took into account the outcome of the market study carried out in WP1. Initially two cartridge designs were done for lyophilized LAMP reagents and with a reaction cell volume of 25uL as it was defined in previous WPs. However, when cartridges were first tested with lyophilized LAMP reagents, it was found out that the lyophilization process inhibited the LAMP reaction. Therefore, a new cartridge design for liquid LAMP reagents had to be done. Over the last months of the project the new cartridges were design and manufactured (see figure given in the annex to this report). At the same time, VITISENS reader was designed and constructed based on the optical configuration, detection instrumentation and shape of the disposable cartridge (picture is shown in the annex). A specific firmware was programmed to include the functionalities and allow the system control with an intuitive user interface through the display. The functionality of the handheld prototype has been tested and the calibration of the fluorimeter was carried out defining the dynamic range of pathogen concentrations required to set up the device calibration curves. All calibration curves have been done with NIST-traceable fluorescein standard. The same fluorescein dilutions were tested with the PCR instrument and Genie II instrument (used by NIB during validation phase) to compare the results. The VITISENS reader showed slightly better limits of detection and quantification than the Genie II. Moreover, the reader´s performance was tested with plant gene DNA and COX master mix (fluorescence before and after the LAMP reaction was measured) given positive results which indicates that the reader is able to detect differences in fluorescence signal before and after the amplification procedure. The heating performance was also checked using a temperature probe inserted in a VITISENS cartridge and connected to a thermometer. Optimization of the VITISENS reader in terms of firmware, sensitivity, etc was done together with the next work package (WP7).

WP7 was focused on VITISENS system validation for FD phytoplasma detection in the laboratory and also in the field. The LAMP assay was first validated by testing infected samples with determined pathogen titres, healthy samples and samples confirmed to be infected with other grapevine pathogens from the in-house collections. The LAMP assay was shown to circumvent the important problem of qPCR, which are the inhibitory effects of plant material (e.g. phenols), accordingly it can be applied to crude plant homogenate as it was described previously in WP3. The sensitivity of the LAMP assay optimized for in-field testing (plant material homogenization and crude extract testing) was shown to be similarly sensitive as classical in-laboratory method. Moreover, the LAMP assay was shown to be specific for FDp and it detects all three different FD types (FD70, FD-C and FD-D), as it is described in WP4. By testing healthy grapevine plants of various cultivars and samples of other phytoplasma hosts, 100 % diagnostic specificity was determined. The LAMP reactions can be repeated several times and on different devices, at different times and by different operators, giving the same result. In-filed procedure was also validated by testing real samples of unknown phytoplasma titre and the results were compared to the results from qPCR. The in-field procedure was shown to be applicable for testing fresh grapevine leaf veins and berries as well as specific, since no cross-reactivity with extracts from healthy plants was observed.

The use and performance of the VITISENS system was also evaluated by testing samples with known titre of the phytoplasma DNA. VITISENS system was found to be user friendly and can be used in-field. The loading of the reaction mix and the sample to the cartridge with single use pipettes is simple and the operation with the reader is intuitive. Grapevine extracts were also used for validation of the VITISENS system. The initial results showed a leaking problem with the cartridges that was later solved in July 2013. Nevertheless, the latest results showed a decrease in the fluorescence detected by the reader after the amplification reactions takes place; therefore further tests would be needed. However, on the other hand, it has been demonstrated that the reader detects fluorescence and the heating instrumentation works as expected as it was previously described in WP6.

Training and Dissemination activities were carried out in the scope of WP8. Main dissemination activities included the maintenance of the project website up-dated and dissemination of the project and its results in several events where the partners assisted as well as publications in different media (specialized magazines, general press, TV broadcaster, scientific papers, etc). These events were not only used to disseminate the project but also to prepare the future exploitation of the VITISENS system. In terms of training, two training sessions took placed during the project lifetime. In these events, the RTD performers supported by training material especially design for these events presented the complete VITISENS system and how to use it. VITISENs system includes the sample collection and preparation protocols, sample introduction to the disposable cartridge and use of the VITISENS reader to perform a test.

The main aim of WP9 was to facilitate the take-up of the project results by the SMEs by ensuring an adequate transfer of the results, promoting foreground protection by IPR as well as prepare the future exploitation of the VITISENS project generated foreground. To this end a constant market watch and technology watch was done over the project duration, knowledge management seminars were carried out.

Potential Impact:

The main objective of VITISENS project is to develop a cost-effective handheld device for rapid in-field detection of Flavescence doreé phytoplasma in grapevines, based on FD DNA extraction and isothermal DNA amplification by means of the innovative LAMP technology performed in a portable hand-held reader designed for non-technical staff.

There are currently no commercially available devices integrating the DNA extraction, amplification and detection steps in a single closed disposable system, which makes this solution a challenge considered feasible by the VITISENS consortium, given the current state of the art. There are accurate but expensive methods to detect FD in the laboratory therefore they are only used to confirm visual inspection of vine symptoms. Moreover, laboratory results are obtained long after the sampling. If during this time infected vineyards are not destroyed or, even worse, are used as grafting material, the epidemic is also being actively spread by man.

The proposed diagnostic tool has the potential to significantly reduce the impact of the FD and maintain a competitive edge in the global market thanks to an accurate, fast and easy detection of FD. In fact, VITISENS will have a positive impact of the sector as a whole, supporting longer term European competitiveness as a knowledge based economy facing new challenges and opportunities in the worldwide scenario.

The end-user of the VITISENS generated foreground will have the following benefits:

- Avoid production losses as well as reduce time and costs, increasing the profit margins
- Reduce use of pesticides against the vector, and in some cases not yet affected by the disease, guarantee the continued FD-free status.
- Maintain the employment
- Gain knowledge in protective measures to avoid the potential loss of priceless genetics, while becoming less pollutant will afford them clear strategic advantage in marketing their products as “environmentally friendly”.
- Nurseries can radically improve the best practices in plant health control methods allowing them to guarantee the value-added FD-free nature of their veins, increasing consumer confidence and attracting new customers.

The led user’s benefits are:

- Consortium lead users can open a new market niche for handheld phytoplasma detectors, increasing their sales.
- The technology generated might also have future potential in being adapted to detect other pathogens and human single nucleotide polymorphism disorders.

Contributions to Regulations and Standards:

One of the main objectives of the new EU Council Regulation (No 479/2008) is to improve production by means of new technologies. VITISENS device will make a positive contribution to the stated objective in the specific area of plant health in collaboration with the European and Mediterranean Plant Protection Organization (EPPO).

On the other hand, FD is listed in the EU2000/29 Council Directive on Harmful organisms considered relevant for the entire Community, as well as in the EPPO A2 quarantine list of pests due to the large potential economic impact of this disease. A certification scheme for grapevines was developed by the EPPO in 1994 and continues to rely on visual inspection as its primary diagnostic method . The procedures to be developed in this proposal could contribute a simple and convenient standardised methodology for advancing EPPO FD detection schemes by means of the VITISENS device. Therefore VITISENS will work to match with the EPPO procedure requirements to obtain the certification during the validation phase of VITISENS.

The project could be used as a platform for the development of best practice guidelines for SMEs in this area, as well as for the creation of new standards for other on-line measurement and control activities. This will ensure standardization in the area of the control of plant pathogen detection throughout Europe.

The use of this technology will also help winegrowers to implement the best available techniques required by the EU, and will also contribute to improve the food safety standards through the pesticide use reduction, which are of concern to consumers. As the technology will allow determining accurately whether a certain vineyard is infected or not, the indiscriminate use of pesticides will be avoided. This will contribute to the European efforts in its global leadership as a driving force for pushing responsible use of pesticides and therefore shift towards an environmentally friendly but still economically competitive use of these substances, meeting at the same time consumer expectations regarding product quality and addressing their health concerns.

The VITISENS project has widespread social impact in the areas of:

• Rural development: Rural areas, representing more than 90% of EU territory and containing more than half of the EU population, face challenges related to growth, jobs and sustainability. VITISENS will facilitate the implementation of recent European rural development programmes and agro-environment schemes that address environmental problems of the sector by encouraging improved and more environmentally friendly procedures.
• Environmental protection and sustainable production: As a complementary means of FD control, pesticide treatments against the vector Scaphoideus titanus are mandatory in many European countries where it is present, even if the disease is not yet established. Apart from the environmental impact and the side-effects on ecosystem components, these measures impede efforts to minimise pesticide use as advised in all national agro-environmental programs. In addition, none of the pesticides used are authorised for sustainable integrated or organic production systems, meaning organic farmers will benefit from better control of FD by limiting blanket use of pesticides which precludes organic certification.
• Promotion of employment: The VITISENS technology is cost-effective and user friendly and allows to increase the knowledge base of winegrowers in these regions while providing them a tool to preserve and help expand their business, thereby supporting employment.
• National and transnational authorities will benefit from this technology when monitoring the evolution and occurrence of the disease. Regular monitoring and surveillance programs of vineyards and mother plants for grafting have become mandatory in several European countries, with significant associated public costs and social damage.
• Health and safety to consumers and EU citizens in general will benefit from reduced need to use pesticides, which will have a significant impact on the health of EU citizens.
• Knowledge and quality of life: The ability to maintain healthy vineyards will improve wine production and the revenues of winegrowers as well as reduce the time required to manage disease problems contributing to the quality of life of the end-users.

The objective of the dissemination activities of the VITISENS was to assure that its results and other non-confidential information (knowledge) would arrive to a wide and relevant audience in order to extend its impact.

Several dissemination actions were carried out during the 28 months of VITISENS, in which non confidential information covering a general project overview as well as the general project objectives and applications of the technology was presented not only to the wine sector but also to a wider audience. The target audiences were divided as potential customers of the technology, the general public and the scientific community in order to adapt the dissemination messages to their expectations and increase the dissemination success but always maintaining coherence and consistency between those messages. One of the main vehicles to share information about VITISENS project was through the official project website, available is different languages (English, German, Italian and French) and up-dated periodically. Moreover, a standardized project presentation was prepared to be used in future dissemination events by the SMEs.

Preparing the VITISENS technology for exploitation and future commercialisation was a must in this project. Since the beginning of the project, the Consortium did a valuable effort to boost the pre-competitive prototype from the VITISENS Project towards a final marketable device. The exploitation strategy has been planned in accordance with the interests of the SMEs, including background management, licences, protection of confidential information and enforcement of copyright has started early in the Project’s life.

Knowledge management through establishment of Intellectual Property Rights (IPR) has been held by the Exploitation Manager, Olivier Zekri (MERCIER). RTD performers have also participated in the IPR and have given advice on exploitation work, but only as observers.

As per the initial agreement between the consortium members, the SME partners will jointly own the Foreground generated during the project in equal shares. The terms and conditions for managing and exploiting the joint Foreground have already been discussed. The future joint ownership agreement will implement the exploitation plans as well as other aspects of the joint ownership.

A list of exploitable knowledge, exploitable products, sector of application and time for commercial use, way of protection and partners involved have been created. So far, no patents relevant to the project goals were found, excepting the use of the LAMP technology patented by the Japanese company Eiken Chemical Co. The research work to be carried out under the project on LAMP is covered by the research exemption principle and thus will not infringe any patent rights. However, the intention of the partners is to use this technology within a new commercial solution, therefore, a license will be needed for the commercial use of LAMP. Partners FORSITE and FERA have already studied the possible options of commercialising VITISENS through an English company which has already negotiate a licence with the Japanese company Eiken Chemical Co. To sell diagnostic kits based on LAMP. Further negotiations with the English company are still required.

A project logo (see illustration in the annex) was created and used in all the dissemination material prepared and since the name “VITISENS” is not used by other companies, the SMEs applied for a Community trade mark to identify the technology on the market which will allow the SMEs to rapidly and effectively protect their commercial sign if desired.

For protecting the other technical aspects of the technology the trade secret will be used. According to most national legislations in the EU, trade secret protection applies to information which has a specific commercial value, e.g. the VITISENS technology and the know-how for its operation.

List of Websites:
www.vitisens.eu

Final Report Summary - VITISENS (COST-EFFECTIVE HAND-HELD DEVICE FOR RAPID IN-FIELD DETECTION OF FLAVENSCENCE DOREE PHYTOPLASMA IN GRAPEVINES)

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