Final Report Summary - VERTIGEEN (INEXPENSIVE AND RELIABLE ON-SITE SOLUTION FOR OLIVE PRODUCERS TO CONTAIN VERTICILLIUM WILT)
Executive Summary:
The European Union leads the global olive oil sector, producing 80% and consuming 70% of the average total world output of over 2.5 million tonnes that guarantee an income of €5 billion per year from olive oil plus another €600 million in table olives. Olive production is distributed into a large number of small and medium enterprises with low profit margins, thus particularly vulnerable to drops in productivity. The competitiveness of EU olive lead market is being currently threatened by the soil-borne fungus Verticillium dahliae responsible for critical damage to the vitality, productivity and yield of more than 200 plants species with crop losses estimated at thousands of millions of dollars worldwide every year, as well as by growing international competition from Tunisia, Turkey, Syria, Morocco and Argentina and steadily olive oil prices drops. In recent years, verticillium wilt is occurring with increasing frequency and severity in most olive growing areas of the Mediterranean basin. Main factors associated with this increase are intensive farming, use of infected but symptomless plant propagation material, planting in infected soils and distribution of the pathogen by wind, irrigation as well as run-off water and the use of contaminated equipment.
VERTIGEEN proposes an innovative, rapid and reliable on-site detection and quantification system of the Verticillium dahliae fungus in soil and plant samples, by the integration and application of new but successfully demonstrated technologies for DNA amplification and electrochemical detection, overcoming the limitations of the state of the art in terms of heterogeneity, portability, price, ease of use and time-to-result.
With an estimated commercial price of €1,000 per device and €10 per test, the system will allow associated olive producers to reduce their losses caused by this pathogen and its spread by means of precise and efficient field interventions within an Integrated Pest Management (IPM) Strategy.
By means of EU-wide strategically planned dissemination actions and highly developed exploitation processes, VERTIGEEN will maximize the competitive benefits for olive growers, olive oil producers and olive tree nurseries SMEs and SME-AGs in all member States.
The consortium provides 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.
Project Context and Objectives:
Olive production is distributed into a large number of small and medium enterprises with low profit margins, thus particularly vulnerable to drops in productivity. The five main EU olive producers -Spain, Italy, Greece, Portugal and France- have more than 11 thousand olive sector SMEs , most of which are members of professional SME Associations (SME-AGs) such as the SME-AG proposers INOLEO, EDOEE and AAR. These play a direct role in the production and distribution of the oil made from their associate’s olives and losses in associate productivity affects their business directly.
The competitiveness of the European olive lead market, already faced with growing international competition from Tunisia, Turkey, Syria, Morocco and Argentina and other adverse factors such as olive oil prices dropping steadily , is currently severely threatened by the soil-borne fungus Verticillium dahliae, responsible for critical damage to the vitality, productivity and yield of more than 200 plant species with crop losses estimated at thousands of millions of dollars worldwide every year. The nature of the infections and the characteristics of the pathogen -a soil habitat, survival structures that persist for years, demonstrated transmission potential and capacity to infect numerous hosts in a continually expanding geographical distribution worldwide, has made Verticillium wilt a chronic economic problem in global crop production. Non-defoliating and defoliating pathotypes of Verticillium dahliae vary in aggressiveness but overall, for commercial olive plantations, this disease is currently considered one of the most serious problems with reported incidences of 30% in some regions of Spain while other studies report olive crop losses ranging from 50 to 89%, depending on olive variety and pathogen aggressiveness .
Currently, there is no single effective treatment for plants affected by Verticillium dahliae, meaning that efficient control of this disease requires an Integrated Pest Management Strategy based on both (a) preventive and (b) eradication measures:
a) Diseased trees are constantly being replaced and new olive groves are continually being planted, making pathogen detection for the choice of verticillium-free planting soil and planting material a critical key point for the integrated preventive management of verticillium wilt in newly established groves. In addition, regular monitoring of the infections in established groves would allow individual infected trees and surroundings to be quickly spot-treated or grubbed out, preventing pathogen spread to the entire grove and eradication of healthy productive olive trees. This would result in improved productivity and cost savings from the investment in the production of healthy olive trees.
b) To protect productive trees in established groves or to disinfect soil before planting, pathogen eradication methods are required. The March 2010 ban on methyl bromide in the EU as a soil fumigant , which was the most effective and widespread method for eliminating this pathogen, due to its considerable side-effects on public health and environment, threatens to drastically increase the incidence of wilt disease and significantly impact olive farmers. Other chemical treatments include Metamsodium, Dazomet or Cholopicin, but these should always be employed at minimum doses in clearly defined areas (focused treatment) where the pathogen has been positively detected and accurately quantified. With timely quantitative analyses the most appropriate treatment can be accurately prescribed based on actual pathogen populations, reducing costs and environmental impact of blanket pesticide use achieving sustainable agriculture practices.
Alternative control methods to reduce the initial inoculum densities in soil, such as crop rotation, irrigation management, soil solarisation or biological control agents, are available but are costly and time consuming, and therefore only suitable for application at small scale on well-defined areas with low density of inoculum. The lack of rapid quantification methods to identify these areas is the main reason why they have not yet been successfully applied in practice4. Attempts have also been made to breed for disease resistance against Verticillium in different crops, but so far resistant clones have only been successfully achieved for tomatoes5.
Moreover, reliable quantification would allow growers to estimate the risk of disease development, and based on risk levels to quickly decide upon the nature and extent of the treatment and to quickly intervene in the orchard, limiting the impact of the disease on their olive groves, increasing crop yield and reducing productivity losses.
Therefore, the development of a new, cost-effective, reliable system for early on-site detection and quantification of Verticillium dahliae such as VERTIGEEN is clearly needed as a first step in the integrated management of verticillium wilt, the only way to effectively fight this disease and minimize the losses caused by this pathogen and its spread. A guide for best practices has been also developed covering methods and timing of sampling for detection and screening as well as intervention protocols.
Scientific Objectives
The overall objective of the research described is to design and develop a method for the early detection and quantification of Verticillium dahliae in soil and in plant using and applying isothermal LAMP DNA amplification coupled to electrochemical detection for routine diagnosis of the Verticillium wilt disease. With this purpose the following measurable specific sub-objectives were defined:
1) A sound sampling strategy for both plants and soil in order to have significant and representative results covering the extension of an average olive tree grove. The most appropriate location (spatial distribution), sampling pattern and scale, season for sampling, sample size and material (e.g. leaves, root tissue, soil aggregates) were selected.
2) A sample processing method for both plants and soil to guarantee maximum test sensitivity (by ensuring maximum efficiency of Verticillium DNA extraction and purification) eliminating potential LAMP inhibitors. The sample obtained must be ready to be introduced in the VERTIGEEN self contained disposable amplification and detection tubes.
Based on these results, a sampling protocol including the optimum testing frequency was established.
The LAMP assay was developed, adapted and optimized for the specific detection of Verticillium dahliae by selecting target DNA sequences for amplification and designing the primers that will start the amplification of such specific sequences. This avoids cross reactivity with other pathogens or sample components.
3) 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 as well as the concentration of the detection reagent (Methylene Blue) were determined.
4) Inoculum levels will be correlated to disease risk levels in olive orchards (no risk, low risk or high risk) with the aim of establishing field intervention protocols for the grower to follow in each case. These have been compiled in a guide of best practices.
Technological objectives:
To develop a homogeneous, simple, rapid, accurate, sensitive, on site electrochemical diagnostic tool for detecting Verticillium dahliae and quantifying infection levels with a cost below €1,000 per unit and €10 per test. The following measurable specific sub-objectives were defined:
1) To fix details regarding technical, biological and design specifications of the VERTIGEEN system in order to adapt it as much as possible to end-user requirements, such as ease of use, measurement time, cost per test, cost of the device, etc.
2) To deliver a simple sample treatment kit adapted to field conditions for sample processing (e.g. roller, crystal beads) with a price below €1.
3) To design and construct a disposable reaction tube where the sample is introduced after being processed with the methods developed for soil and for plant samples, which contains the LAMP and detection reagents, and where the electrochemical measurement will take place once the electrodes connected to the reader are inserted
4) To design and construct the equipment device where the disposable tubes will be inserted containing a simple heating block that reaches the 65ºC required for the LAMP reaction to start in less than 1min. and a simple and inexpensive highly-sensitive multi-channel potentiostat that will allow the quantification of the DNA amplification products by means of the electroactive DNA intercalator Methylene blue. The device provides the results in less than 30 minutes. This reader includes all instrumentation necessary to generate, acquire and process the signal. It must also take into account the end-user requirements such as price, ease of use and robustness.
5) To develop a software package that allows the exchange and management of the data stored in the reader through USB and a firmware that allows the system control with an intuitive user-interface through the display. The management software and a database would be developed to record and strategically establish risk prediction levels based on which indications to the grower will be made.
6) Ensure that each of the components of VERTIGEEN work properly once integrated. 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.
7) 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 VERTIGEEN system can compete with current detection methods in terms of efficacy, specificity, sensitivity and detection limit and to demonstrate that VERTIGEEN 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:
1) To carry out training activities in order to facilitate the take-up of the project results by consortium SME-AGs and SMEs. A detailed plan will be prepared to train the AG members on how to take advantage of the readings of the system
2) To disseminate non-confidential information about the project and its results to a wide and relevant audience in order to maximize the project impact.
3) To promote the exploitation of the foreground generated during the project to the greatest possible advantage for the participating SME-AGs and SMEs
4) To optimize the use of resources and to ensure that all aspects of the EC requirements for communication and reporting are met.
Project Results:
The work carried out along the project was divided into several work packages and activities, R&D, DEMO, OTHER and Management.
WP1: Definition of System Specifications
The consortium successfully completed the market survey. The market survey was performed by means of a questionnaire which compiled the most interesting questions drawn up by each of the project SMEs in English. The final questionnaire was translated to the project SME-AGs languages (Greek, Portuguese and Spanish) as well as Italian; and distributed by each of them within their countries to end-users or any potential customer of the new diagnostic tool. The questionnaire covered 3 main topics: general information about the end user or potential customer, VD infection knowledge and management and interest in a new VD on site detection device. Several dissemination strategies have been used: massive emailing, interviews personally or by phone. Over 500 end-users (farmers, olive oil producers, organizations, etc.) were sought between the 1st of April and the 30th of June 2012. A total of 127 filled questionnaires have been collected, 13 from Spain, 50 from Italy, 9 from Greece and 55 from Portugal. During the General Meeting M6 in York, it was decided to continue this task in order to have more reliable data on the end-users needs and expectations. Over the last months SME-AGs supported by the SMEs in the consortium have collected 37 questionnaires more including 7 laboratories from Spain, making a total of 149 end-users and 7 laboratories. The main points highlighted are the following:
- Most common range of plantation area is up to 20Ha. Fertilizers are used by 56% of the end users, while only 44% uses herbicides. 92% of the end-users harvest every year, 45% make tillage on the soil and only 34% incorporate organic matter to the soil.
- 59% of the end users have had some kind of pest or disease in the last three years and 64% are aware of the VD presence.
- Over 72% of the end users detect the presence of VD based on visual detection methods, and only 9% perform lab analysis. The average time needed to get the results is between 2 and 3 weeks, considered not quick enough by most of the questioned end-users.
- On average, a testing for Verticillium costs 20-50€, but in most of the cases this is funded by public organization.
- Regarding the interest in a new detection tool, over 46% of them stated that it would be necessary to have a quick, cost effective and reliable field diagnosis tests for V. dahliae.
- Another important point was “where the end-user want to use the new diagnostic tool” in order to evaluate some characteristics relative to portability of the device. The questionnaires indicate that 52% of the end users answered directly “in the field” and 17% “in the office or laboratory” or similar facilities.
The most important features of the system are: easy to use, accuracy, measure the degree of infection, quickness, portability and low price (below €1000).
The information gathered was also used to define the technical specifications of the VERTIGEEN system. This information allowed a better adaptation of the system to the end user requirements and demands of the implied sector, ensuring that they are fully met, taking into account the technical feasibility and the experience of the project partners.
WP2: Development of a soil sampling protocol
The aim of this work package was to develop on site DNA extraction methods. In addition, the seasonal aspect of sampling was investigated as well as the most suitable sampling strategy. This included determining the number of subsamples as well as where specifically to take the sample (sample depth and where in relation to the olive tree).
Task 2.1: Selection of suitable soil material and seasonal timing for sampling.
In total, 386 field samples from olive fields and one cotton field in Italy, Greece, Spain and Portugal were taken. Of these samples, 30% were found to be positive. An interesting result from this screen of soil samples was the presence of very high levels of Verticillium dahliae in a cotton field adjacent to an olive field (which was largely negative for Verticillium). This showed the risk from crops such as cotton as an alternative host for the pathogen. This exercise also provided a range of additional soil samples to validate the LAMP assay on.
On the basis of these results, five sites were selected for further in-depth analysis to determine the seasonal timing for sampling and also inform the sampling strategy. This included three sites in Spain and two sites in Portugal. In the case of the Spanish sites, they were sampled twice, once in spring, prior to the summer warm period and once at the end of August. The two sites in Portugal were sampled four times. In each instance, both before and after the summer hot period over two consecutive years. This directly informed seasonal timing for sampling. On the whole relative, levels of abundance of the pathogen and relative incidence was greater before the summer hot period than after suggesting that sampling should be undertaken in spring not summer or autumn.
Task 2.2: Study of the most appropriate sampling pattern
From the initial range of soils that were taken Geostatistical analysis (Krigging) was undertaken at three sites and showed that the pathogen had a highly clustered distribution in soil. More in depth sampling was performed at one site in Portugal taking 103 samples over 1 ha and also taking corresponding disease assessments on the trees from those sampling points. Using indicator Krigging analysis the trees displaying severe symptoms were also present in areas where high level of V. dahliae was detected by qPCR. This showed good evidence that soil levels and symptoms can correspond in olive fields. The indicator Krigging analysis with 103 sampling points confirmed that V. dahliae had a much clustered distribution. However, the Krigging heat maps from this site were used to inform the sampling strategy. Since the pathogen had a highly clustered distribution in all sites sampled, a grid sampling strategy was essential for optimum detection. In addition, analysis of the heat maps showed that although 10 subsamples per ha is likely to detect the pathogen, 25 samples was decided on for practical reasons (i.e. they can form a 5 x 5 grid) and added increased robustness to the sampling strategy. This decision was taken in consultation with the other consortium partners.
The sites in Portugal were also used to inform other aspects of the sampling strategy. These included whether samples should be taken between rows of olive trees or at the tree bases. Tree bases were shown to have significantly higher levels of Verticillium. Therefore, different sampling depths at tree bases were tested to see if differences were present. After removing surface debris, different depths down to 35 cm were tested. Depths from 0 to 9 cm had highest levels of Verticillium on the whole so were selected for the final sampling strategy. This was also the easiest depth to sample.
The direction of the sampling point around the tree base was also tested to see if that affected levels of Verticillium in the soil. Eight sampling points were taken around a series of trees from two sites in Portugal and factors such as gradient, water flow, prevailing wind etc. were considered for each site. No discernible patterns were found and therefore any place around the tree base can be sampled.
Task 2.3: Selection of the most suitable in-field sample processing method
A range of DNA extraction methods were evaluated for potential in field use. These included spin filters, syringe filters and magnetic bead based purification. Magnetic beads showed the most practicality in addition to having a DNA recovery rate approaching laboratory based extraction methods. The practicality of this method was further enhanced by using it with a desktop mini centrifuge. The centrifuge could be purchased relatively cheaply and although added to the initial cost, reduced costs in the long term if large numbers of samples are processed. Using this method took 30 minutes compared to a laboratory scale extraction which can take up to 4 hours. Sensitivity (in terms of DNA recovery) was 10 – 20 times less than the lab extraction but speed and cost was substantially improved. Therefore lower sensitivity could be compensated by taking additional samples. The method had high potential for a successful in field extraction protocol.
WP3: Development of a plant sampling protocol
This WP is devoted to design the optimal plant sampling strategies and processing methods for being performed on-site. The work in this WP is divided into 3 tasks foreseen to be developed within the first 14 months of the project. The work was largely completed in the first project period with the main results included in D3.7 submitted in M14 and updated in M25.
Task 3.1: Selection of suitable plant material and seasonal timing for sampling.
A preliminary sampling protocol based on the literature review (task 3.2-1) was tested successfully during a sampling field trip in southern Spain after the 3-months Technical Meeting. Young branch samples were collected according to the protocol and gave good results when tested by the developed Q-PCR in the laboratory for the presence of Verticillium dahliae (Vd). Collection of root samples however appeared not feasible; and leaves, although collected easily, gave very low percentages of Vd detection. Therefore it was decided that the best sample type for detection of Vd in olive trees are parts from young branches. Examination of samples from individual branches within trees confirms the conclusion from literature that distribution of Vd within infected olive trees may be discontinuous. The percentage of Vd-positive samples per tree varies strongly, from 17% (1 out of 6) till 100% (6 out of 6). However, it was demonstrated that analyses of combined samples from 5 branches per tree gives very reliable results. Samples were collected from 157 olive during sampling sessions repeated at different parts of the year in the period 2012-2014 in Spain (5 times), Italy and Portugal (4 times each) and Greece (once). Vd was detected in samples from all sessions. However, the number of positive samples varied strongly with the year and period of sampling, which is in line with literature reports. Especially after hot summer periods the number of positive samples may be low. Taking into account the seasonality effect on results in different countries and the timing of agricultural practices, the best period for sampling of diseased olive trees for detection of V. dahliae is in late winter or early spring (before pruning of the trees).
Task 3.2: Study of the most appropriate sampling strategy
From a literature survey it was concluded that (a) several samples per tree should be examined because distribution of V. dahliae in diseased trees may be discontinuous; (b) Vd probably is most readily isolated from the wood of current year’s or previous year’s parts of branches in the lower and middle part of the crown; and (c) probably the best period for detection of Vd is in spring and early summer because especially in areas with relatively high summer temperatures the amount of the fungus in the aerial parts of diseased olive trees may vary with the season. The results for individual branch samples show that Q-PCR has a higher sensitivity for detection of Vd in olive samples than the standard plating assay. Moreover, pooling of samples from 5 branches per tree in one analysis resulted in positive test results for all Vd infected trees whereas results for samples from individual branches may vary. Additionally a dilution series showed that the Q-PCR can detect one infected sample in a mix with samples of at least up to 10 healthy samples.
Task 3.3: Selection of the most suitable in-field sample processing method
Q-PCR was used for detection of Vd in the development of sample processing methods and as a reference test in the evaluation of the LAMP PCR. Therefore a PCR soil assay method previously developed by PPO was adapted for use on plant samples. The PPO primers were shown to work well in olive samples and to have a very high specificity for Vd. Also this Q-PCR has a high sensitivity; the threshold level for detection of Vd in olive plant samples appeared to be 0.0001 ng. A laboratory isolation method based on Kingfisher DNA extraction was developed as a reference test for good quality DNA from Vd infected olive material. Also a preliminary protocol for a field method was designed including the use of lateral flow devices (LFD) for capture of DNA from crude plant extracts. The procedure of freeing and extracting DNA from woody samples in the field was shown to be effective for detection of Vd with Q-PCR. The first set of LAMP primers based on the EF sequence (one copy target) developed in WP4 became available in month 13. Preliminary tests of LAMP PCR in the laboratory showed that the assay works at 65 as well as 70˚C but at low concentrations of Vd no positive reactions are found with a threshold level of about 0.1 ng. Also the method of extraction of DNA from plant samples seems to affect the result, suggesting that for LAMP-based detection of Vd the amount and/or quality of the DNA needs to be improved. Therefore in period work was done on further optimization of the LAMP-assay for on-site detection of V. dahliae. A new set of LAMP primers developed by FERA and based on IGS or ITS sequences (multi copy target) was tested. This set increased sensitivity strongly (factor 100-1000) but the sensitivity of the LAMP assay still was less than that of the laboratory Q-PCR. It was demonstrated that the method of DNA extraction strongly influences the results of the LAMP-assay with the method intended to be used in the field (LFD extraction) giving less positive results than the laboratory method (Qiagen extraction). Therefore several options for improving the result have been investigated. It was found that clearing the DNA solution from particulate matter may increase the number of positive results. Both, including a “washing-step” in the procedure for DNA extraction from the lysed sample, or centrifuging the solution and then extracting DNA from the remaining clear fluid using an LFD resulted in improved detection results. Finally it was concluded that, probably because of the low levels of V. dahliae DNA, repeated analysis of the same sample may be needed for a reliable test result with LAMP. These results have been described in detail in D3.7.
The optimal conditions for detection of V. dahliae using LAMP also depend on the characteristics of the device that will be used. Therefore further optimisation of the LAMP procedure for in-field detection of V. dahliae can only be done using the device and tubes developed in WP5. This is part of the work in WP7 (System validation). The above conclusions together with the protocols that have been developed form the starting point for that work.
WP4: development of a LAMP assay for Verticillium dahliae
The main objective of this WP is to develop and optimize a LAMP amplification method for the specific detection of Verticillium dahliae DNA from infected plant material and infected soil, suitable for incorporation into the disposable cartridges. The WP is divided into three tasks foreseen to be developed within the first 15 months of the project. Main results are included in D4.1 submitted in M17. The accomplishment of this task leads to the achievement of Milestone 2.
Task 4.1: Selection of target sequences.
Isolates from target and closely related non-target organisms were obtained. Available sequences for these organisms were either obtained from publically available sequence databases or through DNA sequencing undertaken at Fera. Verticillium sequences were selected for the production of assays that would be specific (only detecting the target pathogen) and sensitive (being able to detect the pathogen to appropriate detectable limits). In addition, a novel sequence with putative specificity to the D-type strain of Verticillium dahliae was identified from sequence databases.
Task 4.2: Design of LAMP primers.
Eight primer sets with putative specificity to Verticillium dahliae were designed in total. Undertaking the LAMP reaction with standard conditions, four of the assays cross-reacted with closely related species. Two assays only had limited cross reaction and therefore alternative reaction conditions were evaluated (reduced primer concentrations and/or reaction temperature) but this did not improve specificity.
In two assays where cross reaction was not observed were taken forward for comprehensive validation with a greater set of isolates. No cross-reaction was observed against the additional isolates. The assay which showed the best sensitivity whilst still retaining specificity was taken forward for further evaluation on field material. The D-type assay was highly specific only detecting isolates of V. dahliae belonging to the D-type.
Task 4.3: Optimization of LAMP protocol.
In order to determine the efficiency of the chosen LAMP primer sets an isolate of Verticillium dahliae was used (isolate V70 collected from olive soils from Spain). The reaction was able to detect approximately 4 pg DNA/µl. Each of the LAMP primer sets developed was optimized independently before being subjected to comparative testing to select the best performing assay. The critical component of a LAMP reaction is the concentration of methylene blue (an intercalating dye used for in the electrochemical detection of the LAMP amplification). With one Verticillium dahliae assay a time to positive penalty of 24% was observed at a concentration of 20 µM methylene blue added compared to no methylene blue added to the master mix. In comparative testing, the most sensitive LAMP primers were compared against two real-time qPCR assays based on primers designed to a single copy gene and a multi-copy gene. The LAMP assay showed equivalent sensitivity to the single copy real-time qPCR assay. The LAMP assay was then tested with five additives and a range of concentrations to determine their ability to mitigate for the presence of inhibitors frequently co-extracted with soil. Of these, skimmed milk powder and bovine serum albumin appeared most effective.
WP5: Development of disposable tube and equipment device
The main objective of this work package was to design, develop and construct the self-contained disposable reaction tubes as well as the integrated VERTIGEEN equipment prototype. The WP was divided into 5 tasks
Task 5.1: Development of disposable reaction tubes. The reaction tube/cartridge was designed and constructed. The design is based on a plastic cartridge where the electrodes are screen printed and the heating resistance integrated. This design allows a simple sample mixture introduction (sample, MB and LAMP primers) and avoids the formation of bubbles. Several micromachining and micromolding techniques were evaluated, bubble formation avoided and several inks for the electrodes were tested. Simulation of electrochemical measurements, optimization of Square Wave Voltametry (SWV) parameters, selection of working electrode material, characterization of signal stability, detection of a sudden decrease of MB concentration and effect of the LAMP reagents on the electrochemical measurements were carried out.
Task 5.2: Development of temperature control system. The heating system includes: (i) a screen-printed resistor to be used as a thermal source. (ii) A contactless precision temperature sensor measuring the reaction temperature. (iii) A high-precision digital temperature controller with a resolution of 0.1ºK (e.g. PID control) which allows (re)adjust the temperature set-point
Task 5.3: Development of instrumentation for electrochemical detection. The electrical schematic design of the whole system was designed and adjusted to the modules assembled.
Task 5.4: Software and user interface. An user friendly interface was developed and the software was programmed and adapted to the SME-AGs requirements. A fluent communication between the SME-AGs and Ateknea allowed the RTD to better adapt the software and interface to their needs. The developed application has two different menus, for end-users/growers and another one for researchers. This differentiation was made in order to allow the researchers to have the possibility of modify different parameters in case they want to continue with research or other porpoises but at the same time, the end-user/growers/associations could just get the final information about the test without possible modification of the testing parameters. The application also allows the user to download a pdf file with the results and other information about the test conditions.
Task 5.5: Integration, characterization and optimization of the prototype performance. All components were assembled and tested before starting the validation. The resistance box was designed and constructed following the requirements of the SME-AGs.
WP6: Correlation between inoculum levels and disease risk levels
This WP is devoted to establishing the correlation between inoculum density in soil as detected by the newly developed VERTIGEEN technology and disease risk. A disease risk category system based on actual data from the participating SME’s will be developed. This information allows growers to evaluate if a site is suitable for establishing new plantations and within existing plantations, based on the risk level, follow a certain intervention protocol (Guide for Best Practices) to be developed as well within this work package. A major part of the work in this WP relies on the detection of V. dahliae using LAMP technology to be developed in other work packages of the project. As this technology became available later than foreseen in the work plan part of the work had to be rescheduled. Based on the work in this work package a System of Disease Risk Categories and a Guide of Best Practices for control of Verticillium wilt in olive have been prepared.
Task 6.1: Survey of literature and other available data. A first scan of the scientific literature for publications concerning disease incidence, disease risk and factors affecting disease incidence of Verticillium wilt was done in period 1. In May 2013 (M16) the 11th International Verticillium Symposium in Göttingen was attended for updating the above survey with recent research findings. In period 2 the scientific literature on Verticillium Wilt of Olive (VWO) has been reviewed extensively for information on control of VWO, epidemiology of VWO (including disease incidence – soil inoculum level relations) and disease classes and threshold levels for VWO. Searching the CAB abstract database using key words connected to these three themes resulted in over 70 useful references. Three groups of factors affect disease levels of Verticillium wilt in olive: 1) pathogen related factors (virulence of isolates present in soil, especially so-called defoliating isolates being highly virulent; and the inoculum level in soil), 2) host related factors (level of susceptibility of specific cultivars, and age of trees) and 3) agronomical factors (including history of a field, susceptible crops being grown on nearby fields or in between the olive trees, irrigation and weed control practices). These factors and their effect on VWO have been described in detail in D6.13 Guide of Best Practices. Information on the relation between inoculum density (ID) in soil and resulting disease incidences (DI) is very limited and fragmented, probably because this relation is strongly influenced by many factors as mentioned above. However, it is very clear that even low amounts of Verticillium in soil can result in serious disease, especially when D-type strains are present. In fact it is concluded that when this type of the pathogen is present in soil no olive should be planted. In addition it is reported that low ID levels of the ND type also may cause substantial disease in olive, especially in highly susceptible cultivars. For that reason in the disease risk system (developed in Task 6.4) 1 ms/g soil is used as threshold level.
Task 6.2: Correlation of LAMP results to ms/g soil. Inoculum density of V. dahliae in soil is traditionally expressed in the number of microsclerotia per gram of soil (ms/g soil), whereas the new VERTIGEEN data will be based on the amount of DNA of V. dahliae (fg DNA/gram of soil) detected by the new LAMP-based VERTIGEEN device. In order to understand what the VERTIGEEN data mean concerning numbers of microsclerotia in soil, the results obtained with the new LAMP-based method have be related to the ms/gram of soil values. At a Technical Meeting in M17 it was agreed to establish first the relation between ms/g soil and fg DNA/g soil as determined by a quantitative real-time PCR (Q-PCR) at the laboratory because at that time the VERTIGEEN device was not yet available and the LAMP detection was still being developed. Later on, when the Vertigeen device is operational, this can be developed further into a relation with the readings from that device. WUR and FERA collected and analysed a set of 34 soil samples with the traditional wet sieving and plating technique as well as with a quantitative real-time PCR laboratory method as developed in WP2. Analysis of the results revealed a clear correlation between the results of both methods. This means that it is very well possible to correlate data expressed in ms/g with data based on the amount of V. dahliae DNA detected in soil. After the Vertigeen device became operational in the last months of the project it turned out that the device in its present state is suitable for testing of soil samples for presence of V. dahliae but not (or not yet) for reliable quantification of the amount of V. dahliae present per gram of soil. Therefore developing a formula for translation of the Vertigeen data into ms/g soil data has not been possible and the Disease Risk Categories had to be related to ms/g soil data.
Task 6.3: Building database of actual ID-DI data from participating SME’s. For this task the soil sampling protocol from WP2 (preliminary version available in M15) and the new LAMP based device for testing of soil samples (preparation of which took more time than expected; see WP4 and WP5) were needed. In a Technical Meeting in M17 the delay and the way to proceed were discussed. It was decided to collect 100 samples from olive field soils, 25 from each participating country, in the summer of 2014 to be analysed later in that year by Vertigeen-LAMP technology. A sampling plan and an enquiry form for collection of data on actual disease index, main agronomical factors and environmental factors were developed by WUR and FERA and discussed at the General Meeting in M24. After the meeting a participant from each of the participating countries translated the form into their national language and sent it with the sampling kits to local olive farmers. Sampling in Portugal and some sampling in Spain was done by WUR and FERA during a sampling trip in August 2014. Together with the samples received directly from farmers this finally resulted in 92 soil samples available for analysis by September 2014. In order to complete the 100 samples for analysis, 8 more samples from Dutch tree nursery soils were included in this collection. For some of the received samples the enquiry form was lacking. As a result data from 83 forms could be included in a database on disease incidence and related main agronomical and environmental factors for specific fields.
The next step was characterization of the amount of V. dahliae in the samples by using the Vertigeen device. For this purpose Ateknea sent a prototype of the machine with about 50 cartridges for testing to both FERA and WUR in Month 33. The machine sent to FERA, unfortunately, did not function properly because what turned out to be a problem in the software. The one sent to WUR was used successfully for some validation experiments with known samples. However, technical problems with the machine and especially the lack of enough cartridges for further validation and testing made it impossible to analyse the collection of field samples. Finally Ateknea has performed this task in M36 after further improving the Vertigeen device. Analysis of the results achieved by Ateknea (see report on D7.15) showed that the device is suitable for reliable detection of V. dahliae. Reliable quantification of the amount of V. dahliae, however, appeared not (yet) possible which made analysis of the database for the ID-DI relation based on the ID of actual fields characterized by VERTIGEEN technology impossible.
Task 6.4: Defining disease risk categories
Data on the relation between soil inoculum level (ID) and disease incidence (DI) in Verticillium wilt of olive (VWO) appeared to be very scarce and fragmented (task 6.1 and 6.2). As a result it was not possible to base threshold levels for risk categories for VWO on information published in the literature. The only sound conclusion in literature concerning this topic is that on soils with the D-type isolates present no olive should be planted. For soils with ND-type isolates of V. dahliae disease risk is lower, but is strongly influenced by many factors as described above (task 6.1). Therefore, in order to make the Disease Risk Category system a robust system, the number of risk categories was limited to three: Low Risk, Limited Risk and High Risk. Based on the available information summarised in task 6.1 it was decided to use as threshold level 1ms/g soil. Low Risk is chosen for soils where no Vd is detected. This category deliberately is not called No Risk because the detection limit of the presently used techniques does not allow the detection of very low levels of V. dahliae. In such soils very low levels of V. dahliae may be present and even with such low levels disease may occur in individual trees especially because the pathogen may be unevenly distributed in soil. Also in long-living crops like olive there is always the risk of introducing the disease during the lifetime of the plantation. The second category is that of Limited Risk (i.e. only ND type of Vd present and ID < 1ms/g). For this category and the High Risk category (D-type of Vd or ND-type with ID > 1ms/g) actual risk levels are strongly influenced by local conditions. Therefore a decision scheme for deciding how to deal with this was designed and included in the Guide of Best Practices.
WP7: System Validation (to be completed by ATEKNEA, input from FERA and WUR will be welcome)
The objectives of WP7 were to prove the system functionality in field conditions and to compare the VERTIGEEN system performance with current detection methods in terms of speed, accuracy, sensitivity and specificity. The validation tests were meant also to demonstrate that the device fulfils the needs of the end-users.
During the initial validation phase the performance of VERTIGEEN device was assessed comparing it with a commercial CH-instrument potentiostat. Different Methylene blue solutions were tested with both devices and good linear correlation were obtained with over 99% of accuracy on both cases.
During the performance assessment of the cartridges several problems were faced, such as electrochemical signal obtained was indirectly proportional to the Methylene blue concentration in the solution, therefore cartridge production process was assessed step by step until the problem was solved. Once the cartridges were functional, a batch of 200 units was produced to proceed with the validation phase.
The next step was to calibrate the device, for that different LAMP experiments in the laboratory were done, electrochemically detecting pure Verticillium DNA. Different experimental conditions were checked, like the methylene blue concentration and the amount of sample needed in each test. The results obtained were good and linear correlations were obtained between the amount of DNA and the signal obtained, with adjustments over 85% in most of the tests.
In order to finalize the calibration, more LAMP experiments were done in the laboratory, but in this case the samples used were DNA extractions from plant and soil samples. After the firsts test, the methodology was changed in order to compensate the matrix effect produced by the samples, obtaining an accuracy of 84%.
Following, ATEKNEA started the Validation of the device. In order to do the laboratory validation, both equipment were sent to WUR and FERA. Different concentrations of pure Verticillium DNA were tested in both RTDs, obtaining good logarithmic correlations. With the results obtained, the final steps and conditions for the LAMP reaction were decided. The experimental conditions for the electrochemically detection were also fixed.
Before the in field validation, the threshold value was set in the device (based on previous experiments), being the detection limit of VERTIGEEN device 2,5ng/uL.
The in field validation was carried out with the DNA extracted from 100 different soil samples, being 91 out of 100 samples in accordance with the standard method (PCR). Of this 91% of accordance, 63 samples are positive and 28 negatives, the other samples are 5 false negatives and 4 false positives.
Furthermore, the hardware and the software were updated during these months of work, according to the problems that appeared with the continuous operation of the device. Also, a second software interface was created, so the non-technical end-users could use the device in a simple and easy way.
Finally, the user manual for the VERTIGEEN device was also prepared. In it there is included general information about the device performance, and a practical and easy-to-use guide on how to run an analysis and how to interpret the results obtained with the device. Also, the Standard Operation Procedure is included in the user-guide.
WP8: Dissemination and Training Activities
Several dissemination activities were planned and performed and all consortium members were actively involved. A project website (www.vertigeen.eu) was created to serve as vehicle of communication and dissemination of the project and its results. A project visual identity (logo, slogan, illustration, colours and lay-out) was also created and used in all the dissemination material prepared so far. Two project brochures providing information about the VERTIGEEN project have been created with the collaboration of all partners and it is being translated into the SME-AGs official languages (Spanish, Portuguese and Greek). One of the brochures reflects the main aim and the scope of the project, explaining the need and the chosen approach to solve the actual problematic. The explanation also includes the benefits that the expected results can provide to the olive sector. The second one explains more in detail the technology and highlights every partner contribution. A general project poster has been also created and has been shown in some dissemination events to which different project partners have attended. As well as the project brochure, the poster has been translated into other European languages. Also a scientific poster has been prepared.
Moreover, a project general presentation was prepared at the end of the project and it is available through SlideShare.
In addition, partners prepared several training materials to be used in external training events (workshops), a power point presentation (first version in English and then translated into the different official languages of the SME-AGs participating in the project) and a video which explains how the soil samples should be taken. This video is available on YouTube and it was used during the sampling requested to the SME-AGs.
WP9: Exploitation Activities
Associations, technical SMEs and end-users have undertaken discussions in periodic project meetings regarding the exploitation potential of the technology. All the associations in the project have stated their interest on the IPR resulting from the project and their motivation for proceeding with the exploitation activities required for ensuring the commercial success of the VERTIGEEN system. SME-AGs have defined the generated foreground and studied the possible protection strategies for each one of them. It was decided that trade secret will be applied to most of the results, but community trade mark for the logo developed as soon as the commercialization is about to start. Patent is not applicable in any of the cases and other strategies are not foreseen.
On the other hand, partners performed market and technology watch which allowed them to define the exploitation strategy. In this context, partners studied the possible licensing for using LAMP technology. Several options were studied, negotiate a license with the Japanese company (Eiken) who hold the LAMP patent and negotiate an exploitation agreement with other companies who already have a license from Eiken (such as Optigene and Prime Diagnostics).
During the second period of the project, technical SMEs supported by the RTDs have calculated the production and selling costs of the complete VERTIGEEN system. Moreover, the potential market has been slightly changed, focusing the efforts in diagnostics laboratory and nurseries and primary customers for the developed technology. The new business model provides a market worth over €10 million, and will provide the each one of the SMEs with over €100.000 the first five year, and the technical SMEs will have a profit of over €2 million after five years.
Commercialization routes have been also studied as well as the role of each one of the SME-AG and SME partner.
All this information has been gathered in one document “Final Business Plan and Plan for Use and Dissemination of Foreground, which also includes the background management and access rights for the different parties.
Potential Impact:
VERTIGEEN addresses a need identified by olive sector SME-Associations for the rapid and reliable on-site detection and future quantification of the Verticillium dahliae fungus in soil and plant samples. The developed technology is based on new but successfully demonstrated technologies: the loop mediated isothermal amplification (LAMP) method and the original in-solution-DNA electrochemical detection technology, overcoming the limitations of the state of the art in terms of heterogeneity, portability, price, easiness to use and time-to-result. With an expected final price of about €1.000 and a price per test of €10, the system would allow the associated olive producers to reduce the losses caused by this pathogen and its spreads by means of precise and efficient field interventions within an Integrated Pest Management (IPM) Strategy.
The competitiveness of the European olive lead market, already faced with growing international competition from Tunisia, Turkey, Syria, Morocco and Argentina and other adverse factors such as olive oil prices dropping steadily , is currently severely threatened by the soil-borne fungus Verticillium dahliae, responsible for critical damage to the vitality, productivity and yield of more than 200 plant species with crop losses estimated at thousands of millions of dollars worldwide every year. The nature of the infections and the characteristics of the pathogen -a soil habitat, survival structures that persist for years, demonstrated transmission potential and capacity to infect numerous hosts in a continually expanding geographical distribution worldwide, has made Verticillium wilt a chronic economic problem in global crop production. Non-defoliating and defoliating pathotypes of Verticillium dahliae vary in aggressiveness but overall, for commercial olive plantations, this disease is currently considered one of the most serious problems with reported incidences of 30% in some regions of Spain while other studies report olive crop losses ranging from 50 to 89%, depending on olive variety and pathogen aggressiveness .
The disease in olive trees was first described in Italy, and afterwards was reported in Spain, Greece, France, Turkey, Syria, Jordan Morocco and California . In recent years, verticillium wilt is occurring with increasing frequency and severity in most olive growing areas of the Mediterranean basin due to intensive farming of highly productive cultivars, planted at high densities and flood irrigated, all of which favour the spread of the pathogen. Other causes of the increased spread of the pathogen include the use of infected but symptomless plant propagation material for the establishment of new olive groves or planting in contaminated soil, which is becoming more common as the pathogen spreads by wind, water or dirty equipment that carries it into clean soil.
Driving consortium SME-AG partners INOLEO, EDOEE, AAR and their memberswere certain that containment and even eradication of this disease is feasible, but only if timely and accurate detection and quantification of the Verticillium fungus in soil and in trees (diseased as well as symptomless) is achieved at a comparatively low price and appropriate effective and sound economic measures are applied within the frame of an Integrated Pest Management strategy. In-field diagnostics SME partners FORSITE and LOEWE; as well as CITOLIVA technological provider and member of INOLEO, confirmed this need. Also olive producers and plant propagation nurseries such as COTEVISA, PELOSI and SAOV, consider a priority the development of innovative ways to defend against this pathogen and are highly committed to the development of the proposed VERTIGEEN technology.
Currently there is no single effective treatment for plants affected by Verticillium Dahliae, meaning that efficient control should be based on preventive and eradication measures. VERTIGEEN consortium not only developed a new, cost-effective, reliable system for early on-site detection of Verticillium Dahliae, but also a Guide of Best Practices in Managing Verticillium wilt in Olive. This guide containing protocols to control the disease in established orchards and prevent contamination of new olive groves or healthy parts of existing groves. This guide is the result of an extensive literature review and contains a decision scheme based on disease risk categories established on the results of work don along the project.
The economic impact of such a tool will affect:
- Specialized olive tree nurseries such as the consortium member COTEVISA, who will be able to assess the condition of sapling, grafts and other planting material before trading benefit from selling value-added certified healthy planting material, increase consumer confidence in their products, improved sales, better competitiveness, employment creation and even opening the door to new export markets.
- Olive growers will gain precise and rapid information, resulting in more efficient interventions. It will also allow to know which areas are affected by Verticillium Dahliae before a new plantation is considered.
The SME-AGs INOLEO, EDOEE and AAR foreseen benefiting from their participation in VERTIGEEN by improving the competitiveness of their associates, who will have early affordable access to a new diagnostic tool that will allow them to significantly reduce the losses caused by this pathogen and consequently to increase their profit margins, preserve employment and maintain a competitive edge in the global market. The AGs will also obtain direct economic benefits from their foreground ownership, via the profit deriving from the sales revenues, estimated at €111.229 for each of the associations in the first 5 years.
Moreover, VERTIGEEN holds the potential for rapid uptake not only in the olive sector but beyond, as in the future it may be adapted to the detection of Verticillium in many other affected agricultural crops such as strawberry, tomato, potato, cotton, vegetables, fruits or various landscape and ornamental trees and shrubs of high relevance in Europe and worldwide, increasing the potential post-project impact for the proposing SME-AGs. Industrial lead user partners FORSITE and LOEWE have also recognized a business opportunity in developing such a tool since apart from its applicability in routine plant pathogen diagnosis, VERTIGEEN can become a valuable tool in human health applications involving single nucleotide polymorphisms (SNP) and for diverse ecological and epidemiological studies (e.g. population dynamics, disease incidence and severity, etc.). These two partners actively supported the commercial exploitation route definition of VERTIGEEN, following agreement with the SME-AGs.
▪ Lead user partner FORSITE as expert developer and manufacturer of on-site diagnostic equipment will benefit from the manufacture of the VERTIGEEN device and disposable test tubes. They will open new markets and income sources with a new portfolio product at an estimated price of €1,000 and cost per test of €10. They expect to open a market niche worth more than €5,9 million in the olive production sector.
▪ Lead user partner LOEWE, which provides a wide range of phytodiagnostic tools will commercialize and distribute the technology, benefiting directly from the sales of VERTIGEEN and by improving their competitiveness offering a new solution to those customers who suffer important economic losses due to the presence of Verticillium dahliae in their orchards and nurseries.
By means of EU-wide strategically planned dissemination actions and highly developed exploitation processes, VERTIGEEN would allow the advancement in the control of this epidemic disease, which depends on the systematic and collective implementation of phytosanitary measures, and would maximize the competitive benefits for olive growers, olive oil producers and olive tree nursery SMEs and SME-AGs in all Member States.
Along the project, SME-AGs, SMEs and RTDs were deeply involved in Dissemination activities and definition of the possible exploitation routes that could be followed after the project ends.
The main dissemination activities carried out during the project include:
- Dissemination throughout the project website
- Dissemination through the partner’s website – linking the project website or even publishing press releases after the project meetings.
- Dissemination through Twitter, some partners used their company Twitter profiles to dissemination events of the projects.
- Publication of press releases in several specialized online media, such as Mercacei Magazine, Interempresas.net Agrodigital.com Sitioliva, Europapress, Mercadosdelvino.es publication for the Dutch Agricultural sector, German institute for Arboriculture, Data Archiving and Networked Services, Greek agricultural press, etc
- Presentations in fairs or symposium of the sector such as VI Simposio Nacional de Olivicultura (2012), Expoliva 2015, Encontro Iberico do Azeite (2013 and 2015), Ovibaje (2013), Andalusian Agrofair (2013), etc.
- Scientific publications (abstracts, poster or papers): 11th International Verticllium Symposium (2013), etc.
- Workshop organized by SME-AGs for their members or other members of the sector.
On the other hand, VERTIGEEN partners dedicated effort as well in the preparation of a Plan for Use and Dissemination of Foreground, which objectives were: To ensure that the SMEs beneficiaries could assimilate the results of the project, to disseminate the results beyond the Consortium to a wider audience, to support the participating SMEs in using and protecting the research results to their best advantage, and to exploit the results and increase the competitiveness of the SMEs beneficiaries. In order to do so, technology watch and market watch was carried out by all partners along the project, deep market study, background and foreground management (including IPR ownership and access rights on the foreground, protection strategies and licensing for future exploitation together with third parties). Moreover, SME-AGs together with the technical SMEs prepared a business case, based on the market analysis carried out previously, production costs of the different parts of the developed technology (sample kits and reader device), taking into account only the potential market in Spain, Portugal, Italy and Greece. Partners have also studied the possible commercialization route taking into account the area of interest of each one of the partners. Technical SMEs will mainly work as manufacture and distribution of the technology. SME-AGs will provide the publicity of the product and collect the royalties gathered from the sales per year.
List of Websites:
www.vertigeen.eu
Contact details (Project Coordinator):
Mr. Albert Nieto
email: albert.nieto@ateknea.com
Tlf: +34 932 049 922
The European Union leads the global olive oil sector, producing 80% and consuming 70% of the average total world output of over 2.5 million tonnes that guarantee an income of €5 billion per year from olive oil plus another €600 million in table olives. Olive production is distributed into a large number of small and medium enterprises with low profit margins, thus particularly vulnerable to drops in productivity. The competitiveness of EU olive lead market is being currently threatened by the soil-borne fungus Verticillium dahliae responsible for critical damage to the vitality, productivity and yield of more than 200 plants species with crop losses estimated at thousands of millions of dollars worldwide every year, as well as by growing international competition from Tunisia, Turkey, Syria, Morocco and Argentina and steadily olive oil prices drops. In recent years, verticillium wilt is occurring with increasing frequency and severity in most olive growing areas of the Mediterranean basin. Main factors associated with this increase are intensive farming, use of infected but symptomless plant propagation material, planting in infected soils and distribution of the pathogen by wind, irrigation as well as run-off water and the use of contaminated equipment.
VERTIGEEN proposes an innovative, rapid and reliable on-site detection and quantification system of the Verticillium dahliae fungus in soil and plant samples, by the integration and application of new but successfully demonstrated technologies for DNA amplification and electrochemical detection, overcoming the limitations of the state of the art in terms of heterogeneity, portability, price, ease of use and time-to-result.
With an estimated commercial price of €1,000 per device and €10 per test, the system will allow associated olive producers to reduce their losses caused by this pathogen and its spread by means of precise and efficient field interventions within an Integrated Pest Management (IPM) Strategy.
By means of EU-wide strategically planned dissemination actions and highly developed exploitation processes, VERTIGEEN will maximize the competitive benefits for olive growers, olive oil producers and olive tree nurseries SMEs and SME-AGs in all member States.
The consortium provides 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.
Project Context and Objectives:
Olive production is distributed into a large number of small and medium enterprises with low profit margins, thus particularly vulnerable to drops in productivity. The five main EU olive producers -Spain, Italy, Greece, Portugal and France- have more than 11 thousand olive sector SMEs , most of which are members of professional SME Associations (SME-AGs) such as the SME-AG proposers INOLEO, EDOEE and AAR. These play a direct role in the production and distribution of the oil made from their associate’s olives and losses in associate productivity affects their business directly.
The competitiveness of the European olive lead market, already faced with growing international competition from Tunisia, Turkey, Syria, Morocco and Argentina and other adverse factors such as olive oil prices dropping steadily , is currently severely threatened by the soil-borne fungus Verticillium dahliae, responsible for critical damage to the vitality, productivity and yield of more than 200 plant species with crop losses estimated at thousands of millions of dollars worldwide every year. The nature of the infections and the characteristics of the pathogen -a soil habitat, survival structures that persist for years, demonstrated transmission potential and capacity to infect numerous hosts in a continually expanding geographical distribution worldwide, has made Verticillium wilt a chronic economic problem in global crop production. Non-defoliating and defoliating pathotypes of Verticillium dahliae vary in aggressiveness but overall, for commercial olive plantations, this disease is currently considered one of the most serious problems with reported incidences of 30% in some regions of Spain while other studies report olive crop losses ranging from 50 to 89%, depending on olive variety and pathogen aggressiveness .
Currently, there is no single effective treatment for plants affected by Verticillium dahliae, meaning that efficient control of this disease requires an Integrated Pest Management Strategy based on both (a) preventive and (b) eradication measures:
a) Diseased trees are constantly being replaced and new olive groves are continually being planted, making pathogen detection for the choice of verticillium-free planting soil and planting material a critical key point for the integrated preventive management of verticillium wilt in newly established groves. In addition, regular monitoring of the infections in established groves would allow individual infected trees and surroundings to be quickly spot-treated or grubbed out, preventing pathogen spread to the entire grove and eradication of healthy productive olive trees. This would result in improved productivity and cost savings from the investment in the production of healthy olive trees.
b) To protect productive trees in established groves or to disinfect soil before planting, pathogen eradication methods are required. The March 2010 ban on methyl bromide in the EU as a soil fumigant , which was the most effective and widespread method for eliminating this pathogen, due to its considerable side-effects on public health and environment, threatens to drastically increase the incidence of wilt disease and significantly impact olive farmers. Other chemical treatments include Metamsodium, Dazomet or Cholopicin, but these should always be employed at minimum doses in clearly defined areas (focused treatment) where the pathogen has been positively detected and accurately quantified. With timely quantitative analyses the most appropriate treatment can be accurately prescribed based on actual pathogen populations, reducing costs and environmental impact of blanket pesticide use achieving sustainable agriculture practices.
Alternative control methods to reduce the initial inoculum densities in soil, such as crop rotation, irrigation management, soil solarisation or biological control agents, are available but are costly and time consuming, and therefore only suitable for application at small scale on well-defined areas with low density of inoculum. The lack of rapid quantification methods to identify these areas is the main reason why they have not yet been successfully applied in practice4. Attempts have also been made to breed for disease resistance against Verticillium in different crops, but so far resistant clones have only been successfully achieved for tomatoes5.
Moreover, reliable quantification would allow growers to estimate the risk of disease development, and based on risk levels to quickly decide upon the nature and extent of the treatment and to quickly intervene in the orchard, limiting the impact of the disease on their olive groves, increasing crop yield and reducing productivity losses.
Therefore, the development of a new, cost-effective, reliable system for early on-site detection and quantification of Verticillium dahliae such as VERTIGEEN is clearly needed as a first step in the integrated management of verticillium wilt, the only way to effectively fight this disease and minimize the losses caused by this pathogen and its spread. A guide for best practices has been also developed covering methods and timing of sampling for detection and screening as well as intervention protocols.
Scientific Objectives
The overall objective of the research described is to design and develop a method for the early detection and quantification of Verticillium dahliae in soil and in plant using and applying isothermal LAMP DNA amplification coupled to electrochemical detection for routine diagnosis of the Verticillium wilt disease. With this purpose the following measurable specific sub-objectives were defined:
1) A sound sampling strategy for both plants and soil in order to have significant and representative results covering the extension of an average olive tree grove. The most appropriate location (spatial distribution), sampling pattern and scale, season for sampling, sample size and material (e.g. leaves, root tissue, soil aggregates) were selected.
2) A sample processing method for both plants and soil to guarantee maximum test sensitivity (by ensuring maximum efficiency of Verticillium DNA extraction and purification) eliminating potential LAMP inhibitors. The sample obtained must be ready to be introduced in the VERTIGEEN self contained disposable amplification and detection tubes.
Based on these results, a sampling protocol including the optimum testing frequency was established.
The LAMP assay was developed, adapted and optimized for the specific detection of Verticillium dahliae by selecting target DNA sequences for amplification and designing the primers that will start the amplification of such specific sequences. This avoids cross reactivity with other pathogens or sample components.
3) 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 as well as the concentration of the detection reagent (Methylene Blue) were determined.
4) Inoculum levels will be correlated to disease risk levels in olive orchards (no risk, low risk or high risk) with the aim of establishing field intervention protocols for the grower to follow in each case. These have been compiled in a guide of best practices.
Technological objectives:
To develop a homogeneous, simple, rapid, accurate, sensitive, on site electrochemical diagnostic tool for detecting Verticillium dahliae and quantifying infection levels with a cost below €1,000 per unit and €10 per test. The following measurable specific sub-objectives were defined:
1) To fix details regarding technical, biological and design specifications of the VERTIGEEN system in order to adapt it as much as possible to end-user requirements, such as ease of use, measurement time, cost per test, cost of the device, etc.
2) To deliver a simple sample treatment kit adapted to field conditions for sample processing (e.g. roller, crystal beads) with a price below €1.
3) To design and construct a disposable reaction tube where the sample is introduced after being processed with the methods developed for soil and for plant samples, which contains the LAMP and detection reagents, and where the electrochemical measurement will take place once the electrodes connected to the reader are inserted
4) To design and construct the equipment device where the disposable tubes will be inserted containing a simple heating block that reaches the 65ºC required for the LAMP reaction to start in less than 1min. and a simple and inexpensive highly-sensitive multi-channel potentiostat that will allow the quantification of the DNA amplification products by means of the electroactive DNA intercalator Methylene blue. The device provides the results in less than 30 minutes. This reader includes all instrumentation necessary to generate, acquire and process the signal. It must also take into account the end-user requirements such as price, ease of use and robustness.
5) To develop a software package that allows the exchange and management of the data stored in the reader through USB and a firmware that allows the system control with an intuitive user-interface through the display. The management software and a database would be developed to record and strategically establish risk prediction levels based on which indications to the grower will be made.
6) Ensure that each of the components of VERTIGEEN work properly once integrated. 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.
7) 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 VERTIGEEN system can compete with current detection methods in terms of efficacy, specificity, sensitivity and detection limit and to demonstrate that VERTIGEEN 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:
1) To carry out training activities in order to facilitate the take-up of the project results by consortium SME-AGs and SMEs. A detailed plan will be prepared to train the AG members on how to take advantage of the readings of the system
2) To disseminate non-confidential information about the project and its results to a wide and relevant audience in order to maximize the project impact.
3) To promote the exploitation of the foreground generated during the project to the greatest possible advantage for the participating SME-AGs and SMEs
4) To optimize the use of resources and to ensure that all aspects of the EC requirements for communication and reporting are met.
Project Results:
The work carried out along the project was divided into several work packages and activities, R&D, DEMO, OTHER and Management.
WP1: Definition of System Specifications
The consortium successfully completed the market survey. The market survey was performed by means of a questionnaire which compiled the most interesting questions drawn up by each of the project SMEs in English. The final questionnaire was translated to the project SME-AGs languages (Greek, Portuguese and Spanish) as well as Italian; and distributed by each of them within their countries to end-users or any potential customer of the new diagnostic tool. The questionnaire covered 3 main topics: general information about the end user or potential customer, VD infection knowledge and management and interest in a new VD on site detection device. Several dissemination strategies have been used: massive emailing, interviews personally or by phone. Over 500 end-users (farmers, olive oil producers, organizations, etc.) were sought between the 1st of April and the 30th of June 2012. A total of 127 filled questionnaires have been collected, 13 from Spain, 50 from Italy, 9 from Greece and 55 from Portugal. During the General Meeting M6 in York, it was decided to continue this task in order to have more reliable data on the end-users needs and expectations. Over the last months SME-AGs supported by the SMEs in the consortium have collected 37 questionnaires more including 7 laboratories from Spain, making a total of 149 end-users and 7 laboratories. The main points highlighted are the following:
- Most common range of plantation area is up to 20Ha. Fertilizers are used by 56% of the end users, while only 44% uses herbicides. 92% of the end-users harvest every year, 45% make tillage on the soil and only 34% incorporate organic matter to the soil.
- 59% of the end users have had some kind of pest or disease in the last three years and 64% are aware of the VD presence.
- Over 72% of the end users detect the presence of VD based on visual detection methods, and only 9% perform lab analysis. The average time needed to get the results is between 2 and 3 weeks, considered not quick enough by most of the questioned end-users.
- On average, a testing for Verticillium costs 20-50€, but in most of the cases this is funded by public organization.
- Regarding the interest in a new detection tool, over 46% of them stated that it would be necessary to have a quick, cost effective and reliable field diagnosis tests for V. dahliae.
- Another important point was “where the end-user want to use the new diagnostic tool” in order to evaluate some characteristics relative to portability of the device. The questionnaires indicate that 52% of the end users answered directly “in the field” and 17% “in the office or laboratory” or similar facilities.
The most important features of the system are: easy to use, accuracy, measure the degree of infection, quickness, portability and low price (below €1000).
The information gathered was also used to define the technical specifications of the VERTIGEEN system. This information allowed a better adaptation of the system to the end user requirements and demands of the implied sector, ensuring that they are fully met, taking into account the technical feasibility and the experience of the project partners.
WP2: Development of a soil sampling protocol
The aim of this work package was to develop on site DNA extraction methods. In addition, the seasonal aspect of sampling was investigated as well as the most suitable sampling strategy. This included determining the number of subsamples as well as where specifically to take the sample (sample depth and where in relation to the olive tree).
Task 2.1: Selection of suitable soil material and seasonal timing for sampling.
In total, 386 field samples from olive fields and one cotton field in Italy, Greece, Spain and Portugal were taken. Of these samples, 30% were found to be positive. An interesting result from this screen of soil samples was the presence of very high levels of Verticillium dahliae in a cotton field adjacent to an olive field (which was largely negative for Verticillium). This showed the risk from crops such as cotton as an alternative host for the pathogen. This exercise also provided a range of additional soil samples to validate the LAMP assay on.
On the basis of these results, five sites were selected for further in-depth analysis to determine the seasonal timing for sampling and also inform the sampling strategy. This included three sites in Spain and two sites in Portugal. In the case of the Spanish sites, they were sampled twice, once in spring, prior to the summer warm period and once at the end of August. The two sites in Portugal were sampled four times. In each instance, both before and after the summer hot period over two consecutive years. This directly informed seasonal timing for sampling. On the whole relative, levels of abundance of the pathogen and relative incidence was greater before the summer hot period than after suggesting that sampling should be undertaken in spring not summer or autumn.
Task 2.2: Study of the most appropriate sampling pattern
From the initial range of soils that were taken Geostatistical analysis (Krigging) was undertaken at three sites and showed that the pathogen had a highly clustered distribution in soil. More in depth sampling was performed at one site in Portugal taking 103 samples over 1 ha and also taking corresponding disease assessments on the trees from those sampling points. Using indicator Krigging analysis the trees displaying severe symptoms were also present in areas where high level of V. dahliae was detected by qPCR. This showed good evidence that soil levels and symptoms can correspond in olive fields. The indicator Krigging analysis with 103 sampling points confirmed that V. dahliae had a much clustered distribution. However, the Krigging heat maps from this site were used to inform the sampling strategy. Since the pathogen had a highly clustered distribution in all sites sampled, a grid sampling strategy was essential for optimum detection. In addition, analysis of the heat maps showed that although 10 subsamples per ha is likely to detect the pathogen, 25 samples was decided on for practical reasons (i.e. they can form a 5 x 5 grid) and added increased robustness to the sampling strategy. This decision was taken in consultation with the other consortium partners.
The sites in Portugal were also used to inform other aspects of the sampling strategy. These included whether samples should be taken between rows of olive trees or at the tree bases. Tree bases were shown to have significantly higher levels of Verticillium. Therefore, different sampling depths at tree bases were tested to see if differences were present. After removing surface debris, different depths down to 35 cm were tested. Depths from 0 to 9 cm had highest levels of Verticillium on the whole so were selected for the final sampling strategy. This was also the easiest depth to sample.
The direction of the sampling point around the tree base was also tested to see if that affected levels of Verticillium in the soil. Eight sampling points were taken around a series of trees from two sites in Portugal and factors such as gradient, water flow, prevailing wind etc. were considered for each site. No discernible patterns were found and therefore any place around the tree base can be sampled.
Task 2.3: Selection of the most suitable in-field sample processing method
A range of DNA extraction methods were evaluated for potential in field use. These included spin filters, syringe filters and magnetic bead based purification. Magnetic beads showed the most practicality in addition to having a DNA recovery rate approaching laboratory based extraction methods. The practicality of this method was further enhanced by using it with a desktop mini centrifuge. The centrifuge could be purchased relatively cheaply and although added to the initial cost, reduced costs in the long term if large numbers of samples are processed. Using this method took 30 minutes compared to a laboratory scale extraction which can take up to 4 hours. Sensitivity (in terms of DNA recovery) was 10 – 20 times less than the lab extraction but speed and cost was substantially improved. Therefore lower sensitivity could be compensated by taking additional samples. The method had high potential for a successful in field extraction protocol.
WP3: Development of a plant sampling protocol
This WP is devoted to design the optimal plant sampling strategies and processing methods for being performed on-site. The work in this WP is divided into 3 tasks foreseen to be developed within the first 14 months of the project. The work was largely completed in the first project period with the main results included in D3.7 submitted in M14 and updated in M25.
Task 3.1: Selection of suitable plant material and seasonal timing for sampling.
A preliminary sampling protocol based on the literature review (task 3.2-1) was tested successfully during a sampling field trip in southern Spain after the 3-months Technical Meeting. Young branch samples were collected according to the protocol and gave good results when tested by the developed Q-PCR in the laboratory for the presence of Verticillium dahliae (Vd). Collection of root samples however appeared not feasible; and leaves, although collected easily, gave very low percentages of Vd detection. Therefore it was decided that the best sample type for detection of Vd in olive trees are parts from young branches. Examination of samples from individual branches within trees confirms the conclusion from literature that distribution of Vd within infected olive trees may be discontinuous. The percentage of Vd-positive samples per tree varies strongly, from 17% (1 out of 6) till 100% (6 out of 6). However, it was demonstrated that analyses of combined samples from 5 branches per tree gives very reliable results. Samples were collected from 157 olive during sampling sessions repeated at different parts of the year in the period 2012-2014 in Spain (5 times), Italy and Portugal (4 times each) and Greece (once). Vd was detected in samples from all sessions. However, the number of positive samples varied strongly with the year and period of sampling, which is in line with literature reports. Especially after hot summer periods the number of positive samples may be low. Taking into account the seasonality effect on results in different countries and the timing of agricultural practices, the best period for sampling of diseased olive trees for detection of V. dahliae is in late winter or early spring (before pruning of the trees).
Task 3.2: Study of the most appropriate sampling strategy
From a literature survey it was concluded that (a) several samples per tree should be examined because distribution of V. dahliae in diseased trees may be discontinuous; (b) Vd probably is most readily isolated from the wood of current year’s or previous year’s parts of branches in the lower and middle part of the crown; and (c) probably the best period for detection of Vd is in spring and early summer because especially in areas with relatively high summer temperatures the amount of the fungus in the aerial parts of diseased olive trees may vary with the season. The results for individual branch samples show that Q-PCR has a higher sensitivity for detection of Vd in olive samples than the standard plating assay. Moreover, pooling of samples from 5 branches per tree in one analysis resulted in positive test results for all Vd infected trees whereas results for samples from individual branches may vary. Additionally a dilution series showed that the Q-PCR can detect one infected sample in a mix with samples of at least up to 10 healthy samples.
Task 3.3: Selection of the most suitable in-field sample processing method
Q-PCR was used for detection of Vd in the development of sample processing methods and as a reference test in the evaluation of the LAMP PCR. Therefore a PCR soil assay method previously developed by PPO was adapted for use on plant samples. The PPO primers were shown to work well in olive samples and to have a very high specificity for Vd. Also this Q-PCR has a high sensitivity; the threshold level for detection of Vd in olive plant samples appeared to be 0.0001 ng. A laboratory isolation method based on Kingfisher DNA extraction was developed as a reference test for good quality DNA from Vd infected olive material. Also a preliminary protocol for a field method was designed including the use of lateral flow devices (LFD) for capture of DNA from crude plant extracts. The procedure of freeing and extracting DNA from woody samples in the field was shown to be effective for detection of Vd with Q-PCR. The first set of LAMP primers based on the EF sequence (one copy target) developed in WP4 became available in month 13. Preliminary tests of LAMP PCR in the laboratory showed that the assay works at 65 as well as 70˚C but at low concentrations of Vd no positive reactions are found with a threshold level of about 0.1 ng. Also the method of extraction of DNA from plant samples seems to affect the result, suggesting that for LAMP-based detection of Vd the amount and/or quality of the DNA needs to be improved. Therefore in period work was done on further optimization of the LAMP-assay for on-site detection of V. dahliae. A new set of LAMP primers developed by FERA and based on IGS or ITS sequences (multi copy target) was tested. This set increased sensitivity strongly (factor 100-1000) but the sensitivity of the LAMP assay still was less than that of the laboratory Q-PCR. It was demonstrated that the method of DNA extraction strongly influences the results of the LAMP-assay with the method intended to be used in the field (LFD extraction) giving less positive results than the laboratory method (Qiagen extraction). Therefore several options for improving the result have been investigated. It was found that clearing the DNA solution from particulate matter may increase the number of positive results. Both, including a “washing-step” in the procedure for DNA extraction from the lysed sample, or centrifuging the solution and then extracting DNA from the remaining clear fluid using an LFD resulted in improved detection results. Finally it was concluded that, probably because of the low levels of V. dahliae DNA, repeated analysis of the same sample may be needed for a reliable test result with LAMP. These results have been described in detail in D3.7.
The optimal conditions for detection of V. dahliae using LAMP also depend on the characteristics of the device that will be used. Therefore further optimisation of the LAMP procedure for in-field detection of V. dahliae can only be done using the device and tubes developed in WP5. This is part of the work in WP7 (System validation). The above conclusions together with the protocols that have been developed form the starting point for that work.
WP4: development of a LAMP assay for Verticillium dahliae
The main objective of this WP is to develop and optimize a LAMP amplification method for the specific detection of Verticillium dahliae DNA from infected plant material and infected soil, suitable for incorporation into the disposable cartridges. The WP is divided into three tasks foreseen to be developed within the first 15 months of the project. Main results are included in D4.1 submitted in M17. The accomplishment of this task leads to the achievement of Milestone 2.
Task 4.1: Selection of target sequences.
Isolates from target and closely related non-target organisms were obtained. Available sequences for these organisms were either obtained from publically available sequence databases or through DNA sequencing undertaken at Fera. Verticillium sequences were selected for the production of assays that would be specific (only detecting the target pathogen) and sensitive (being able to detect the pathogen to appropriate detectable limits). In addition, a novel sequence with putative specificity to the D-type strain of Verticillium dahliae was identified from sequence databases.
Task 4.2: Design of LAMP primers.
Eight primer sets with putative specificity to Verticillium dahliae were designed in total. Undertaking the LAMP reaction with standard conditions, four of the assays cross-reacted with closely related species. Two assays only had limited cross reaction and therefore alternative reaction conditions were evaluated (reduced primer concentrations and/or reaction temperature) but this did not improve specificity.
In two assays where cross reaction was not observed were taken forward for comprehensive validation with a greater set of isolates. No cross-reaction was observed against the additional isolates. The assay which showed the best sensitivity whilst still retaining specificity was taken forward for further evaluation on field material. The D-type assay was highly specific only detecting isolates of V. dahliae belonging to the D-type.
Task 4.3: Optimization of LAMP protocol.
In order to determine the efficiency of the chosen LAMP primer sets an isolate of Verticillium dahliae was used (isolate V70 collected from olive soils from Spain). The reaction was able to detect approximately 4 pg DNA/µl. Each of the LAMP primer sets developed was optimized independently before being subjected to comparative testing to select the best performing assay. The critical component of a LAMP reaction is the concentration of methylene blue (an intercalating dye used for in the electrochemical detection of the LAMP amplification). With one Verticillium dahliae assay a time to positive penalty of 24% was observed at a concentration of 20 µM methylene blue added compared to no methylene blue added to the master mix. In comparative testing, the most sensitive LAMP primers were compared against two real-time qPCR assays based on primers designed to a single copy gene and a multi-copy gene. The LAMP assay showed equivalent sensitivity to the single copy real-time qPCR assay. The LAMP assay was then tested with five additives and a range of concentrations to determine their ability to mitigate for the presence of inhibitors frequently co-extracted with soil. Of these, skimmed milk powder and bovine serum albumin appeared most effective.
WP5: Development of disposable tube and equipment device
The main objective of this work package was to design, develop and construct the self-contained disposable reaction tubes as well as the integrated VERTIGEEN equipment prototype. The WP was divided into 5 tasks
Task 5.1: Development of disposable reaction tubes. The reaction tube/cartridge was designed and constructed. The design is based on a plastic cartridge where the electrodes are screen printed and the heating resistance integrated. This design allows a simple sample mixture introduction (sample, MB and LAMP primers) and avoids the formation of bubbles. Several micromachining and micromolding techniques were evaluated, bubble formation avoided and several inks for the electrodes were tested. Simulation of electrochemical measurements, optimization of Square Wave Voltametry (SWV) parameters, selection of working electrode material, characterization of signal stability, detection of a sudden decrease of MB concentration and effect of the LAMP reagents on the electrochemical measurements were carried out.
Task 5.2: Development of temperature control system. The heating system includes: (i) a screen-printed resistor to be used as a thermal source. (ii) A contactless precision temperature sensor measuring the reaction temperature. (iii) A high-precision digital temperature controller with a resolution of 0.1ºK (e.g. PID control) which allows (re)adjust the temperature set-point
Task 5.3: Development of instrumentation for electrochemical detection. The electrical schematic design of the whole system was designed and adjusted to the modules assembled.
Task 5.4: Software and user interface. An user friendly interface was developed and the software was programmed and adapted to the SME-AGs requirements. A fluent communication between the SME-AGs and Ateknea allowed the RTD to better adapt the software and interface to their needs. The developed application has two different menus, for end-users/growers and another one for researchers. This differentiation was made in order to allow the researchers to have the possibility of modify different parameters in case they want to continue with research or other porpoises but at the same time, the end-user/growers/associations could just get the final information about the test without possible modification of the testing parameters. The application also allows the user to download a pdf file with the results and other information about the test conditions.
Task 5.5: Integration, characterization and optimization of the prototype performance. All components were assembled and tested before starting the validation. The resistance box was designed and constructed following the requirements of the SME-AGs.
WP6: Correlation between inoculum levels and disease risk levels
This WP is devoted to establishing the correlation between inoculum density in soil as detected by the newly developed VERTIGEEN technology and disease risk. A disease risk category system based on actual data from the participating SME’s will be developed. This information allows growers to evaluate if a site is suitable for establishing new plantations and within existing plantations, based on the risk level, follow a certain intervention protocol (Guide for Best Practices) to be developed as well within this work package. A major part of the work in this WP relies on the detection of V. dahliae using LAMP technology to be developed in other work packages of the project. As this technology became available later than foreseen in the work plan part of the work had to be rescheduled. Based on the work in this work package a System of Disease Risk Categories and a Guide of Best Practices for control of Verticillium wilt in olive have been prepared.
Task 6.1: Survey of literature and other available data. A first scan of the scientific literature for publications concerning disease incidence, disease risk and factors affecting disease incidence of Verticillium wilt was done in period 1. In May 2013 (M16) the 11th International Verticillium Symposium in Göttingen was attended for updating the above survey with recent research findings. In period 2 the scientific literature on Verticillium Wilt of Olive (VWO) has been reviewed extensively for information on control of VWO, epidemiology of VWO (including disease incidence – soil inoculum level relations) and disease classes and threshold levels for VWO. Searching the CAB abstract database using key words connected to these three themes resulted in over 70 useful references. Three groups of factors affect disease levels of Verticillium wilt in olive: 1) pathogen related factors (virulence of isolates present in soil, especially so-called defoliating isolates being highly virulent; and the inoculum level in soil), 2) host related factors (level of susceptibility of specific cultivars, and age of trees) and 3) agronomical factors (including history of a field, susceptible crops being grown on nearby fields or in between the olive trees, irrigation and weed control practices). These factors and their effect on VWO have been described in detail in D6.13 Guide of Best Practices. Information on the relation between inoculum density (ID) in soil and resulting disease incidences (DI) is very limited and fragmented, probably because this relation is strongly influenced by many factors as mentioned above. However, it is very clear that even low amounts of Verticillium in soil can result in serious disease, especially when D-type strains are present. In fact it is concluded that when this type of the pathogen is present in soil no olive should be planted. In addition it is reported that low ID levels of the ND type also may cause substantial disease in olive, especially in highly susceptible cultivars. For that reason in the disease risk system (developed in Task 6.4) 1 ms/g soil is used as threshold level.
Task 6.2: Correlation of LAMP results to ms/g soil. Inoculum density of V. dahliae in soil is traditionally expressed in the number of microsclerotia per gram of soil (ms/g soil), whereas the new VERTIGEEN data will be based on the amount of DNA of V. dahliae (fg DNA/gram of soil) detected by the new LAMP-based VERTIGEEN device. In order to understand what the VERTIGEEN data mean concerning numbers of microsclerotia in soil, the results obtained with the new LAMP-based method have be related to the ms/gram of soil values. At a Technical Meeting in M17 it was agreed to establish first the relation between ms/g soil and fg DNA/g soil as determined by a quantitative real-time PCR (Q-PCR) at the laboratory because at that time the VERTIGEEN device was not yet available and the LAMP detection was still being developed. Later on, when the Vertigeen device is operational, this can be developed further into a relation with the readings from that device. WUR and FERA collected and analysed a set of 34 soil samples with the traditional wet sieving and plating technique as well as with a quantitative real-time PCR laboratory method as developed in WP2. Analysis of the results revealed a clear correlation between the results of both methods. This means that it is very well possible to correlate data expressed in ms/g with data based on the amount of V. dahliae DNA detected in soil. After the Vertigeen device became operational in the last months of the project it turned out that the device in its present state is suitable for testing of soil samples for presence of V. dahliae but not (or not yet) for reliable quantification of the amount of V. dahliae present per gram of soil. Therefore developing a formula for translation of the Vertigeen data into ms/g soil data has not been possible and the Disease Risk Categories had to be related to ms/g soil data.
Task 6.3: Building database of actual ID-DI data from participating SME’s. For this task the soil sampling protocol from WP2 (preliminary version available in M15) and the new LAMP based device for testing of soil samples (preparation of which took more time than expected; see WP4 and WP5) were needed. In a Technical Meeting in M17 the delay and the way to proceed were discussed. It was decided to collect 100 samples from olive field soils, 25 from each participating country, in the summer of 2014 to be analysed later in that year by Vertigeen-LAMP technology. A sampling plan and an enquiry form for collection of data on actual disease index, main agronomical factors and environmental factors were developed by WUR and FERA and discussed at the General Meeting in M24. After the meeting a participant from each of the participating countries translated the form into their national language and sent it with the sampling kits to local olive farmers. Sampling in Portugal and some sampling in Spain was done by WUR and FERA during a sampling trip in August 2014. Together with the samples received directly from farmers this finally resulted in 92 soil samples available for analysis by September 2014. In order to complete the 100 samples for analysis, 8 more samples from Dutch tree nursery soils were included in this collection. For some of the received samples the enquiry form was lacking. As a result data from 83 forms could be included in a database on disease incidence and related main agronomical and environmental factors for specific fields.
The next step was characterization of the amount of V. dahliae in the samples by using the Vertigeen device. For this purpose Ateknea sent a prototype of the machine with about 50 cartridges for testing to both FERA and WUR in Month 33. The machine sent to FERA, unfortunately, did not function properly because what turned out to be a problem in the software. The one sent to WUR was used successfully for some validation experiments with known samples. However, technical problems with the machine and especially the lack of enough cartridges for further validation and testing made it impossible to analyse the collection of field samples. Finally Ateknea has performed this task in M36 after further improving the Vertigeen device. Analysis of the results achieved by Ateknea (see report on D7.15) showed that the device is suitable for reliable detection of V. dahliae. Reliable quantification of the amount of V. dahliae, however, appeared not (yet) possible which made analysis of the database for the ID-DI relation based on the ID of actual fields characterized by VERTIGEEN technology impossible.
Task 6.4: Defining disease risk categories
Data on the relation between soil inoculum level (ID) and disease incidence (DI) in Verticillium wilt of olive (VWO) appeared to be very scarce and fragmented (task 6.1 and 6.2). As a result it was not possible to base threshold levels for risk categories for VWO on information published in the literature. The only sound conclusion in literature concerning this topic is that on soils with the D-type isolates present no olive should be planted. For soils with ND-type isolates of V. dahliae disease risk is lower, but is strongly influenced by many factors as described above (task 6.1). Therefore, in order to make the Disease Risk Category system a robust system, the number of risk categories was limited to three: Low Risk, Limited Risk and High Risk. Based on the available information summarised in task 6.1 it was decided to use as threshold level 1ms/g soil. Low Risk is chosen for soils where no Vd is detected. This category deliberately is not called No Risk because the detection limit of the presently used techniques does not allow the detection of very low levels of V. dahliae. In such soils very low levels of V. dahliae may be present and even with such low levels disease may occur in individual trees especially because the pathogen may be unevenly distributed in soil. Also in long-living crops like olive there is always the risk of introducing the disease during the lifetime of the plantation. The second category is that of Limited Risk (i.e. only ND type of Vd present and ID < 1ms/g). For this category and the High Risk category (D-type of Vd or ND-type with ID > 1ms/g) actual risk levels are strongly influenced by local conditions. Therefore a decision scheme for deciding how to deal with this was designed and included in the Guide of Best Practices.
WP7: System Validation (to be completed by ATEKNEA, input from FERA and WUR will be welcome)
The objectives of WP7 were to prove the system functionality in field conditions and to compare the VERTIGEEN system performance with current detection methods in terms of speed, accuracy, sensitivity and specificity. The validation tests were meant also to demonstrate that the device fulfils the needs of the end-users.
During the initial validation phase the performance of VERTIGEEN device was assessed comparing it with a commercial CH-instrument potentiostat. Different Methylene blue solutions were tested with both devices and good linear correlation were obtained with over 99% of accuracy on both cases.
During the performance assessment of the cartridges several problems were faced, such as electrochemical signal obtained was indirectly proportional to the Methylene blue concentration in the solution, therefore cartridge production process was assessed step by step until the problem was solved. Once the cartridges were functional, a batch of 200 units was produced to proceed with the validation phase.
The next step was to calibrate the device, for that different LAMP experiments in the laboratory were done, electrochemically detecting pure Verticillium DNA. Different experimental conditions were checked, like the methylene blue concentration and the amount of sample needed in each test. The results obtained were good and linear correlations were obtained between the amount of DNA and the signal obtained, with adjustments over 85% in most of the tests.
In order to finalize the calibration, more LAMP experiments were done in the laboratory, but in this case the samples used were DNA extractions from plant and soil samples. After the firsts test, the methodology was changed in order to compensate the matrix effect produced by the samples, obtaining an accuracy of 84%.
Following, ATEKNEA started the Validation of the device. In order to do the laboratory validation, both equipment were sent to WUR and FERA. Different concentrations of pure Verticillium DNA were tested in both RTDs, obtaining good logarithmic correlations. With the results obtained, the final steps and conditions for the LAMP reaction were decided. The experimental conditions for the electrochemically detection were also fixed.
Before the in field validation, the threshold value was set in the device (based on previous experiments), being the detection limit of VERTIGEEN device 2,5ng/uL.
The in field validation was carried out with the DNA extracted from 100 different soil samples, being 91 out of 100 samples in accordance with the standard method (PCR). Of this 91% of accordance, 63 samples are positive and 28 negatives, the other samples are 5 false negatives and 4 false positives.
Furthermore, the hardware and the software were updated during these months of work, according to the problems that appeared with the continuous operation of the device. Also, a second software interface was created, so the non-technical end-users could use the device in a simple and easy way.
Finally, the user manual for the VERTIGEEN device was also prepared. In it there is included general information about the device performance, and a practical and easy-to-use guide on how to run an analysis and how to interpret the results obtained with the device. Also, the Standard Operation Procedure is included in the user-guide.
WP8: Dissemination and Training Activities
Several dissemination activities were planned and performed and all consortium members were actively involved. A project website (www.vertigeen.eu) was created to serve as vehicle of communication and dissemination of the project and its results. A project visual identity (logo, slogan, illustration, colours and lay-out) was also created and used in all the dissemination material prepared so far. Two project brochures providing information about the VERTIGEEN project have been created with the collaboration of all partners and it is being translated into the SME-AGs official languages (Spanish, Portuguese and Greek). One of the brochures reflects the main aim and the scope of the project, explaining the need and the chosen approach to solve the actual problematic. The explanation also includes the benefits that the expected results can provide to the olive sector. The second one explains more in detail the technology and highlights every partner contribution. A general project poster has been also created and has been shown in some dissemination events to which different project partners have attended. As well as the project brochure, the poster has been translated into other European languages. Also a scientific poster has been prepared.
Moreover, a project general presentation was prepared at the end of the project and it is available through SlideShare.
In addition, partners prepared several training materials to be used in external training events (workshops), a power point presentation (first version in English and then translated into the different official languages of the SME-AGs participating in the project) and a video which explains how the soil samples should be taken. This video is available on YouTube and it was used during the sampling requested to the SME-AGs.
WP9: Exploitation Activities
Associations, technical SMEs and end-users have undertaken discussions in periodic project meetings regarding the exploitation potential of the technology. All the associations in the project have stated their interest on the IPR resulting from the project and their motivation for proceeding with the exploitation activities required for ensuring the commercial success of the VERTIGEEN system. SME-AGs have defined the generated foreground and studied the possible protection strategies for each one of them. It was decided that trade secret will be applied to most of the results, but community trade mark for the logo developed as soon as the commercialization is about to start. Patent is not applicable in any of the cases and other strategies are not foreseen.
On the other hand, partners performed market and technology watch which allowed them to define the exploitation strategy. In this context, partners studied the possible licensing for using LAMP technology. Several options were studied, negotiate a license with the Japanese company (Eiken) who hold the LAMP patent and negotiate an exploitation agreement with other companies who already have a license from Eiken (such as Optigene and Prime Diagnostics).
During the second period of the project, technical SMEs supported by the RTDs have calculated the production and selling costs of the complete VERTIGEEN system. Moreover, the potential market has been slightly changed, focusing the efforts in diagnostics laboratory and nurseries and primary customers for the developed technology. The new business model provides a market worth over €10 million, and will provide the each one of the SMEs with over €100.000 the first five year, and the technical SMEs will have a profit of over €2 million after five years.
Commercialization routes have been also studied as well as the role of each one of the SME-AG and SME partner.
All this information has been gathered in one document “Final Business Plan and Plan for Use and Dissemination of Foreground, which also includes the background management and access rights for the different parties.
Potential Impact:
VERTIGEEN addresses a need identified by olive sector SME-Associations for the rapid and reliable on-site detection and future quantification of the Verticillium dahliae fungus in soil and plant samples. The developed technology is based on new but successfully demonstrated technologies: the loop mediated isothermal amplification (LAMP) method and the original in-solution-DNA electrochemical detection technology, overcoming the limitations of the state of the art in terms of heterogeneity, portability, price, easiness to use and time-to-result. With an expected final price of about €1.000 and a price per test of €10, the system would allow the associated olive producers to reduce the losses caused by this pathogen and its spreads by means of precise and efficient field interventions within an Integrated Pest Management (IPM) Strategy.
The competitiveness of the European olive lead market, already faced with growing international competition from Tunisia, Turkey, Syria, Morocco and Argentina and other adverse factors such as olive oil prices dropping steadily , is currently severely threatened by the soil-borne fungus Verticillium dahliae, responsible for critical damage to the vitality, productivity and yield of more than 200 plant species with crop losses estimated at thousands of millions of dollars worldwide every year. The nature of the infections and the characteristics of the pathogen -a soil habitat, survival structures that persist for years, demonstrated transmission potential and capacity to infect numerous hosts in a continually expanding geographical distribution worldwide, has made Verticillium wilt a chronic economic problem in global crop production. Non-defoliating and defoliating pathotypes of Verticillium dahliae vary in aggressiveness but overall, for commercial olive plantations, this disease is currently considered one of the most serious problems with reported incidences of 30% in some regions of Spain while other studies report olive crop losses ranging from 50 to 89%, depending on olive variety and pathogen aggressiveness .
The disease in olive trees was first described in Italy, and afterwards was reported in Spain, Greece, France, Turkey, Syria, Jordan Morocco and California . In recent years, verticillium wilt is occurring with increasing frequency and severity in most olive growing areas of the Mediterranean basin due to intensive farming of highly productive cultivars, planted at high densities and flood irrigated, all of which favour the spread of the pathogen. Other causes of the increased spread of the pathogen include the use of infected but symptomless plant propagation material for the establishment of new olive groves or planting in contaminated soil, which is becoming more common as the pathogen spreads by wind, water or dirty equipment that carries it into clean soil.
Driving consortium SME-AG partners INOLEO, EDOEE, AAR and their memberswere certain that containment and even eradication of this disease is feasible, but only if timely and accurate detection and quantification of the Verticillium fungus in soil and in trees (diseased as well as symptomless) is achieved at a comparatively low price and appropriate effective and sound economic measures are applied within the frame of an Integrated Pest Management strategy. In-field diagnostics SME partners FORSITE and LOEWE; as well as CITOLIVA technological provider and member of INOLEO, confirmed this need. Also olive producers and plant propagation nurseries such as COTEVISA, PELOSI and SAOV, consider a priority the development of innovative ways to defend against this pathogen and are highly committed to the development of the proposed VERTIGEEN technology.
Currently there is no single effective treatment for plants affected by Verticillium Dahliae, meaning that efficient control should be based on preventive and eradication measures. VERTIGEEN consortium not only developed a new, cost-effective, reliable system for early on-site detection of Verticillium Dahliae, but also a Guide of Best Practices in Managing Verticillium wilt in Olive. This guide containing protocols to control the disease in established orchards and prevent contamination of new olive groves or healthy parts of existing groves. This guide is the result of an extensive literature review and contains a decision scheme based on disease risk categories established on the results of work don along the project.
The economic impact of such a tool will affect:
- Specialized olive tree nurseries such as the consortium member COTEVISA, who will be able to assess the condition of sapling, grafts and other planting material before trading benefit from selling value-added certified healthy planting material, increase consumer confidence in their products, improved sales, better competitiveness, employment creation and even opening the door to new export markets.
- Olive growers will gain precise and rapid information, resulting in more efficient interventions. It will also allow to know which areas are affected by Verticillium Dahliae before a new plantation is considered.
The SME-AGs INOLEO, EDOEE and AAR foreseen benefiting from their participation in VERTIGEEN by improving the competitiveness of their associates, who will have early affordable access to a new diagnostic tool that will allow them to significantly reduce the losses caused by this pathogen and consequently to increase their profit margins, preserve employment and maintain a competitive edge in the global market. The AGs will also obtain direct economic benefits from their foreground ownership, via the profit deriving from the sales revenues, estimated at €111.229 for each of the associations in the first 5 years.
Moreover, VERTIGEEN holds the potential for rapid uptake not only in the olive sector but beyond, as in the future it may be adapted to the detection of Verticillium in many other affected agricultural crops such as strawberry, tomato, potato, cotton, vegetables, fruits or various landscape and ornamental trees and shrubs of high relevance in Europe and worldwide, increasing the potential post-project impact for the proposing SME-AGs. Industrial lead user partners FORSITE and LOEWE have also recognized a business opportunity in developing such a tool since apart from its applicability in routine plant pathogen diagnosis, VERTIGEEN can become a valuable tool in human health applications involving single nucleotide polymorphisms (SNP) and for diverse ecological and epidemiological studies (e.g. population dynamics, disease incidence and severity, etc.). These two partners actively supported the commercial exploitation route definition of VERTIGEEN, following agreement with the SME-AGs.
▪ Lead user partner FORSITE as expert developer and manufacturer of on-site diagnostic equipment will benefit from the manufacture of the VERTIGEEN device and disposable test tubes. They will open new markets and income sources with a new portfolio product at an estimated price of €1,000 and cost per test of €10. They expect to open a market niche worth more than €5,9 million in the olive production sector.
▪ Lead user partner LOEWE, which provides a wide range of phytodiagnostic tools will commercialize and distribute the technology, benefiting directly from the sales of VERTIGEEN and by improving their competitiveness offering a new solution to those customers who suffer important economic losses due to the presence of Verticillium dahliae in their orchards and nurseries.
By means of EU-wide strategically planned dissemination actions and highly developed exploitation processes, VERTIGEEN would allow the advancement in the control of this epidemic disease, which depends on the systematic and collective implementation of phytosanitary measures, and would maximize the competitive benefits for olive growers, olive oil producers and olive tree nursery SMEs and SME-AGs in all Member States.
Along the project, SME-AGs, SMEs and RTDs were deeply involved in Dissemination activities and definition of the possible exploitation routes that could be followed after the project ends.
The main dissemination activities carried out during the project include:
- Dissemination throughout the project website
- Dissemination through the partner’s website – linking the project website or even publishing press releases after the project meetings.
- Dissemination through Twitter, some partners used their company Twitter profiles to dissemination events of the projects.
- Publication of press releases in several specialized online media, such as Mercacei Magazine, Interempresas.net Agrodigital.com Sitioliva, Europapress, Mercadosdelvino.es publication for the Dutch Agricultural sector, German institute for Arboriculture, Data Archiving and Networked Services, Greek agricultural press, etc
- Presentations in fairs or symposium of the sector such as VI Simposio Nacional de Olivicultura (2012), Expoliva 2015, Encontro Iberico do Azeite (2013 and 2015), Ovibaje (2013), Andalusian Agrofair (2013), etc.
- Scientific publications (abstracts, poster or papers): 11th International Verticllium Symposium (2013), etc.
- Workshop organized by SME-AGs for their members or other members of the sector.
On the other hand, VERTIGEEN partners dedicated effort as well in the preparation of a Plan for Use and Dissemination of Foreground, which objectives were: To ensure that the SMEs beneficiaries could assimilate the results of the project, to disseminate the results beyond the Consortium to a wider audience, to support the participating SMEs in using and protecting the research results to their best advantage, and to exploit the results and increase the competitiveness of the SMEs beneficiaries. In order to do so, technology watch and market watch was carried out by all partners along the project, deep market study, background and foreground management (including IPR ownership and access rights on the foreground, protection strategies and licensing for future exploitation together with third parties). Moreover, SME-AGs together with the technical SMEs prepared a business case, based on the market analysis carried out previously, production costs of the different parts of the developed technology (sample kits and reader device), taking into account only the potential market in Spain, Portugal, Italy and Greece. Partners have also studied the possible commercialization route taking into account the area of interest of each one of the partners. Technical SMEs will mainly work as manufacture and distribution of the technology. SME-AGs will provide the publicity of the product and collect the royalties gathered from the sales per year.
List of Websites:
www.vertigeen.eu
Contact details (Project Coordinator):
Mr. Albert Nieto
email: albert.nieto@ateknea.com
Tlf: +34 932 049 922