Community Research and Development Information Service - CORDIS

FP7

CO-FREE Report Summary

Project ID: 289497
Funded under: FP7-KBBE
Country: Germany

Final Report Summary - CO-FREE (Innovative strategies for copper-free low input and organic farming systems)

Executive Summary:
The CO-FREE project aimed to develop innovative methods, tools and concepts for the replacement of copper in European organic and low input fruit, grapevine, potato, and tomato production systems by (i) providing alternative compounds, (ii) ‘smart’ application tools and (iii) by integrating these tools into traditional and novel copper-free crop production systems. CO-FREE aimed to identify strategies to develop (v) ‘smart’ breeding goals by the development of crop ideotypes and (vi) foster consumer acceptance of novel disease-resistant cultivars by consumers and retailers. The innovations and production systems were (iv) evaluated in a multi-criteria assessment with respect to agronomic, ecological and economic performance. In the course of the project, farmers, advisors, the plant protection industry, policy makers and researchers as well as stakeholders of the European organic and low input sector (food supply chain, retailers, producer associations) were closely involved to ensure rapid development, dissemination and adoption of the copper replacement/reduction strategies as soon as they became available.
In CO-FREE a total of 17 alternative compounds was investigated for which modes of action, formulations, and application strategies were explored in the lab and field. During the lifetime of CO-FREE, one active substance was approved under EC regulation 1107/2009, two registration dossiers have been submitted and three are in preparation. Furthermore, for three additional alternative compounds, efficacy was demonstrated under field conditions but additional R&D is necessary. Most CO-FREE candidates exhibited unproblematic ecotoxicological profiles in detailed studies on non-target organisms (beneficial arthropods, aquatic and soil indicator organisms). Costs for registration, however, are high and require a substantial initial investment by SMEs. This means that, together with the facts that (i) copper has a broad spectrum of activity, (ii) it is unlikely that one compound will have the potential to completely replace copper in all crops, (iii) the alternative compounds in the best cases had similar efficacy as copper and (iv) new compounds have to be safeguarded to remain effective over time, it is likely that different candidate compounds are necessary to further reduce/replace copper. Thus a range of products will be required for practice, to which CO-FREE has contributed strongly with a number of candidate compounds with a technology readiness level of 8, which provide the foundation for the development of new products for the market.
Practical Decision Support Systems (DSS) are one of the key approaches to copper reduction and yield security. Within CO-FREE, DSS for grape (downy mildew and black rot) and potato (late blight) have been optimized or developed from scratch. The beta versions of the DSS are ready for implementation into farming practice.
Robust/resistant varieties are a major contribution to copper replacement and their availability is constantly increasing. However, introduction into markets remains a challenge. In CO-FREE, strategies were developed to improve market introduction of new varieties, with a focus on potato. The variety approach is valuable in potato and tomato, for example, but feasibility also depends on crop and region, and especially in the case of grapevine, is severely limited by consumer expectations and the legal framework.
Advanced self-regulating cultivation techniques (agroforestry and Very Low Input Systems (VLIPS)) have been explored in grapevine and/or apple, both of which showed potential for promotion of biodiversity and reduction of copper use. However, these experimental production systems are not yet ready for implementation at scale. The potential of resilient systems in the context of reducing copper is not (yet) explored at length nor fully exploited. Substantial R&D investments are needed to develop these experimental systems into feasible mainstream options. In many cases, this will need at least 20 years. In CO-FREE, a partial proof of concept was provided in selected model systems and current limits were identified.
In conclusion, depending on the crop, a range of measures were developed and are ready to be implemented and used, while others need some more time; for example, for several of the alternative compounds investigated in CO-FREE, the earliest registration is likely to be completed from 2022. Also, new varieties need time to be adopted by farmers, retailers and consumers, and communication and commitment along the whole value chain is essential. Results indicated that strategies including the use of alternative compounds as one component together with DSS and further measures will be the way forward to further reduce/replace copper. However, the replacement and reduction of copper is currently slowed down beyond the technical potential by, for example, the legal frameworks, associated costs or lack of markets.

Project Context and Objectives:
Project description
The CO-FREE project aimed to develop innovative methods, tools and concepts for the replacement of copper in European organic and low input fruit, grapevine, potato, and tomato production systems by (i) providing alternative compounds, (ii) ‘smart’ application tools and (iii) by integrating these tools into traditional and novel copper-free crop production systems. CO-FREE aimed to identify strategies to develop (v) ‘smart’ breeding goals by the development of crop ideotypes and (vi) foster consumer acceptance of novel disease-resistant cultivars by consumers and retailers. The innovations and production systems were (iv) evaluated in a multi-criteria assessment with respect to agronomic, ecological and economic performance. In the course of the project, farmers, advisors, the plant protection industry, policy makers and researchers as well as stakeholders of the European organic and low input sector (food supply chain, retailers, producer associations) were closely involved to ensure rapid development, dissemination and adoption of the copper replacement/reduction strategies as soon as they became available.

Objectives
The objectives of CO-FREE are mirrored in the content and approaches of the work packages (WPs). The objectives cover the different aspects outlined above and lead via a continuous flow and feedback between each other to the development of copper-free production systems.

Objective 1 – Development of novel alternative compounds of microbial and plant origin
The development of alternative compounds involves a multi-step process from identification of a candidate compound over involvement of a company, to marketing and finally implementation in plant protection. As an outcome of ENDURE (Köhl et al, 2010), nine steps were characterized in this process, of which the early steps include the determination of targeted diseases and markets, the screening and identification of suitable compounds, database mining, and efficacy testing in bioassays. In later steps, aspects such as preliminary assessment of mass production potential, pilot formulations, up-scaling mass production in pilot plants, field testing and integration in cropping systems are needed. The alternative compounds to be further developed in CO-FREE all have reached one or more of the advanced steps of the ENDURE scale, and their potential to be successfully developed for the market is confirmed. In particular, the choice for compounds was based on the following criteria: (i) proven efficacy against at least one of the major copper-controlled diseases, with specific, clearly identified aspects remaining to be improved in the frame of the project (e.g rainfast formulation, clarification of mode of action for optimization of the application parameters), (ii) novelty, and (iii) involvement of a leading/innovative SME company ensuring further development and marketing. Care was taken that compounds from different origin (i.e. plants and microorganisms) and different modes of action (i.e. fungicidal or bactericidal activity and induced resistance) were included in order to achieve an optimal and broad approach.

Objective 1 was addressed by WPs 2 and 3 and facilitated a rapid development and market introduction of cutting-edge novel alternative compounds from microbial origin (Trichoderma atroviride SC1 & protein extract SCNB1, the oligosaccharidic complex COS-OGA, yeast-based derivatives, bacterial cultures/metabolites from Xenorhabdus sp. and Aneurinibacillus migulanus, the fungal antagonist Cladosporium cladosporioides H39) and from plant origin sage, liquorice, PLEX, seaweed by providing gap-filling research to leading/innovative SMEs. In the course of the project six additional promising candidate compounds from CO-FREE partners were included. The specific objectives were:
• to optimize field application protocols for inducers of resistance and biocontrol agents based on improved knowledge of the interaction between plant, pathogen and environment (data ready stepwise in months 12, 24, 36, 44);
• to characterize the active compounds in the plant extracts and their mechanism of action (data ready stepwise in months 12, 24, 36, 44);
• to determine the spectrum of activity of products of microbial origin and plant origin (data ready stepwise in months 10, 22, 34);
• to develop suitable formulation to maximize the efficacy of resistance inducers and products from microbial origin (data ready in months 10, 12, 24, 34, 36, 44);
• to formulate the active compounds of plant extracts in order to increase efficacy, UV stability, rainfastness and shelf-life (data ready stepwise in months 10, 22, 34);
• to optimize/upscale the production process and reduce production costs (data ready stepwise in months 12, 24, 36, 44);
in order to finally develop novel copper-alternative compounds of plant and microbial origin (finally ready in month 48).

For each compound, CO-FREE focused on a specific sub-set of such objectives depending on the identified knowledge gaps as indicated in the WPs. In order to confirm the efficacy of the formulated compounds, small scale tests under controlled conditions on potato and tomato against P. infestans, on grape against P. viticola and on apple against V. inaequalis were carried out.

Objective 2 –Optimization of use of alternative products and management tools
The optimization of use of alternative compounds and management tools aimed at achievement of maximum disease control by combining the project-developed alternative compounds from objective 1 into disease control strategies in organic and low input/IPM farming systems. These were used together with agronomic tools, such as disease tolerant varieties and optimized use of application equipment. Growers were provided with tools (computer aided and technical notes) in decision making for timing and optimization of application of such compounds. In this process, results from WP 4 were used together with results from outside the project for building of disease control strategies to be finally tested in the different crops. The work to address objective 2 was performed in WP4. The specific objectives were:
• to develop disease control strategies which optimize and combine the application of copper alternative compounds from objective 2 with pre-existing knowledge and/or knowledge generated by other parallel research projects (strategies developed stepwise in months 6, 9, 12, 21, 24, 33, 36, 44);
• to adjust spray techniques to the characteristics of the alternative compounds (data ready stepwise in months 10, 22, 34);
• to assess the feasibility of substituting copper with alternative compounds in fungicide resistance management (ready in month 48);
• to develop decision support systems (DSSs) exemplarily for an annual (potato) and a perennial crop (grape), which consider the plant (level of susceptibility), the pathogen (level of virulence), the yield expectations (only for potato) and the optimized use of alternative compounds (based on mode of action, persistence, intrinsic efficacy) to help growers in implementing the diseases control strategies (ready in month 48);

Objective 3 –Development of copper-free fruit production systems
For achievement of objective 3, novel techniques and technologies (see objectives 1 and 2) were evaluated in the context of different copper-free apple production systems. These apple production systems included (i) traditional, high density organic and IPM plantations of scab-susceptible cultivars in the main apple production areas in Europe, (ii) high-density, high yield production systems with scab-resistant cultivars, (iii) high-yield production systems that include all preventive techniques to lower disease pressure, as well as (iv) innovative agroforestry based apple production systems that combine apple production and arable crops. The work concerning objective 3 was covered in WP5. The assessments of trials included (i) yield, quality, (ii) pests and diseases with a special focus on occurrence of apple scab and secondary diseases (iii) impact on non-target organisms and ecological services of the system, as well as economic performance. The specific objectives were:
• to evaluate novel tools and techniques to control key pathogens without copper in traditional scab-susceptible and scab-resistant high-density apple orchards (results ready stepwise in months 12, 24, 36, 44, 48)
• to evaluate novel techniques within advanced high productivity apple production systems that include all known preventive measures (including resistant cultivars, reduced planting density (ready in month 48)
• to develop and assess the performance and environmental impact of highly diversified low input agroforestry apple production systems (ready in month 48)

Objective 4 –Development of copper-free grapevine production systems
In order to develop copper-free grapevine production systems, novel techniques and technologies (see objectives 1 and 2) were used and evaluated. These grapevine production systems included (i) traditional vineyards with downy mildew (Plasmopara viticola) with susceptible Vitis vinifera cultivars in the Atlantic (France), Mediterranean (Greece, France) and continental (Northern Italy, Switzerland) wine production areas in Europe, and (ii) vineyards planted with interspecific, downy mildew tolerant cultivars (France, Switzerland). Assessments addressed (i) yield, quality, (ii) pests and diseases with a special focus on occurrence of secondary diseases, (iii) ecological impact on non-target organisms and ecological services of the systems as well as economic performance. The work to address objective 4 was covered in WP6. Specific objectives were:
• to evaluate novel tools and techniques to control key pathogens without copper in traditional downy mildew and black-rot susceptible vineyards (results ready stepwise in months 12, 24, 36, 44, 48)
• to evaluate novel techniques within advanced high productivity grapevine production systems that include all known preventive measures (including disease-tolerant cultivars) (ready in month 48)
• to explore the potential of downy mildew tolerant production systems with respect to environmental impact and ecosystem services (ready in month 48)

Objective 5 –Development of copper-free annual crops (potato, tomato) production systems
Copper plays a major role in organic cultivation of potato and tomato as stand-alone control of late blight (P. infestans), while in integrated/low-input farming it is used as tank-mix with synthetic fungicides. Objective 5 aimed at the evaluation of novel production tools and strategies for potato and field tomato, free of copper use. The integration of novel alternative compounds, decision support systems (DSS) and of cultivars ideotypes, not only chosen for resistance towards late blight, but also for other agronomic factors (yield, quality, nutrient use efficiency), is important and was addressed. Assessments included (i) yield and quality, (ii) diseases (including P. infestans and Alternaria solani), (iii) ecological impact on non-target organisms, and economic performance. The work to address objective 5 was covered in WP7. Specific objectives were:
• to evaluate novel alternative compounds and strategies in traditional organic and low-input potato and field tomato production systems (results ready stepwise in months 12, 24, 36, 44, 48)
• to develop potato ideotypes and associated management systems to meet the demands of the market and to compensate for agronomic deficiencies (results ready stepwise in month 12, 36, 48)

Objective 6 –Ecological impact assessment of novel copper-free techniques and production systems
Generally, it can be expected that compounds of natural origin, as used in CO-FREE and addressed in objective 1, degrade fast in the environment and thus may only have minor ecological impact. However, this needs to be proven by eco-toxicological studies in the laboratory, and in the field. The results of these studies serve also for the SMEs for justification to invest into the development of a full dossier required for registration of alternative compounds. Since novel copper-free strategies as described under objectives 3, 4, and 5 are supposed to have positive effects on the environment, eco-toxicological studies are needed to prove and quantify the added ecological value of such systems.
Objective 6 was a horizontal activity addressed in WP8. The overall aims of objective 6 were to (i) investigate eco-toxicological side-effects of formulated novel alternative compounds and strategies addressed in objective 1 in initial laboratory studies, and (ii) to quantify positive impacts on the environment for novel strategies and crop production systems addressed in objectives 3, 4 and 5. The specific objectives were:
• to evaluate the potential toxic effects of the novel alternative compounds in the laboratory on beneficial arthropods species (results ready stepwise in month 35, 48)
• to evaluate the potential adverse effects of the novel alternative compounds in the laboratory on non-target aquatic indicator organisms (results ready stepwise in month 35, 48)
• to evaluate potential effects of the novel alternative compounds in the laboratory on earthworm species as soil indicator organisms (results ready stepwise in month 35, 48)
• to evaluate negative effects of selected alternative compounds and to quantify positive environmental effects of novel strategies and production systems in the field (protocol ready in month 5; results ready stepwise in month 36, 48)

Project Results:

Work package 2: Development of novel copper-alternative compounds of microbial origin
Task 2.1 Development of Trichoderma atroviride SC1, protein extract SCNB1 and Lysobacter capsici AZ78 (FEM)
Experiments under controlled conditions were conducted in order to study the mechanism of action of three copper alternatives against grapevine downy mildew [Plasmopara viticola] and tomato late blight [Phytophthora infestans]. A fungal (Trichoderma atroviride SC1) and a bacterial (Lysobacter capsici AZ78) biocontrol agent were analyzed, as well as a protein extract (SCNB1). Dosage and timing of SC1, SCNB1 and AZ78 treatments were optimized to maximize the efficacy against downy mildew and late blight. Results indicated that weekly preventive treatments are required in order to achieve sufficient efficacy against the two diseases.
Trichoderma atroviride SC1. SC1 acts by inducing grapevine resistance against downy mildew and showed slight direct toxicity against Pl. viticola sporangia. SC1 formulation was optimized for field applications.
Protein extract SCNB1. SCNB1 showed complex mechanism of action: a mild direct toxicity against Pl. viticola sporangia, induction of defense-related genes in grapevine and improvement of bacterial population on grapevine leaves. The formulation of SCNB1 was optimized.
Lysobacter capsici AZ78. Concentration and timing was optimized. The application of AZ78 (108cfu/ml) one day before pathogen inoculation (P. infestans and P. viticola) reduced the pathogen attacks to the same extent as a copper based fungicide. Furthermore, the improved efficacy reached through the combination of AZ78 and low doses of a copper based fungicide prompted its development as a novel plant protection product. During this work, the capacity of AZ78 to produce lytic enzymes and release compounds toxic against phytopathogenic oomycetes was shown.
The effect of temperature on the plant protection efficacy of AZ78 was assessed and the product was assessed in field trials. A prototype formulation for the application in field was developed. The additives used in the formulation protected the AZ78 cells under field conditions guaranteeing its survival and, as a consequence, its control of P. viticola was similar to a copper based fungicide.
During this project, grapevine and downy mildew interactions with these biocontrol agents and resistance inducers were also better characterized with novel molecular tools. We demonstrated that AZ78 needs to compete with indigenous communities to properly display biocontrol properties, and this parameter should be considered to optimize the use of biocontrol agents under field conditions. Also, abiotic stress exposure should be taken into account to maximize the efficacy of resistance inducers under field conditions.
Task 2.2 Development of the oligosaccharidic complex COS-OGA (FYTO, FiBL)
Experiments were conducted to (1) optimize field application protocols for the oligosaccharidic complex treatments and (2) set up new formulations to maximize efficacy of the oligosaccharidic complex on solanaceae against P. infestans and on grapevine against P. viticola under controlled conditions.
Optimization of the field application protocols
The accumulation effect of the elicitation signal has been demonstrated on tomato plants and the persistence of the signal was observed for at least 22 days after one single spraying.
Temperature and humidity seem to have no major effect on elicitation of tomato plants.
Rainfalls simulations were carried out on tomato plants previously sprayed by the oligosaccharidic complex. The elicitation signal was not affected by rainfall. Showing that the oligosaccharidic complex triggers cell response very fast after which it can be washed off from the leaf surface.
Gene expression caused by formulation 928B7 was analysed in grapevine seedlings under controlled conditions. Plants treated with the reference-inducer BABA showed enhanced expression levels in selected genes (Gluc, PR1, STS) as compared to water-treated plants whereas formulation 928B7 showed enhanced expression-levels as compared to water-treated plants in two of the examined genes (STS, LOX9).
Innovative application strategies were explored in field trials with grapevines and apples by combining COS/OGA treatments with preventive treatments with contact fungicides.
In conclusion, formulation 928B7 is now a well developed product that contributes to disease reduction in several crops, including apples if used in a refined disease control strategy. However, its potential to reduce copper use on grapevines is limited.
Setting up of new formulations to maximize efficacy of the oligosaccharidic complex
Based on transcriptomics (on tomato and potato plants) and proteomics (on potato plants), two different formulations were investigated (928B7 and 6715B). If 928B7 clearly triggers SAR response, the mode of action of 6715B appears to be much more complex.
The efficacy of these two formulations against P. infestans was assessed in controlled conditions on potato plants. Formulation 928B7 poorly reduced P. infestans symptoms (about 30% efficacy), whereas formulation 6715B offered an almost total protection against late blight (99% efficacy).
Task 2.3 Development of yeast-based derivates (ALD, FiBL)
Experiments were conducted to optimize field application protocols for yeast derivative treatments and to set up new formulations to maximize efficacy of yeast derivatives on grapevines against P. viticola under controlled conditions.
The impact of yeast derivative treatments on expression of resistance-related genes was quantified as a basis to optimize application strategies. Both, yeast-based derivate ALD1901 as well as the reference inducer BABA significantly reduced disease severity caused by P. viticola. Plants treated with the reference-inducer BABA showed enhanced expression levels in three of the selected genes (Gluc, PR1, STS) as compared to water-treated plants. When compared to ALD1901-treated plants, expression levels of BABA-treated plants were enhanced in three more genes (LOX9, NCED and PR17).
A total of 3 bioassays with 9-12 different treatments each were conducted to quantify the impact of application time before challenge infection, incubation time, and inoculum density on efficacy of ALD1901.
In order to optimize the efficacy of ALD1901 for treatment of grapevines under field conditions, the combination of ALD1901 with a range of 4 reference additives was evaluated in various concentrations under lab conditions. The combination of ALD1901 with 4 standard organic contact fungicides was also evaluated in order to quantify any compatibility issues if ALD1901 is to be integrated in practice-oriented crop protection strategies.
The test substance ALD1901 and ALD1901 & Heliosol and ALD1901 combined with a low dose of copper was evaluated against downy mildew. In a field season that was very conducive to downy mildew, resistance inducers, if applied alone, in general failed to protect grapevine leaves and grape bunches effectively against grapevine downy mildew.
Based on lab experiments conducted in 2012 and 2013 and the field experiment conducted in 2012, a selected strategy that implements ALD1901 both alone and in combination with other plant protection products was evaluated.
In conclusion, the optimized formulation of yeast based derivative treatments contributes to disease reduction in several crops, especially against powdery mildews. However, its potential to reduce copper use in grapevines proved to be very limited in the season that it was tested.
Task 2.4 Development of H39 (DLO)
Experiments were conducted to study the interaction between H39 and apple scab (Venturia inaequalis) in vitro, under controlled conditions and under field conditions. The main objective was to determine the developmental stage of V. inaequalis that is most vulnerable for the antagonistic activity of H39. Based on results obtained in vitro and under controlled conditions, field trials were conducted with applications of H39 at different degree hours before and after natural infection of V. inaequalis. Results of the trials indicated that C. cladosporioides H39 reduced V. inaequalis sporulation even if applied after a (forecasted) infection event of V. inaequalis. The effect of such treatments after infection events was stronger compared to effects of treatments shortly before infection events.
The timing of antagonist applications was studied in a series of field trials in organic and conventional apple orchards outside the CO-FREE project by partners collaborating with DLO. Spray schedules were fixed as calendar sprays during the initial field trials and were subsequently developed further to stake account of forecasting models. In such schedules, H39 applications were positioned 1 to 2 days after a forecasted infection event, based on the results obtained within CO-FREE Task 2.4. The results of these field trials including results obtained in CO-FREE Task 2.4 by DLO have been published as peer-reviewed article in Plant Disease in 2015.
Task 2.5 Development of Aneurinibacillus migulanus, Xenorhabdus and Photorhabdus sp. and Lysobacter capsici (ENE, JKI, FEM)
Work on the development of the following microbial agents was carried out: the gram-negative enterobacteriaceae Xenorhabdus bovienii, X. nematophila and Photorhabdus luminescens, all symbiotically associated with commercially produced entomopathogenic nematodes; the spore-forming bacterium Aneurinibacillus migulanus (syn. Bacillus brevis) and, in the last 18 months of the project, on the bacterium Lysobacter capsici (AZ78).
Development of Xenorhabdus sp. and Photorhabdus luminescens
The four bacteria symbionts of entomopathogenic nematodes suppressed the zoospore hatch of P. infestans at concentrations as low as 0.1% fermentate. One of the isolates (X. bovienii EN02) was further tested in detached tomato-leaf assays and the best isolate was finally tested in a whole plant assay on two tomato varieties. Disease suppression was 90 to 100% (statistically significant) after treatment with a 20% or 5% aqueous suspension of a freshly grown X. bovienii EN02 for both tomato varieties tested.
Development of A. migulanus
Former research on the bacterium A. migulanus had shown production of two bioactive compounds, the cyclopeptide Gramicidin S and a peptide surfactant, in shaken flasks but it failed to do so in bioreactors. To elucidate the mechanisms responsible for this difference, the cultivation in shaken flask cultures was optimized.
The biosurfactant of A. migulanus was not determined directly. Instead, the surfactant activity of the bacteria broth was tested by a droplet-spreading test. In this experimental series, it became evident that a heat treatment of the bacteria broth did enhance the biosurfactant activity.
The optimization of the growing medium for A. migulanus was done using a genetic algorithm. With the best performing medium composition, yields of 1e10 viable spores per ml were obtained in shaken flasks and in pilot bioreactors of 30 l and 200 l. The spores were spray-dried to obtain the wettable powder for field experiments. The attachment to the leaves and the rain fastness can be improved by adding suitable spreaders and stickers to the spray-suspension.
Development of Lysobacter capsici
Due to rearrangement of funding, an additional bacterium was included in the last 18 months of the project. This was a gram-negative bacterium, known to survive well under refrigerated conditions if the supernatant is removed and replaced by sterile Ringer solution (Partner 3, FEM). This type of formulation is difficult to scale up, since it would require a separation of bacteria from the supernatant in large amounts under sterile conditions. Several alternative procedures were therefore assessed for the formulation of the bacteria. The suitability of the formulation was assessed by measuring the viability of the bacteria in the stored product.
The composition of the production medium for the bacterium was optimised successfully. Like the medium for A. migulanus, it is based on cheap raw-materials to render the production cost effective.
Further work should aim to define the best harvest time and water activity to obtain good storage stability under non-sterile conditions since maintaining sterile conditions on a large scale would be expensive and prone to failures.

Work package 3: Development of novel copper-alternative compounds of plant origin
Task 3.1: Development of sage extract (FEM, CER)
Ethanol extract of sage, Salvia officinalis, obtained with Soxhlet method, has shown high activity against P. viticola, its activity is similar or better than the efficacy of copper and it considerably reduces infections of downy mildew.
The possibility to extend the use of sage extract in plant protection against other fungi (P. infestans and V. inaequalis) was evaluated. The interesting ability to control P. infestans on tomato confirmed the antifungal activity of sage extract against oomycetes.
In the CO-FREE project a simple method to extract the active compounds from dried sage leaves, reducing the high production costs, was developed. The sage extract was dried and milled resulting in a powder form which is cheaper and easy to use.
The efficacy of sage extract obtained with the new method against P. viticola was tested under greenhouse conditions. The effectiveness of the new powder extract was similar to the old ethanol extract. This new industrial method of production developed in CO-FREE allows large amounts of sage extract to be obtained, with reduced risks for the operator in the phase of production and halved the costs.
To optimize the crude extract, and to find cheaper sources (including industrial synthesis) the active molecules in sage were characterized via chromatography separation and GC-MS analysis. Two molecules have shown a good efficacy against downy mildew (Molecule A and B), especially if used in combination.
To improve the rain fastness of the new sage extract, it was formulated in collaboration with partner CER. However, addition of adjuvants always resulted in a reduced efficacy against the diseases or did not affect the rain fastness. On the other hand, the combination of the extract with a natural product used to create a biofilm, showed promising results in laboratory and greenhouse. Unfortunately, these results were not confirmed in the field experiment in 2014, a year with extreme rain fall in Italy.
Today new technologies such as nano- or microencapsulation could be a winning opportunity to formulate the extract of sage or other plants. This approach could be the solution to reduce wash-off, however each step that improves and modifies a product generally increases the final cost.
To find new cheaper sources of the identified active natural compounds, the effectiveness of the natural compounds of seven plants belonging to labiatae, asteraceae and pinaceae families were tested against P. viticola in laboratory and greenhouse trials. Three of them showed a good efficacy similar to copper and sage. Moreover, the combination of these plant extracts, seemed to allow a substantial reduction in the amount of raw materials used. Additionally, the combination some of these plant extracts with S. officinalis increased the activity of sage extract against P. viticola and reduced it´s wash-off. Further, a plant, belonging to solanaceae family, that produced both the secondary metabolite of interest and a high amount of biomass in a short time was identified.
The idea to synthesize the secondary metabolites of S. officinalis with a cell culture technique failed, due to difficulties optimizing efficient production and extraction protocols, high industrial costs and the use of eco-unfriendly substances or solvents.
We conclude that the possibility of wide future use of these plant extracts is possible; however, some obstacles (optimized formulation and plant production in terms of active ingredients content) still need to be overcome before a commercial product can be produced.
Task 3.2: Development of Liquorice-based extract (TRIF, JKI)
Treatments with ethanolic licorice extract (Glycyrrhiza glabra) has shown to be highly active against P. infestans infection on tomatoes and Pseudoperonospora cubensis infection on cucumber. Initial trials using the raw extract on apples against V. inaequalis also showed promising results. The overall results of the indoor trials showed that the activity of crude licorice extract against oomycete pathogens is similar to the efficacy of copper and significantly reduces infections by downy mildews. During the CO-FREE project an additional efficacy confirmation under controlled conditions on grapes against P. viticola was carried out by partner FEM. However, when applying the crude extract in the field, efficacies are much lower. Outside of the CO-FREE project an improved formulation which increases efficacy, UV stability and shelf-life was successfully established (project funded by the German Central Innovation Programme for SMEs (ZIM) of the Federal Ministry for Economic Affairs and Energy, KF 3113801MD3). Thus TRIF maximized the opportunity to finalize a commercial product.
A high variation within the G. glabra accessions in their corresponding chemical profiles was found. By the end of the project, two reliable licorice suppliers, able to supply in tonnes with economic feasible production costs, were located.
Within CO-FREE, a proof of concept regarding the extraction process in terms of active ingredients, yield, shelf life of the extract (> 2 years), etc., was developed by TRIF as a successful scale-up from laboratory scale to a pilot scale.
For the characterization of the active molecules in the extract TRIF collaborated with the Italian Institute of Biomolecular Chemistry, Catania. In total, 15 components have been identified and quantified. In in vitro trials against P. infestans, one fraction showed the best efficacy, and the corresponding 3 compounds were identified and confirmed to be the same as reported earlier by JKI.
A general optimization can be applied by either optimizing the extraction process outlined above and/or by purification of the raw extract by increasing the amount of the active molecules afterwards. In principle, purification of the extract was possible, but this approach was not the final solution in terms of feasibility and cost. The optimization goal in terms of extraction, upscaling and purification is still ongoing. Down stream processing, linking raw material properties, process optimization and product attributes, as an integral approach to identify and adopt the specific requirements of processing systems will be used and will be the next step of TRIF.
In previous studies, there was evidence that the high potential of licorice extract in controlling oomycetes might be not only due to its unquestioned direct effect on the pathogen, but also to an indirect mode of action. Based on the results in Task 3.2 of CO-FREE, fractions of the raw extract should be tested for their assumed resistance inducing effects.
Via DAB (Diaminobenzidin) staining it could be shown that like in cucumber the treatment of healthy tomato leaves with licorice extract or its active fractions (F4=sterols and terpenoids (not fungitoxic); F6=acidic substances incl. flavonoids (fungitoxic)), lead to an accumulation of ROS (reactive oxygen species like H2O2) in the treated tissue. All immune response pathways in plants start with such an accumulation of ROS. Since it was not clear, by which signal transduction pathway, licorice extract and F4 would act, the work focused primarily on identification of possible pathways involved. Thus, PR- genes (pathogenesis related genes) of the three main immune pathways (jasmoinc acid: PIN2, salicylic acid: PR1a, GlucA; ethylene: NP24, PR1b1, ERF4) were analyzed in tomato plants (cv. `Red Robin´) treated with crude extract and with F4. Results indicated that it is most likely, that the induced effects in the plants after treatment respond to the jasmonic acid pathway. Furthermore, a cross talk between the pathways has to be assumed.
Task 3.3: Development of PLEX (AKI, FiBL)
PLEX is an ethanolic plant extract. It has shown high activity under controlled conditions against P. viticola and V. inaequalis, causal agents of grapevine downy mildew and apple scab. The efficacy of this extract was similar to the efficacy of copper treatments (80-95%) for concentrations between 1000 and 2500 ppm.
Furthermore, the extract showed a good activity against Fusarium sp. and Microdochium sp. (IC50 between 300 and 1000 ppm) in vitro. This confirms its antifungal activity against ascomycetes and its potential broad spectrum activity.
Bioguided fractionation and LC-DAD-MS analysis of the PLEX-extract were carried out to identify active compounds, in order to optimize PLEX production and formulation. Two active compounds were identified in three active fractions (F2.1, F2.2, and F2.3).
These two compounds were quantified in several PLEX samples in order to optimize the harvest period. They were extracted and analysed by LC-DAD and active compounds were quantified by external calibration.
According to these preliminary results, the best harvest period for the location investigated appeared to be from July to November.
Process development and extract formulation were then realised to optimize PLEX efficacy under field conditions. A production upscale is under development in order to optimize the extraction process in larger amounts to provide formulated extract for field test season.
The efficacy of PLEX crude extracts and PLEX formulated with several adjuvants was tested under controlled conditions. The addition of some adjuvants allowed the development of a formulated product, with a high efficacy and good rain fastness.
Field experiments with this formulation have shown a high efficacy of the product against P. viticola and V. inaequalis. Further developments are necessary (production process and formulation optimization) but PLEX appeared to be a potential alternative to the use of copper for the treatment of these cultures.
Task 3.4: Development of seaweed-based extracts (BIOAT, JKI, FiBL)
The aim in CO-FREE was to optimize BioAtlantis seaweed based formulations towards a level of efficacy that matches copper based fungicides currently available in the market. A seaweed-based extract manufactured by BioAtlantis can enhance biotic stress tolerance, achieving two to three fold reductions in diameters of Alternaria induced lesions. This seaweed formulation 19-01 was used in the first phase of CO-FREE. At the initial stage of the project, we examined the mechanism of improved plant self-defence (peroxidase activity) elicited by 19-01 in a tomato- Alternaria interaction in a growth chamber facility at BioAtlantis. The study observed and quantified an upregulated peroxidase activity at the protein level in 19-01 treated tomatoes after challenge inoculation with a toxin from a fungal pathogen, Alternaria alternate.
In-house trials using 19-01 was subsequently performed by CO-FREE partners, JKI (cucumber/downy mildew) and FiBL (grape/downy mildew) in WPs-3.
As an initial step, plant self-defence inducing property of pure seaweed based extracts was checked in CO-FREE. It was demonstrated that pure seaweed-based plant priming (formulation 19-01) triggered systemic signaling response and up-regulation of peroxidase only when challenged by external stimuli such as pathogen infection. However, the maximum efficacy level obtained via triggering plant self-defence was only 49.3% against downy mildew in controlled conditions. In order to maximize the efficacy, pure seaweed extracts were combined with other naturally derived extracts at the BioAtlantis research laboratory. Thus, a range of potential seaweed based blends were developed and were subjected to testing against various plant-pathosystems in the CO-FREE platform. Out of 36 different seaweed based blends, four formulations were highly effective in controlling downy mildew of grapevine caused by P. viticola (In-house testing under high disease pressure). It was further proven that its efficacy was not lost even after artificial rain simulation of 25mm. Out of four promising seaweed based blends, two chemically distinct formulations will be further subjected to field level trials to prove their efficacy under different climatic and disease pressure conditions before it can be commercialized.

Work package 4: Optimization of use of alternative products and management tools
Task 4.1 Optimization of application strategies based on project-generated and pre-existing knowledge (FiBL, JKI)
A database (DB) on the characteristics of the novel copper alternative compounds investigated in WPs2 and 3 was established based on available knowledge, either from literature or from experts and data generated within the CO-FREE consortium. As agreed by the partners in the consortium agreement, priority was given to IP protection in line with the requirements of the involved industry partners. The DB consists of confidential areas to which only the partners providing the data and the industry providing the product have access. The coordinator and the deputy coordinator have full access to the data base as agreed by the partners. At the end of CO-FREE, the DB included a large number of datasets with stand-alone applications and strategies built from alternative compounds (variations in formulation, concentration and combination) plus the respective references and controls. Datasets on alternative compounds from 2012 to 2015 comprised in grapevine 91 entries, in apple 41 entries, in potato 40 entries and in tomato 68 entries. The database proved to be a very valuable tool to track the outcomes of the individual trials in different locations.
Task 4.2 Optimization of use of existing application equipment for alternative compounds (JKI)
In this task, three finally formulated CO-FREE test products (licorice extract, Aneurinibacillus migulanus, seaweed extract) were foreseen to be tested in a spray lab to optimize the use of existing application equipment in tomato against P. infestans. During the project, all of the alternative compounds were further optimized, but no end-formulated product was available in time, so that objectives had to be adapted in part.
Results for preliminary formulations of licorice extract, gained in the frame of a German national project (BÖLN 09OE101) have been produced and are available. Two preliminary formulations of seaweed extract were investigated in this task for determination of suitable nozzle type and water amount. In addition, the more stable formulation was tested with regard to rain fastness.
Determination of ideal nozzle type and application amount
The optimal leaf foliage coverage was achieved by an application rate of 800l/ha for the licorice formulations (BÖLN 09OE101) and 1200l/ha for seaweed formulations. The selected nozzle type for the preliminary licorice formulations was Lechler Air-injector compact nozzle IDK 12004, whereas the preferred nozzle type for the preliminary seaweed formulations was the standard nozzle EVS 80 04.
Determination of the best application amount for preliminary licorice formulations
The best control of P. infestans on potted tomato plants (disease severity 3%) could be achieved with an application rate of 800l/ha. Further increasing of the application rate to 1200l/ha led to a decreasing efficacy (disease severity 30%).
Investigation of rain fastness of preliminary seaweed formulations
The formulations 19-16B and 19-16C of seaweed extract were tested in trials on tomato for their rain fastness. Both seaweed formulations significantly controlled the infestation with P. infestans without rain. The effect was comparable to copper treatment (19-16C) or even better (19-16B).
Seaweed extract 19-15B and copper showed a sufficient rain fastness in treated tomato `cv. Red Robin´, whereas the rain fastness of 19-16C was low. However, in 1915-B a mild phytotoxic effect could be observed which was absent in 19-16C.
Subsequently based on this investigation, the preliminary formulation 19-16-C, which showed significant disease control without rain, was re-formulated to overcome the rain fastness issue in CO-FREE.
Task 4.3 Management of fungicide-resistant pathogen populations by alternative compounds in low input/IPM strategies (JKI)
Pathogen strains of P. infestans (2 sensitive and 2 resistant to metalaxyl) were examined regarding their sensitivity towards the alternative compounds under lab conditions.
The results showed that most of the CO-FREE test products under investigation, which exhibited fungitoxic effects were to some extent active against different P. infestans strains. Moreover, three CO-FREE test products were efficient in controlling both, resistant and sensitive strains of P. infestans at increased concentration rates. This is of importance for their performance in the field, where in different regions and also in different sites, different pathogen strains will occur.
The results demonstrated that most of the tested alternative compounds are generally suited for use instead of copper in resistance management strategies against P. infestans.
Task 4.4: Management tools to support growers and extension services in applying disease control strategies: decision support systems in grape (BIFA)
The model
A dynamic population model for the development of downy mildew epidemics in vineyards was developed. The model is based on published information. Multi dimensional fractional boxcar trains are used to simulate the dispersion in the flow of the population through the successive biological stages. Population models simulate the development and growth of a disease population in a crop what enables to quantify the actual infection risk during successive infection events.
Validation
Between 2008 and 2014 observations were made on the development of the downy mildew epidemic by partners from CO-FREE (FEM, FiBL, BPI) and others in untreated vineyards in Bulgaria, Austria, North Italy, on Sicily, Spain, Switzerland, Greece, England, Belgium, The Netherlands and Quebec. These locations represented very different climatic conditions. Observations were made on standard European grape varieties, as well as on less susceptible interspecific varieties. On each location on 5 to 12 dates during the growing season observations were made on the incidence of downy mildew.
Conclusions
(i) In 97% of the cases the first infection was observed on the day it was predicted, or later. This means that fungicide treatments can safely be omitted until the model signalizes the first infection event. Depending on the location, this can result in a considerable reduction in number of fungicide applications.
(ii) After first infection has occurred, the observed epidemic closely resembled the simulation results. This means that simulated infection moments and their severity can be used to judge the necessity to apply a fungicide.
(iii) Infections indicated by the model a slight infection risk in most cases did not lead to symptoms on more robust interspecific grape varieties, meaning this can be used as threshold to distinguish between treating European grape varieties and less susceptible interspecific varieties.
(iv) Additional work is necessary to implement the use of scouting results as feedback for the simulations. This would further localize the simulations and adjust the forecasted infection risk for next rain events, to effectiveness of previous treatments in this vineyard.
Public accessibility of the model
During the project, the downy mildew model was re-coded in Java to enable public accessibility on a cloud based platform. www.rimpro.eu is a platform for pest and disease models to be used as decision support systems (DSS). Both, the Grape Downy Mildew Model and an experimental model on Grape Black Rot (Guignardia bidwellii) that was developed within the COFREE project, are now available for use on this platform.
Task 4.5: Management tools to support growers and extension services in applying disease control strategies: decision support systems for organic potato production systems (UKA/ZEPP)
From 2012 to 2014 an improved DSS for P. infestans in organic potatoes was developed in the frame of the CO-FREE project. This work included as primary steps the development of an ontogenetic model (SIMONTO) based on the BBCH scale of potatoes, and its validation. Additionally, the soil mineral nitrogen dynamics in organic potatoes and nitrogen contents of leaves and tuber correlated with the BBCH scale were modelled and integrated in the exiting DSS Öko-SIMPHYT (www.isip.de ISIP-Informationssystem integrierte Pflanzenproduktion). This development was based on intensive data mining in existing data sets. The trials were run from 2012 to 2015; varieties with different maturity (very early, early and mid-early) were included, different nutrition levels and a huge number of sequential harvests were performed to improve the DSS. Validation trials were done by UKA, JKI, IPP and INRA and finally the new models were implemented in a website of ISIP.
The decision support system Öko-SIMPHYT recommends a copper application strategy to control late blight in potatoes. Öko-SIMPHYT is available for registered famers and advisors in the internet.
DSS Öko-SIMPHYT currently contains two models:
1. SIMBLIGHT1: predicts the outbreak of P. infestans epidemics and recommends the date to start copper applications (depending on temperature, humidity, soil moisture, variety, emergence date, potato production density in region, site)
2. Öko-SIMPHYT3: recommends an application rate (kg/ha and year Cu (a.i.)) and treatment intervals between copper treatments (depending on temperature, humidity, precipitation, variety, foliage development)
CO-FREE-part
The last part of the output is the CO-FREE-part which shows information on the termination of copper spraying depending of the site and the other information which was individually given by the user. The main feature is the simulated or assessed date of the begin of flowering (in case that the user corrected the button) and the termination date.

Work package 5: Development of copper-free fruit production systems
Task 5.1 Evaluation of novel copper alternatives, tools and strategies in traditional organic apple production systems (LAI, ITAB/GRAB)
Within CO-FREE, open-field trials were carried out on apples in Italy and France from 2013 to 2015. The trials aimed to evaluate the efficacy of novel plant protection products against the major fungal diseases of apple, and their crop safety. In some trials, single compounds were found to be comparable to copper treatment. However, in many cases, the new products tested did not show levels of disease control, which can be considered comparable to the commonly used reference fungicides based on copper, lime sulphur, hydrogen carbonates, and acidified clays. This emphasizes the importance of supporting the former products in the ongoing approval and registration procedures on both, a European and national level, in order to have them available for crop protection without any additional restrictions. Nevertheless, it must be pointed out that not all the novel products from CO-FREE that have been tested in laboratory and greenhouse trials, have yet been evaluated also in the open field on apple due to limited availability at their respective developmental stage. Thus, further field trials will be necessary.
Task 5.2 Evaluation of advanced high yield, apple production systems (FiBL)
The potential and limits of very low input and no input apple production systems was successfully explored. Generally, the advanced systems performed well but the extremely difficult climatic situation in 2012 and in 2013 also demonstrated the limits of preventive strategies: In 2012, the first major outbreak of virulent V. inaequalis populations has been observed. Control strategies were subsequently adapted in order to limit the further establishment of virulent populations in 2013 and beyond. In a fungicide-free environment additional diseases developed, as we found in the case of Marssonina coronaria, a disease that causes increasing problems in Southern Germany, Northern Italy and Switzerland, especially in low input orchards. Our study demonstrated that drastic copper reduction up to replacement is feasible in advanced apple production systems. However, disease control by copper-free fungicides is essential to preserve resistance to V. inaequalis and to control secondary diseases such as sooty blotch as well as M. coronaria.
Since 2009, M. coronaria leaf blotch disease has become a problem in low input apple production in Middle Europe resulting in early leaf fall and reduced yield and quality of the fruits. Due to difficulties in unambiguously assigning early leaf spots to M. coronaria leaf blotch, a quantitative fluorogenic PCR (TaqMan) was developed for testing suspicious leaf and fruit samples for the presence of M. coronaria. This test has proven to be a valuable tool for early diagnosis of unidentified leaf spots and to provide support for deciding the appropriate preventive and direct measures to control spread of the disease.
Varietal resistance to pathogens is a key element in the design of very low input production systems (VLIPS). In order to support selection of cultivars for fruit and fruit juice production and to optimise plant protection strategies on these cultivars it is important to know their susceptibility to M. coronaria. In this study 16 commercially important or newly developed apple cultivars were screened for susceptibility to M. coronaria. One year old saplings were inoculated using conidia grown in liquid medium and kept at 23°C and under high humidity. The severity of any resulting infection was scored 3 times using a simple scale adapted to visual symptoms. Several cultivars showed good initial resistance to the disease, including Gala, Ariane and Elstar. However, at the last scoring, 21 days after inoculation, Gala had become one of the most severely infected cultivars, suggesting that resistance to the disease is complex, and involves a number of mechanisms.
In conclusion, VLIPS have proven great potential to reduce copper use as well as insecticides. However, the state-of-the-art VLIPS as tested in CO-FREE is not yet ready for wide scale adoption by professional apple growers even though the most advanced components of preventive disease and pest control were implemented.
Task 5.3 Evaluation of novel agroforestry-based apple production systems (ORC)
Novel land use systems that integrate woody species into the agricultural landscape have the potential to balance productivity with protection of the environment and the maintenance of ecosystem services. Although the potential of agroforestry-based agricultural systems has been demonstrated in principle, information of usefulness in the context of European low-input production systems is lacking. Integrating top fruit production into an agroforestry system, where woody species are integrated with crop production, may have a beneficial effect on the control of plant pathogens such as scab (V. inaequalis). However, the introduction of such systems into European high-yielding traditional apple production systems will meet substantial obstacles as the approach affects not only agronomic performance but also well-established fruit production traditions. This research aimed to evaluate an apple/arable agroforestry approach as a sustainable strategy for reducing copper inputs in organic and low input systems using two case studies; Wakelyns Agroforestry, Suffolk, UK and Whitehall Farm, Cambridgeshire, UK.
Research focused on five elements that are likely to be impacted by an agroforestry systems approach to no-copper use:
(i) yield and quality of apples, (ii) emergence of primary and secondary pests and diseases, (iii) impact on ecosystem services and functionality, (iv) the economics of agroforestry-based apple production and (v) impact on management activities
The two case study systems provided contrasting approaches to agroforestry-based apple production in terms of scale and design. The low density, high diversity approach at Wakelyns Agroforestry seemed to have benefits in terms of reducing disease levels, and could work well in a diverse, potentially small-scale system such as a market garden, where apples could contribute to direct marketing channels such as vegetable box schemes or farm shops. The commercial silvoarable system at Whitehall Farm showed that an agroforestry approach per se is not successful at reducing scab levels. However, if combined with careful selection of resistant varieties and, if possible, mixed planting of varieties, the other benefits that agroforestry brings, particularly to arable systems, may make this approach attractive to arable farmers looking to diversify their enterprises or protect their farms against environmental problems.

Work package 6: Development of copper-free grapevine production systems
Task 6.1: Evaluation of novel copper strategies in traditional Vitis vinifera-based organic and IPM grapevine productions systems (INRA, ITAB/GRAB, FEM, BPI)
The strategies to control downy mildew (P. viticola) in grapes were evaluated under field conditions in Greece (BPI) and Italy (FEM) in experimental vineyard under commercial-like conditions and in France (ITAB/GRAB/IFV) under commercial conditions in organic vineyards. In addition, the ecological effects of copper were investigated in France in an experimental vineyard (INRA). Amounts of copper given below, indicate the use per year.
Commercial-like vineyards - FEM, BPI
During years 2012, 2013 and 2014, alternative compounds were selected and in 2014 and 2015 were included, according to their mechanism of action, in strategies aiming to effectively control grapevine downy mildew. Since none of the products was able in the previous years to fully control the disease, in the strategies a limited number of treatments (max three) with low dosages of copper were combined with the experimental products.
In 2014, strategies using the most effective products according to their mechanism of action and the forecasted meteorological conditions at the time of application were tested.
The strategies were set up according to the following criteria:
(i) To increase basal plant self-protection, the resistance inducer was used from the beginning of the season at least weekly, preferably in combination with the treatments until mid-summer
(ii) Bacteria were applied any time, but not when temperature was higher than 30°C
(iii) Plant extracts were used for secondary infections before a short period of rain or dew
(iv) Copper or products with sufficient rainfastness were used in case of heavy rain
In 2015, strategies combining the most effective products according to their mechanism of action with low dosages of copper at the most risky stage of grapevine for the disease (before and after bloom) were tested.
The strategies were set up according to the following criteria:
(v) Alternative compounds were applied according to their mechanism of action taking in account disease models and weather forecast
(vi) Alternative compounds were used from the beginning of the season according to models, but at least weekly
(vii) Copper was applied at low doses at the period of highest susceptibility (max three treatments before and after bloom)
(viii) Low dosage of copper (15-20g Cu metal/hl) was applied on all vineyards on wet plants (sporulation) in long periods of rain to slow down the epidemic.
Results FEM
In 2014, the disease pressure was extremely high and some infections happened without the possibility to reapply the treatment, because of many days of continuous rain. All products, including the copper reference did not control the disease at sufficient level and the trial was stopped in July. However, with the low copper strategy (treatments with a resistance inducer, followed by 1.5kg/ha and year of Cu and afterwards use of a plant extract) ensured a protection of the vineyard comparable to the copper reference (7kg/ha and year of Cu).
In the 2015 season, the disease pressure was not as high as in the former years. The tested plant extracts and the milk derivate applied with only 1.5kg/ha and year of Cu showed an effectiveness against downy mildew, statistically similar to the copper reference (8kg/ha and year of Cu).
Results BPI
In 2014, the disease pressure was extremely high. All products, including the copper reference (7kg/ha and year Cu) did not achieve downy mildew reduction at sufficient level hence the trial was stopped in July. Reduction in percentage leaf area infected over the season was 61% for the full copper reference. However, the low copper strategy (in addition to alternative compounds only 0.7kg/ha and year of Cu was applied once), resulted in protection of the vineyard not significantly different to the full copper dose.
In the 2015 season, the disease pressure was lower than in former years in Greece, similar to Italy. The plant extracts, the milk derivate and the tested bacterium showed a good effectiveness against downy mildew, statistically similar to the copper reference. Thus, these alternative compounds in combination with 0.7kg/ha and year of Cu were as effective as 7.7kg/ha and year applied in total in the copper reference plots.
Commercial and commercial-like organic vineyards – ITAB/GRAB/IFV
During 2013, 2014 and 2015, alternative compounds to be tested in the field against grape downy mildew were selected. They were chosen according to the French national criteria, i.e. only products with an ADE permission (“Autorisation de Distribution pour Expérimentation” delivered by national authorities) were assessed. Thus, only a limited number of alternative compounds from CO-FREE, which had ADE permission, could be tested. Trials have been carried out in two sites in France in commercial (ITAB/GRAB) and commercial-like (ITAB/IFV) organic vineyards.
The alternative products were assessed as stand-alone if their potential efficacy described by the company was supposed sufficient, and associated in a strategy with low copper dose if their potential efficacy was supposed not sufficient. A low copper dose treatment was set-up for comparison.
In strategies, the alternative products were applied weekly from the beginning of the season. In 2013 and 2014, the application of a low copper dose was realised weekly in association with the alternative products in order to slow down the epidemic. In 2015, the application of a low copper dose was foreseen according to the climatic risk. Because no risk occurred at the ITAB/GRAB site in 2015, no low copper dose was applied.
Results ITAB/GRAB/IFV
In 2013, the disease level was high at both trial sites and best efficacy was observed for copper-based treatments. The same level of efficacy was observed for the low copper dose (600g/ha and year of Cu) and the reference (1600g/ha and year of Cu). No effect of the alternative products as stand-alone and additional effects (in combination with low copper) were observed. One of the test products was associated with a higher abundance of predatory mites. At the ITAB/IFV site, the best efficacy was observed for the copper reference. No effect and additional effect of the alternative compounds tested as stand-alone or in low copper strategy, respectively, was observed.
In 2014 at ITAB/GRAB site, the disease level was moderate. The best efficacy was observed for copper-based treatments, where the same level of efficacy was observed for the low copper dose (900g/ha and year of Cu) and the reference (3600g/ha and year of Cu). No effect and additional effect of the alternative compounds tested as stand-alone and with low copper dose, respectively, was observed. One of the test products had seemed to slow down the epidemic (observed in mid-August), however no significant effect was demonstrated. At the ITAB/IFV site, climatic conditions were very favourable to high infection risks in July. Also here, no effect and additional effect of the compounds tested as stand-alone or in low copper strategy, respectively, was observed.
In 2015 at the ITAB/GRAB site, the disease level was low. Alternative compounds as stand-alone or with low doses of copper had no effect in controlling downy and powdery mildew. No negative effect of the test products on predatory mites was observed. At the ITAB/IFV site, climatic conditions were unfavourable to disease development but after artificial inoculation and fogging, infection occurred. Again, the best efficacy was observed for copper-based treatments. However, an interesting effect of two low copper strategies was observed at the end of July on leaves. Here, disease severity was significantly lower than in the untreated control and higher than the reference. However, no control of the disease was observed on the grape bunches.
Ecological evaluation of copper treatments on leaf communities - INRA
In order to investigate the ecological evaluation of copper treatments and especially the target and non-target effects on leaf communities, initial plans were modified for environmental constraints. The strong mildew incidence in 2014 on the experimental plots meant that this research was carried out on adjacent vinyards. The results tended to show a higher biodiversity and higher numbers of pathogens and insects in the non-treated rows, especially the control rows without grass. For instance, in the three families of mites present in vine rows (phytoseiidae, tydeidae, tetranychidae), the number of individuals was significantly higher in control rows without grass than (i) in control rows with grass (ii) in rows with full copper and (iii) in rows with half copper treatments. Altogether, there was a weak effect of copper on the insect community. However, there was a bigger effect of the absence of grass on the number of insects. It could be expected that without grass between the rows, insects could find a refuge in the vine stocks.
Task 6.2 Evaluation of copper-free crop protection strategies in disease tolerant grapevine production systems (FiBL, INRA)
Trials Switzerland - FiBL
In 2014, 2 inducers of resistance (yeast-based product and COS-OGA) were compared to an untreated control, and the standard strategy with acidified clay + sulphur. Treatments starting before flowering and ended at veraison. In 2015, 2 novel experimental plant-based fungicides (LAR-16, SB T36.48) were compared to an untreated control and a reference treatment with acidified clay and sulphur. Treatments started before flowering and ended at veraison. Disease and pest assessments comprised P. viticola, O. tuckeri, Botrytis cinerea and Drosophila suzukii.
To quantify the epidemic of P. viticola, disease assessments were conducted in sentinel plots throughout the seasons in the experiments described above, as well as in the commercial vineyard planted with Vitis vinifera varieties (`Riesling´ x `Sylvaner´ or `Pinot Noir´) treated according to recommended FiBL standards (max. 4kg copper/ha and year) as well as in the untreated plots of the GEP efficacy trial (cv. `Riesling´ x `Sylvaner´) at the FiBL pesticide screening vineyard.
Disease assessments were conducted according to EPPO guidelines and included P. viticola and O. tuckeri mainly. Weather data were collected within the vineyard and infection risks by P. viticola were calculated by the Vitimeteo Decision Support System (www.agrometeo.ch).
Results FiBL
A range of potential copper alternatives were tested on interspecific varieties in 2014 and 2015. The usage of acidified clay preparations in tank mixture with wettable sulphur provided excellent control of both, P. viticola and O. tuckeri. In contrast, neither of the inducers of resistance provided sufficient protection until harvest. In contrast, the two different botanical-based pilot fungicides provided control in the period when they were applied but not until harvest probably due to limited persistence and rain fastness. The varieties tested did significantly differentiate in their susceptibility to P. viticola and O. tuckeri. One of the purposes of this study was to explore whether current commercially available interspecific grapevine cultivars differ with respect to their capacity to build up resistance to fungal attack if elicited with known inducers of resistance. The yeast based inducer of resistance seemed to provide some protection against O. tuckeri but the effect needs to be verified to draw any further conclusions.
The experiments clearly demonstrated that no compromises can be made regarding the efficacy level of copper alternatives. Lack of persistence and limited rain fastness limit the uses of copper alternatives even if used on moderately resistant cultivars.

Trials France - INRA
Three grape production systems have been compared for their performance and their productivity. A first system (RES), was planted with grape varieties resistant to downy and powdery mildew (muscadine genes). A so called BIO system relied on full doses copper treatments, and a so called INT system received ¼ dose of copper as compared to BIO. The latter two systems were planted with cv. `Merlot´. Data were analyzed by intersystem comparisons and compared to averages of vine growers from the DEPHY vine growers regional network (similar cultivars, similar pathogen pressure and climatic conditions). A multi-criteria assessment of the performances was used for each system, using indicators focusing on different parameters: (i) production - yield, wine quality, pesticide residues on grapes and in wine, (ii) agronomic - strength and nutritional status of the vines, presence of pests, (iii) environmental - pesticides use, fuel use and (iv) economic - working time, production costs.
Results INRA
Both, RES and INT systems based on low (almost no) copper application presented rather good results except for the cost and labour investment in the RES system. This was due to the low price expected for the wine, and due to grape pruning which is not yet mechanized because of the experimental vineyard. Results confirmed that low, or almost no copper in grape systems are promising options for the future.

Work package 7: Development of copper-free annual crops (potato, tomato) production systems
Task 7.1 Evaluation of novel copper alternatives and tools and strategies in traditional organic potato production systems (IPP, JKI, INRA, UKA)
Results IPP
In 2012 and in 2013 no significant differences were noted after treatment of the susceptible cv. `Ditta´ with some alternative products. However, in 2013 highest yield was reached after treatments with the tested elicitors applied together with half dose of copper (3.0kg/ha and year). No influence of alternative products on presence of ladybirds was observed.
In Poland in 2014, the performance of a potato cv. medium resistant to P. infestans (`Sante´) treated with alternative compounds (CO-FREE strategy) was compared to a susceptible cv. (`Ditta´) treated with 3.5kg/ha and year Cu (reference strategy). Here, no copper reference was tested. Disease pressure was high and highest total yields were obtained in the CO-FREE strategy with stand-alone treatments with either a microbial product (EN-03) or a resistance inducer (6715B).
In 2015, alternative compounds as stand-alone treatments or followed by low doses of copper were compared for their performance in the cv. `Ditta´ and the resistant cv. (`Lord´). In `Ditta´, combined treatments with EN-03 and 6715B reduced infections on leaves by P. infestans as well as the resistance inducer followed by three copper sprays with 250g/ha and year of Cu each. The highest yield in `Ditta´ was found for the resistance inducer in combination with the low doses of copper. In `Lord´ treatments with 6715B combined with EN-03 were tested. They did not reduce disease severity. However, the treatments had a positive impact on yield compared to the control.
Results JKI
In field trials CO-FREE test products were tested against late blight (P. infestans) in three different potato varieties (`Ditta´, `Jelly´ and `Allians´) in Brandenburg (Germany) from 2012-2015. To indicate the spray start, simulations with the DSS Öko-SIMPHYT were used in all years. The alternative compounds were preventively applied 4 to 6 times at intervals of 7 to 10 days. In 2013 and 2014 a new low-dose copper product ‘Cuprozin progress’ was used. Additionally, phosphonates were tested against late blight in the last two years and phosphonate residues in tubers were analysed in 2014. Colorado potato beetle was controlled by a single treatment of ‘Spinosad’ every year, except in 2013 when two treatments in cv. `Allians´ were necessary. In 2013, 2014, 2015 with the beginning of tuber growth, a weekly harvest was carried out to estimate the daily increase. In September of each year, each potato variant was harvested.
In 2012, no difference in leaf infection with P. infestans between the tested products COFREE24 and COFREE25 were found in the cv. `Ditta´. Considering diseased leaf area by Alternaria spp., COFREE25 showed a significant efficacy of 14%, which was about 50% of the efficacy after copper treatment (26%). The more resistant cv. `Jelly´ showed similar results (not statistically significant) at a lower level. The copper applications delayed late blight infestation for about 6 days. The surplus in yield compared to the untreated control was 6t/ha and statistically significant (α = 0.1). The tested CO-FREE products had not yet contributed satisfactorily to yield stability, but showed yield increases of up to 4t/ha (not statistically significant). Field trials with the cv. `Jelly´ showed negative yields in all of the treatments. Here, severe late blight infestation took place later, and the treatments with test products did not result in enough of a delay of infection to affect the yield. Under these circumstances the stress of any treatment could lead to yield losses in comparison to the untreated control.
In 2013, a hailstorm destroyed the whole experiment and tuber growth was entirely stopped. An evaluation of the different treatments was not possible. But yields showed that the earlier cv. `Ditta´ profited from the faster development and can realise higher yields under extreme conditions than `Allians´.
In 2014, the mean yields were appropriately high (untreated control (UC) `Ditta´ = 39.3t/ha and UC `Allians´ 37.4t/ha). The testproduct COFREE24 showed no statistically significant influence on Alternaria spp. and Phytophthora infestations. Copper treatment with either 2kg/ha and year Cu or 0.5kg/ha and year Cu + phosphonates in the variety `Ditta´ resulted in 30% and 21% efficacy, respectively. The fosetyl-aluminum content in potato tubers [mg/kg] of cv. `Ditta´ were analysed by Eurofins-lab. Throughout the season, the mean fosetyl-aluminum content was about half the European maximum residue level for poatao tubers (which is 30mg/kg phosphonic acid (H3PO3)).
The year 2015 was a dry year with lower yields than 2014 (UC `Ditta´ = 25.7t/ha and UC `Allians´ 28.9t/ha). No late blight occurred in the trials and leaf losses were due to Alternaria spp. infections. The copper treatment as well as the combination of 19-21E+EN03 showed a tendency (not significant) of a low effect on Alternaria spp..
Results INRA
CO-FREE test products were tested for their efficacy against late blight in 2012-2015 in a high disease pressure, oceanic environment in western France. The trials involved several potato cultivars with different levels of field resistance to the disease. The results showed that the alternative products performed significantly less well than the copper reference under high disease pressure, and were not able to significantly reduce the disease spread. They also showed that, contrary to initial expectations, cultivars responded in different ways and with different intensities to defense stimlulation by potential elicitors. Some disease reduction was observed with all products in cv. `Desiree´, whereas product-specific responses were observed in the other three cultivars, irrespective of their intrinsic resistance level. This suggests that the use of alternative products with a plant defense stimulating mode of action can be advised in a cultivar-specific way, and under low to moderate disease pressure. These products should therefore be thought as part of complex, integrated control strategies rather than as 'copper replacement' products.
These main findings were included as part of a video prepared together between WP7 and WP9 of CO-FREE and pointed to the use and acceptability of resistant potato cultivars to combat late blight in organic systems. This video was on display at the EXPO in Milano, 2015.
Results UKA
A 2-factorial field trial was performed in 2014 at the experimental farm (Domäne Frankenhausen) in Germany with factor 1: variety (resistant cv. `Vitabella´/tolerant cv. `Alians´) and factor 2: disease control (F6715B/copper hydroxid/control) with 4 replicates in a randomised design. It was shown that the resistant variety had the strongest impact on both disease and yield, including a slight yield increase with F6715B. Copper was best performing in the more susceptible variety.
P. infestans developed moderately during the season. First incidence of late blight was observed by July 3, 2014. The strongest effect on the disease progress was related to the variety. `Vitabella´ was almost without disease symptoms throughout July independent of any treatment while in `Allians´ the disease could develop to almost 100 percent in the control plots by the end of July. The copper treatment which was in total 1kg, resulted in a clear delay of the disease progress. In contrast, the disease progress curve in the control treatment was around 3 days ahead in the development of the disease. The alternative product slightly delayed the disease progression between July 3rd to 14th. The reduction of the disease resulted in no significant increase in yield in any of the treatments. The increase in yield was around 5% in `Allians´ with the copper treatment, while in `Vitabella´ also F6715 showed a stimulating effect of around 6% more yield compared to the control. In conclusion, the results showed that the use of a resistant variety is a very valuable alternative to copper.
Task 7.2 Development of potato ideotypes and integration into management systems for copper-free potato production system (INRA, LBI, ORC, UKA, JKI)
The aim of WP 7.2 in CO-FREE was to identify potato ideotypes (i.e. ´ideal cultivars’) suitable for low input systems with no or little use of copper. It was planned as a three-stage process: (i) a conceptual study, aiming at defining from expert knowledge ideotypes suited for different conditions (climatic, production systems, etc.), (ii) experiments to test these ideotypes in large fields under contrasted conditions, (iii) data analysis, to validate or not the performance of ‘adapted’ vs. ‘non adapted’ ideotypes in the different environments.
Results
Six different questionnaires were received, corresponding to different countries and production situations: two answers from Germany (continental climate, ware production), two from France (oceanic climate, seed + ware production), and one each from the Netherlands (cool oceanic climate, ware production) and UK (oceanic climate, ware production). These answers thus cover a wide range of organic potato production in the northern half of Europe. Although the absolute number of questionnaires received was small, it should be noted that each respondent indeed indicated that the questionnaire he/she sent represented a synthesis of the opinions of several persons consulted in the process of completing the questionnaire. Therefore, we regard the trends emerging from the analysis as stronger than the absolute sample size might at first suggest.
Biotic constraints
The main pests perceived as limiting for organic potato production in Europe were foliage and tuber blight due to P. infestans, and several skin diseases (common scabs due to Streptomyces spp, black scurf due to Rhizoctonia solani and silver scurf caused by Helminthosporium solani). Interestingly, animal pests (Colorado potato beetle, aphids or nematodes) were never seen as major threats, similarly, quarantine pathogens (nematodes, Colorado beetle, Ralstonia spp. and Clavibacter spp).
Abiotic constraints
The main abiotic stress facing organic potato crops in Europe was the absolute supply of nitrogen. The enquiry revealed also that potassium availability becomes an increasing issue. By contrast, neither water nor phosphorus were regarded as main problems.
Potential control measures
The first line of reasoning when trying to design ideotypes for low-input, copper-free potatoes was of course to analyze which type and level of resistance is available to the main pests targeted by copper. Resistance to late blight in foliage and tubers is recognized to exist in germplasm by enquiry respondents, but its efficacy is viewed as limited, probably because of repeated breakdown of major-gene, high level resistance. This lever is thus regarded as difficult to use because of this, resulting in a moderate average score for performance in new design. Resistance to viruses (PVY, PVX) is also known to exist, but there is a mixed appreciation of performance and ease of use: high in France and NL, low in Germany. No resistance is identified against other major pests, such as R. solani.
What are the consequences of using resistance? There was a general concern about durability and/or efficacy if used alone, generating doubt as to a ‘genetics only’ type of solution. Respondents thus suggested the use of DSS for applications of Cu/alternative products, early planting/pre-sprouting (favoring plant vigour), and general hygiene measures in storage and with machinery. These suggestions were not specific to any region, and raised a general consensus among respondents, irrespective of local production situations.
Task 7.3 Evaluation of novel alternative compounds and copper-free strategies on low input and organic field tomato production systems (BPI, ITAB/Sonito)
Field trials in tomato were performed under conditions conducive to high disease development in mediterranean France and in southern Greece. The trials were conducted according to EPPO standards in complete randomized block designs on current late blight-susceptible tomato cultivars. Integration of alternative compounds and DSS were used. Assessments included efficacy of alternative compounds against late blight caused by P. infestans, yield quantity and quality, and impact of the tested products on non-target organisms. Trials were done in field IPM tomato production systems (BPI-Greece) and in field organic tomato systems (ITAB/SONITO-France).
Research carried out in both countries was divided into two parts: (i) in years 1-3, single alternative compounds were evaluated in experiments in commercial/experimental farms (ii) in years 2-4, novel strategies proposed under CO-FREE or inspired by the producer’s survey and the results of experiments in years 1-3, were evaluated in order to validate the strategies with respect to agronomic and economic performance and technical feasibility. Economic performance was estimated based on a common protocol. Up to two strategies (low copper strategy and no copper strategy) were evaluated per year and site.
In conclusion, (i) there are promising results (trials in Greece) that indicate that copper use could be reduced with the use of novel CO-FREE compounds considering the efficacy achieved under extremely high disease pressure (100% crop damage in controls -‘worst case scenario’), (ii) it should be noted that due to the limited time and number of trials conducted, only a limited number of new CO-FREE products was tested in the tomato/Phytophthora system in the field and (iii) optimization with respect to formulation, application technique etc. in most cases was not yet finalized hence it is considered that in the following years new formulations with improved efficacy will be produced and be commercially available.
A questionnaire for a producer survey was developed by ITAB and was adjusted and used also by BPI. The surveys were carried out during the first two years of the project and were supported by regional tomato producer’s organizations. The information was gathered from both organic (France) and low input IPM tomato producers (Greece), in order to identify the most cost-efficient ‘low copper management systems’ used in practice. For the most efficient components/strategies, an in-depth qualitative investigation was conducted on the selected farms that were included in the survey. The analysis of the data gathered, provided the baseline for the evaluation of strategies developed in other work packages in the frame of CO-FREE.
The survey conducted in France in organic farms highlighted that, among the production of the farms surveyed, tomato for industry was a high-income production, contributing to secure the income of the farm. Tomato production highly depended on the use of copper. The capacity of innovation in new cultural practices was partially restricted downstream by the specifications of the purchaser. Farmers were mostly in a ´substitution strategy´: change one product by another, rather than a ´redesign approach´. However, growers were willing to substitute copper with effective alternatives that would not add significant cost to their production. They were reluctant to convert to organic tomato production (only 40% replied yes to the relevant question), although they recognized the environmental benefits of conversion. Their decision for converting would depend mainly upon market demands (tomato canning industry) and cost of production. Health issues related to the use of conventional pesticides was another regulating factor for their decision but to a lesser extent. Although they were using fungicides and copper, they were willing (73% positive answers) to test new alternative products.

Work package 8: Horizontal activity 1: Ecological impact assessment of novel copper-free techniques and production systems
Task 8.1: Impact of alternative compounds on beneficial arthropods in the laboratory (BPI)
The potential toxic effect of ten alternative compounds from CO-FREE to eight non-target arthropods was assessed in the laboratory using the dry residue exposure method (‘worst case scenario’). The non-target arthropods included the tier I risk assessment indicator species (Aphidius rhopalosiphi, Typhlodromus pyri) and another six species of natural enemies relevant to main pests in tomato, grapevine, apple and potato i.e. Coccinella septempuctata, Macrolophus pygmaeus, Cryptolaemus montrouzieri, Nephus includens, Nesiodiocoris tenuis, Orius laevigatus.
Since toxicity of the CO-FREE compounds was expected to be low, limit tests were conducted at a rate equivalent to four times the recommended field dose rate (RFDx4) based on the maximum recommended application number of the CO-FREE compounds in the efficacy field trials. Hence, if the corrected mortality in the limit test rate was lower than 50%, no other rate was tested. In the opposite case, the LR50 was estimated.
The effect of all tested CO-FREE compounds based on % corrected mortality was harmless to slightly harmful (corrected mortality <30%) to all the non-target predators tested at a rate which was equivalent to four times the RFD except for the case of one compound which was harmful to C. montrouzieri at RFDx4, moderately harmful at RFDx2 but harmless or slightly harmful at RFD.
Task 8.2: Toxicity assessment of alternative compounds on non-target aquatic indicator organisms in the laboratory (BPI)
The toxicity of eleven alternative compounds has been assessed on two non-target aquatic indicator organisms in the laboratory. Ten of the tested samples exhibited low toxicity on the alga Pseudokirchneriella subcapitata and eight of them exhibited low toxicity on the crustacea Daphnia magna. Three compounds were more toxic to Daphnia magna and one compound to Pseudokirchneriella subcapitata.
Task 8.3: Toxicity assessment of alternative compounds on non-target soil indicator organisms in the laboratory (BPI)
The potential toxicity of ten alternative compounds has been assessed on two non-target soil indicator organisms (Eisenia fetida and Enchytraeus albidus).
No significant mortality occurred and no adverse effects on growth were observed for all tested compounds on Eisenia fetida. No significant adverse change of reproduction was recorded for five of the tested compounds, while statistically significant adverse effects on reproduction (p<0.05) was observed for three of them at the highest tested concentration of 1000mg/kg dry weight. The amount of one alternative compound was not sufficient to carry out the definitive test for the estimation of EC50 and NOEC values for Eisenia fetida.
No significant mortality occurred for all tested compounds on Enchytraeus albidus. In addition, no significant adverse effects on reproduction were recorded for nine of the tested compounds at the highest tested concentration of 1000mg/kg dry weight, while for one compound the NOEC value was estimated to be 500mg/kg dry soil.
Task 8.4: Protocol to assess the ecological impact of novel alternative compounds, strategies and production systems in the field (BPI, ITAB/GRAB, LAI, INRA, ORC)
Assessments of the CO-FREE products on beneficial arthropods were conducted in the efficacy experimental fields of tomato and grapevine in Greece (2012-2014), grapevine and apple in France (2013-2015) and apple in northern Italy (2013-2015) based on relevant protocols established in the frame of the project (CO-FREE handbook). Predatory mites, especially phytoseidae, seemed to be the best indicator group to assess the impact on beneficial arthropods mainly due to the small size of plots corresponding to small number of insect predators and spiders. No negative effect of the tested alternative compounds was observed on phytoseid mites in grapevine (Greece, France) and apple fields (France, northern Italy). In plum tomato (Greece) only the incidence of generalist predators and parasitized aphids could be recorded since the numbers of beneficial insects in the plots did not allow statistical analysis. An increased number of collembola, which is a biodiversity index insect group, was observed in the plots of some CO-FREE test products possibly due to feeding on the compounds.
Impact assessment of an agroforestry/arable production system on beneficial arthropods and insect pollinators in eastern England showed comparable total hymenoptera abundance between the agroforestry cultivation and an orchard. However, at the genus level, there were significantly more Lassioglossum and Bombus in the orchard than in the agroforestry and more predatory and parasitic wasps in the agroforestry system.

Work package 9: Horizontal activity 2: Socio-economic impact assessment of copper-free production systems and strategies to foster consumer acceptance of new varieties
Task 9.1 Socio-economic impact assessment of copper-independent production systems (Lead: LBI)
The economic impact of minimization of copper use was assessed for four typical commercial production systems (potato, tomato, apple and grape) by partial budgeting. In this, the incremental effects on costs and returns of copper minimization compared to current practices (including the use of copper), were estimated, based on expert estimates.
The analysis showed that minimization of copper use by replacement with alternative PPP’s is expected to be impossible or to have a major negative economic impact in most production systems, except for IPM-tomato production, mainly because of negative effects on (marketable) yields. Even if copper replacement strategies including resistant varieties are used, the economic impact was expected to be negative for most situations. Only for potato production and medium yielding grape production systems, the economic impact was expected to be positive if resistant varieties are used.
Sensitivity analysis showed that changes in the costs of disease control were not of major importance, with the exception of tomato production. It also showed that price effects might be highly important: if price premiums of 10% for ‘copper-free’ products will be attained, the economic impact of copper replacement by alternative PPP-spraying becomes positive for apple and potato production in low risk situations, besides IPM-tomato production. For strategies including resistant varieties, a 10% price premium was more than sufficient to attain a positive economic impact in all production systems.
It is concluded that besides the development of good resistant varieties with comparable yields and qualities as current varieties, major marketing efforts, along with the introduction of ‘copper-free’ crop products and varieties, are an essential element of a strategy to minimize the use of copper.
Task 9.2 Development of a strategy to foster consumer acceptance of new varieties (lead: LBI)
In 2015, at BioFach (world’s leading trade fair for organic products) in Nuremberg, Germany, a workshop was organized entitled: “Strategies for market acceptance of new resistant cultivars - from obstacles to chances”. The workshop aimed at an exchange of examples of successful initiatives for joint collaborations to market resistant cultivars in crops such as potato, apple and grapes. This led to a discussion with market parties as to what extent such new cultivars are acceptable or not, and how marketing tools can be developed for the next steps forward. First-hand experience of organic market trials with new potato, grapevine and apple cultivars were reported and discussed. Ca. 40 participants including traders, retailers (health food stores and supermarkets), consumers, growers and breeders were present. We aimed to identify key elements for future success by collating experience from past and ongoing efforts in Europe.
A critical note was made on experimenting with pilots as a tool to test introduction of new cultivars in a shop. It is important to also monitor repeat sales, as without repeat sales the introduction has failed. One important key element that was put forward by a retailer involved in pilots with late blight resistant cultivars in the Netherlands was that the retailer often lacks knowledge about the problems growers have to deal with. The retailer made consumers part of the solution and introduced the new cultivars in his shop together with posters, flyers and new recipes. Other retailers introduced the apple of the month to promote new cultivars. It was also stressed by a consumer that new cultivars enlarge the diversity of cultivars, which is in line with the fact that biodiversity is the base of a sound organic production. Her advice was not to stress too much the need for new cultivars to prevent diseases but make it attractive to consumers by telling that your retailer cares about biodiversity.

Work package 10: Horizontal activity 3: Dissemination of new knowledge and novel practices
During the project duration of CO-FREE, partners published 18 peer reviewed articles and had more than 100 oral and poster presentations at conferences and stakeholder meetings. Field trials were presented to farmers on 13 open field days and in further events an exchange with farmers, breeders, advisors, consumers, industry and other stakeholdes was realized. A video was produced under the lead of LBI and INRA focussing on the issue of breeding and consumer acceptance of new cultivars. CO-FREE was presented by JKI and FiBL at EXPO Milan at the EU-pavillon in 2015 (live-streamed) where the video was shown. Contacts and discussions with policy makers took place in different meetings. These comprised e.g. the participation of JKI and FiBL in expert discussions on copper in Germany in all years of the project and participation of FiBL in DG Sanco’s "low risk substance" expert group that developed a guidance document for improved registration procedures for botanicals (2013-2015). The final CO-FREE conference was held as part of the Science Forum at BioFach (world’s leading trade fair for organic products) in Nuremberg, Germany in 2016. The aim was to give an update on the current situation and results (from in- and outside of CO-FREE) of the topics, novel products, tools, resilient cropping systems, and strategies to reduce or replace copper in the future. Eight talks were held by scientists and stakeholders, followed by a general discussion.
The CO-FREE website will be maintained and continually updated with new publications and information derived from CO-FREE. This is particularly the case for novel copper alternatives, where publication is restricted until all IP protection is ensured.

Conclusions of the CO-FREE project
Substantial progress has been made in the development of copper reduction and replacement strategies, elucidating the potential but also the limits of individual techniques. Copper remains difficult to replace, not only due to the techniques but also due to legal limitations and costs involved. From a present perspective, immediate phasing out of copper would at present create unbearable risks and costs (e.g. for tools or risk compensation). Thus, phasing out of copper is currently not feasible generally. However, in the four systems under investigation in CO-FREE, further reduction is achievable in most crops, and possibilities are seen highest in potato>apple>tomato>grapevine. The key to the reduction/replacement of copper is the use of smart management strategies, which comprise a variety of approaches. Already, farmers are looking more for resilient varieties and combine them with reduced copper applications, and CO-FREE is contributing to further copper reduction by having identified a number of candidate compounds and improved DSS and by having investigated strategies to be used in the crops.
The activities of CO-FREE strongly contributed to further copper reduction/replacement, in order to maintain or enlarge EU organic and low input production areas. Novel advanced low input farming systems, alternative compounds and new tools have the potential to reduce the total pesticide use by more than 50%, depending on crop and region.

Potential Impact:
Potential impact and main dissemination activities and exploitation of results
The expected impacts of Work Programme 2011 (WP2011) were to (i) reinforce the EU’s science base in the bio-economy by building on Knowledge Based Bio Economy and (ii) by addressing the challenges facing society as was highlighted in Europe 2020 strategy. The work programme was designed to improve food security and safety for Europe and beyond and to strengthen communication, dissemination and knowledge transfer to ensure rapid uptake and valorisation of results by stakeholders and (SME-) industries. With special emphasis on low input and organic production systems, the programme aimed at increasing sustainability of production systems by improving crop health and crop protection.
The main expected impacts listed for topic KBBE.2011.1.2-06: Strategies to replace copper-based products as plant protection products (PPPs) in low input and organic farming systems were to (i) deliver alternative plant protection compounds and appropriate/improved management methods to reduce the dependency on copper based fungicides/products in organic and low-input integrated pest management systems where very few chemical alternatives may be used, (ii) deliver a positive impact on the environment by replacing copper use and to support EU policy to reduce chemical inputs in crop production, (iii) to meet the needs of the agricultural sector across different regions in Europe, (iv) provide results that are of interest and potential benefit to SMEs, (v) provide a beneficial economic impact to the sector concerned, and (vi) ensure a strong link between academia and end-users.
CO-FREE provided a significant contribution towards the main impacts intended by the European Union since the main objective of CO-FREE was to deliver to farmers, advisors and the scientific community improved and innovative methods, tools and concepts useful for replacement of copper in organic and low input production systems. The project ensured that these outputs had a positive impact on the environment, continued security and profitability of organic and low-input food production, and improved perception of food and farming by civil society.
In CO-FREE, the potential and limits of advanced copper-free production systems have been identified by providing alternative compounds, providing ‘smart’ application tools and by integrating these tools into traditional and novel copper-reduced crop production systems. These copper-reduced apple, grapevine, potato and tomato production systems have been evaluated in a multi-criteria assessment with respect to agronomic, ecologic and economic performance. Providing technical solutions for plant production is a prerequisite for change, but not sufficient to achieve a major shift in current European organic and low input production systems exposed to competitive economic environment. CO-FREE has therefore also looked into strategies to identify ‘smart’ breeding goals by development of crop ideotypes and foster consumer acceptance of novel disease-resistant cultivars by consumers and retailers. By involving farmers, advisors, plant protection industry, policy makers and researchers as well as the stakeholders of the European organic and low input sector (food supply chain, retailers, producers associations), CO-FREE has taken care of a rapid development, dissemination and adoption of the copper replacement/reduction strategies.

Impact on delivering alternative plant protection compounds and appropriate/improved management methods to reduce the dependency on copper based fungicides/products in organic and low-input integrated pest management systems where very few chemical alternatives may be used.
CO-FREE facilitated the development of copper-alternative compounds of plant and microbial origin to reduce/replace copper within traditional production systems by optimizing formulations, up-scaling of production, extended elucidation of modes of action, identification of active compounds (WP2-3). Use of alternative compounds and reduction of copper use were to be optimized by means of novel ‘smart’ management tools in WP4. These novel tools were then integrated into existing production systems (WP5-7). In addition, development/exploration of the potential of novel copper-free production systems with increased intrinsic tolerance to pests and diseases were investigated (WP 5-7). In particular, the following achievements were made:
Alternative compounds:
In CO-FREE a total of 17 alternative compounds was investigated and in different formulations, application doses etc. tested in lab and field. During the life time of CO-FREE, two registration dossiers have been submitted and three are in preparation. Additional three dossier submissions are foreseen in the next four years, since additional RTD is needed. For three other alternative compounds, submission has not yet been decided. Currently, one active substance developed by a CO-FREE partner (COS-OGA (FytoSave), partner FYTO) is approved under EC regulation 1107/2009 and listed in Annex I (currently for control of powdery mildews in cucurbits and fruity solanaceae). Thus, from the 17 compounds under investigation, one has been introduced to the market and eight are designated to be further developed for market introduction. Costs for registration, however, are high and require a substantial initial investment by SMEs. Since most alternative candidates are likely to cost more than copper, the return of investment (RoI) is crucial and may be a limiting factor. However, the Technology Readyness Level (TRL) of several CO-FREE compounds was shown to be at least 8, i.e. their effectiveness was proven under field conditions, indicating that they have best prerequisites for a future market. All of 11 tested CO-FREE candidates exhibited mainly unproblematic ecotox profiles in detailed studies on non-target organisms (arthoropods, aquatic and soil indicator organisms).
This, together with the facts that (i) copper has a broad spectrum of activity, (ii) that it is unlikely that one compound will have the potential to completely replace copper in all crops, (iii) the alternative compounds in the best cases had similar efficacy as copper and (iv) that new compounds have to be safeguarded to keep effectiveness over time, it can be postulated that different candidate compounds are necessary to further reduce/replace copper, thus a range of products will be required for practice. CO-FREE has contributed strongly to further copper reduction possibilities with a number of candidate compounds which high TRL and which will be further developed aiming at development of new products for the market. This however needs some more time, and the best case scenario for market introduction for the next candidates will be 2022.
Management tools/Decision Support Systems (DSS)
Practical DSS are one of the key approaches to copper reduction and replacement. For the management of several key diseases in organic crops reliable DSS are available to growers and advisers. In apple, DSS are already best practice. DSS should be accurate and easy to use to make alternative strategies as effective and reliable as copper schedules. Within CO-FREE, DSS for grape (downy mildew and black rot; Bio Fruit Advies, Netherlands) and potato (late blight, University of Kassel/ZEPP, Germany) have been optimzed or developed from scratch.

Grapevine
Grape downy mildew DSS was developed on the basis of the RIMpro model (Bio Fruit Advies). The model was validated and optimized. For blackrot on grapes, a new model was developed. Users (producers, consultants and scientist) can connect their ‘on-farm’ weather stations to the system, and use the model output in their daily decision-making on crop protection. Currently, over 1500 weather stations are connected worldwide. Both, the Grape Downy Mildew Model and the experimental model on Grape Black Rot (Guignardia bidwellii), are available for use on the cloud-based platform www.rimpro.eu. The grapevine models are expected to have a positive impact on yield security if copper alternatives are to be used.

Potato
An improved DSS for Phytopththora infestans in organic potatoes was developed in the frame of CO-FREE (University of Kassel and ZEPP, Germany). This work included as primary steps the development of an ontogenetic model (Simonto) based on the BBCH scale of potatoes, and its validation. Additionally the soil mineral nitrogen dynamics in organic potatoes and nitrogen contents of tuber and leaves were modelled and integrated in the exiting DSS Öko-SIMPHYT (www.isip.de ISIP-Informationssystems integrierte Pflanzenproduktion). Potato varieties with different maturity (very early, early and mid-early) were included, different nutrition levels and a huge number of sequential harvests were performed to improve the DSS. The data were intensively validated and finally the new models were implemented in a website of ISIP. The new model has potential for copper reduction in potato, since it estimates if treatments late in the season are necessary or not for yield security.

Resilient production systems
Varieties
Experience showed that breeding of disease resistant cultivars leads to copper-free cultivation and acceptable yields. Robust/resistant varieties are thus a major contribution to copper replacement and their availability is constantly increasing. Bottlenecks for their use in practice include the adoption by representatives of the whole value chain. For successful introduction of new cultivars, it is necessary to meet the minimum criteria of growers, traders and consumers.
In CO-FREE, strategies based on pilots were identified to overcome such bottlenecks and thus improve market introduction of new varieties. It was shown that for the successful introduction of new cultivars, there must be a recognised need - a pull factor. For creating of such pull factors, it is important to involve other chain players. Here, a shared vision & language among chain players and common culture is necessary. New concepts need to fit in existing structures, and adoption needs time. Furthermore, the usefulness of a variety approach differs largely depending on crop and region (apple, grapevine, tomato).

Apple
In CO-FREE, progressive cultivation techniques (agroforestry and Very Low Input Systems (VLIPS)) have been explored, both of which showed potential for promotion of biodiversity and reduction of copper use. However, these experimental production systems are not ready for implementation in commercial table apple production at scale due to shortcommings in control of of secondary emerging diseases control.
As a prerequisite to low input apple production, scab resistant cultivars are needed and available. However emergence of virulent scab populations necessitates control strategies and limits the use. Thus, in apple, a copper ban will affect yield security due to lack of control of other (emerging) diseases and the necessity to prevent the spread of virulent scab populations.
A number of alternative PPPs, DSS and strategies are available at present. CO-FREE demonstrated that especially in early season and under cold and wet weather conditions there is still a lack of alternatives.
Grapevine
Use of tolerant varieties, like PiWi (German: pilzwiderstandsfähig), reduces the number of treatments needed per season (16 treatments- in traditional varieties vs. 4 treatments in PiWi). The usage of acidified clay preparations in tank mixture with wettable sulphur provided excellent control of both, P. viticola and Oidium tuckeri. The varieties tested did significantly differentiate in their susceptibility to both pathogens. The experiments also clearly demonstrated that no compromises can be made regarding the efficacy level of copper alternatives. Lack of persistence and limited rain fastness limit the uses of copper alternatives even if used on moderately resistant cultivars.
PiWi cultivation provides an excellent means for copper reduction in areas where planting is legal and a market can be developed. For the foreseeable future, commercial organic grapevine production will rely on susceptible varieties and thus depend on highly efficient fungicides, including copper.
Nevertheless, in Vitis vinifera novel PPPs have the potential to reduce copper substantially (eg. 25% to 70%.) if combined in a refined strategy. There is space for improvement of e.g. formulations and application etc.. After all, complete copper replacement is not likely at the current state of knowledge (unless in combination with e.g. phosphonates). Moreover, no information is available regarding the occurrence of emerging diseases if copper is omitted.

Potato
In potato, copper replacement and yield security can only be achieved by consequent use of resistant cultivars, and thanks to incessant activities since Blight-MOP, increasing numbers of resistant/robust varieties are available on the market. Thus, potato could be the first crop where copper replacement can be technically feasible. Nevertheless, this will need time and efforts to finally be adopted in practice. As mentioned above, DSS will contribute to copper reduction in susceptible cultivars. Furthermore, CO-FREE alternative compounds may contribute to resistance management to prevent resistance breakdown of cultivars. Here, the marketing concept developed in CO-FREE will be essential to bridge the gap between providing R&D and novel varieties and the introduction into the market chain not only in potato but also in other crops

Conclusion
The potential of resilient systems in the context of reducing copper is not (yet) explored at length nor fully exploited. Usage of resistant cultivars is straight-forward and provides opportunities for copper reduction. Innovative systems are often focused on promotion of biodiversity, and pest control. CO-FREE clearly demonstrated that depending on the crop, disease control poses a huge challenge. Thus, substantial R&D investments are needed to develop the current experimental systems into feasible main stream options. In many cases, this will need at least 20 years. In CO-FREE, a partial proof of concept was provided in selected model systems and the current limits were identified.
Overall, in can be stated that in order to bridge the gap until sustainable alternatives are available on the market, copper still is needed for organic and low-input farming. However, improved application of low dosages of copper can be realized, including the use of resistant/tolerant varieties and advanced DSS. By the time when alternative(s) are available, correct application time according to mechanism of action needs to be considered, and under high level of disease risk, copper may still be needed. The inclusion of low-dosage copper strategies (use of alternative compounds, resistant varieties and DSS; see above) will be the way forward towards further copper reduction/replacement.


Positive impact on the environment by replacing copper use and by supporting EU policy to reduce chemical inputs in crop production.
Extensive copper fungicide use since the 19th century has led to accumulation in the top soil layers in many areas in Europe, mainly in vineyards, orchards and hops fields. Earlier surveys indicated that levels currently found in agricultural soils reached levels that are toxic to earthworms under laboratory conditions. The EFSA concluded based on the peer review of the pesticide risk assessment of copper (EFSA, 2008) that copper affects soil biota and is considered highly toxic to aquatic organisms. In the meantime, newer investigations indicated that the effects of copper, which is only partly available when bound to soil, most likely have been overestimated to some extent. Nevertheless, further copper reduction is expressed goal of both, organic farming and the EC.
The main uses of copper in organic farming systems in Europe currently include grapevine, pome and stone fruit, olive, potato, and some vegetable crops. In the EU/EFTA countries, copper use is still allowed (i.e. all countries except for The Netherlands, Denmark, Norway, and Sweden). The annual copper use depends on the crop/disease complex, pedo-climatic conditions, and the legal framework. In Germany, in 2013, an average of 1.4kg/ha and year of Cu were used in organic fruit or potato cultivation, while in grapes an average of 2.3kg/ha and year were recorded. However, the EC allows up to 6kg/ha and year of Cu and in many European countries amounts of copper up the limit are used. Indications for copper use in olive trees vary substantially between countries, but on an average, it is estimated that 20% of the area is treated with 2 kg copper/ha and year. The total potential to reduce copper in European organic farming systems is estimated to range between 800 and 1200 tons per year. In conventional, IPM and low-input farming systems copper is also still widely used, mainly due to low costs and in order to avoid or delay the development of resistance against single site fungicides.
Thus, alternative strategies are expected to have substantial positive impact on the environment. For example, both, state-of-the-art organic and IPM apple production systems receive 20-30 pesticide treatments (pest, disease and weed control) in current disease-susceptible apple orchards. A survey in Switzerland indicated a reduction by up to 45% if scab-resistant apple cultivars are planted. It is anticipated that in advanced low input orchards, pesticide use can be further reduced while maintaining high yield and quality levels. The introduction of advanced grapevine production systems has a similar potential for reduction of pesticide use.
Ecological impact
In CO-FREE, the overall ecological impact of novel alternative compounds has been assessed at the level of individual field experiments under WP8. However, the environmental impact is the result of all measures taken, including pest and disease control and soil management. All copper-free/reduced production systems evaluated under WP 5-7 have therefore been assessed by a multi-criteria assessment according to a procedure given in a handbook developed under horizontal activity in WP8.
The ecotoxicological profile of most of the tested substances showed no or low toxic effects on non-target insect, aquatic and soil indicator organisms in laboratory studies. Assessments of the CO-FREE products on beneficial arthropods were conducted in the efficacy experimental fields of tomato, grapevine and apple, based on relevant protocols established in the frame of the project (CO-FREE Handbook). Predatory mites, especially Phytoseidae, seemed to be the best indicator group to assess the impact on beneficial arthropods mainly due to the small size of plots corresponding to small number of insect predators and spiders. No negative effect of the tested CO-FREE compounds was observed on phytoseid mites in grapevine (Greece, France) and apple fields (France, northern Italy). In plum tomato (Greece) only the incidence of generalist predators and parasitized aphids could be recorded since the numbers of beneficial insects in the plots did not allow statistical analysis. An increased number of collembola, which is a biodiversity index insect group, was observed in the plots of some CO-FREE test products possibly due to feeding on the compounds.
Furthermore, a partial proof of concept was provided in selected resilient model systems (VLIPS, agroforestry). VLIPS excelled in providing higher biodiversity in VLIPS orchards whereas marketable yield was limited due to insufficiently controlled (emerging) plant diseases. Impact assessment of an agroforestry/arable production system on beneficial arthropods and insect pollinators in Eastern England showed comparable total hymenoptera abundance between the agroforestry and orchard. However, at the family level, there were significantly more Lassioglossum and Bombus in the orchard than in the agroforestry and more predatory and parasitic wasps in the agroforestry.
EC-policy on phasing out of copper
EC-policy is discussing phasing out of copper in the foreseeable future. As soils have accumulated copper over decades, only a minimal direct impact on mitigation/remediation of soil contamination can be expected from a project proposed under topic KBBE.2011.1.2-06. The main impact of CO-FREE therefore consisted in providing tools and technologies that build the fundament for a continuation and further progress of organic production of grapevine, fruit, and other key commodities. The successful replacement/reduction of copper use affects at least 240’000 ha in current European organic farming systems and will also build the grounds for further increase of the production area.
As discussed in the final CO-FREE conference, immediate phasing out of copper would at present create unbearable risks and costs (e.g. for tools or risk compensation). Thus, phasing out of copper is currently not feasible generally. However, in the four systems under investigation in CO-FREE, further reduction is achievable in most crops, and possibilities are seen highest in potato>apple>tomato>grapevine. The key to reduction/replacement of copper is the use of smart management strategies, which comprise a variety of approaches. Already, farmers are looking more for resilient varieties and combine them with reduced copper applications, and CO-FREE is contributing to further copper reduction by having identified a number of candidate compounds and improved DSS and by having investigated strategies to be used in the crops.
The activities of CO-FREE substantially contributed to further copper reduction/replacement and will thus help to preserve or enlarge EU organic and low input production areas. Novel advanced low input farming systems, alternative compounds and new tools have the potential to reduce the total pesticide use by more than 50%, depending on crop and region.
As mentioned above, potato could be the first crop where copper replacement can be technically feasible. The potential of resistant cultivars and difficulties in market introduction were already recognized as a result of Blight-MOP. This approach was consequently further developed. In CO-FREE, a range of new candidate substances were identified and further developed. A great impact can be seen in the use of resistant cultivars, which are already available. In order to stabilize the resistance over time, further measures are necessary, including the use of PPPs. The integration of a new model in the existing DSS Öko-SIMPHYT, as carried out in CO-FREE, will contribute to further copper reduction, since the model estimates if treatments late in the season are necessary or not for yield security. The use of alternative compounds will furthermore support the long lasting of resistance features in robust varieties. However, some more years will be required for further development of the compounds investigated. Nevertheless, in potato several measures are already available which positively impact environmental aspects After the market introduction of further alternative compounds, the impact will be even higher, since copper may then be completely omittable. Moreover, CO-FREE identified strategies to overcome bottlenecks for introduction of new varieties on the market and refined breeding strategies based on close stakeholder involvement.
Overall, CO-FREE tackled various important aspects along the production chain, from breeding, over cultivation and marketing, thus contributing strongly to a successful implementation towards copper replacement in potato and other crops. Public awareness was increased by devising new and innovative communication schemes. Nevertheless, it will still need time and efforts to finally adopt all measures at scale in practice.

The second crop in the list above is apple, where copper is used to stabilize yield. Here, varieties, DSS and already marketed alternative PPPs are available, which strongly contribute to a positive impact on the environment. As outlined above, CO-FREE compounds need to be further investigated in this system and the emergence of virulent scab populations limits the use of resistant varieties. However, in CO-FREE, progressive cultivation techniques without copper (agro-forestry and VLIPS) have been explored with partial proof of concept and interesting results to build on. Here, substantial R&D investments are needed to develop the current experimental systems into feasible mainstream options. In many cases, this may need however at least 20 years. In conclusion, the dependency on copper for scab control was reduced but emerging diseases necessitate the provision of copper availability as fall-back option in case of emergency.

In tomato, copper usage is presently very high. Former trials demonstrated the potential to reduce copper in tomato by 50%. Two CO-FREE candidate compounds were identified with the potential to further substantially reduce copper use, thus having further positive impact on the environment. In tomato, robust varieties are lacking and breeding for resistance is urgently needed.
In grape, tolerant varieties are available and provide substantial potential for copper reduction. However, cultivation of resistant cultivars is not legal in many key regions and a market would need to be developed. Nevertheless, for niche markets, this can be a good option. DSS have been further refined in the project for downy mildew and a pilot model was introduced for black rot in grape. Furthermore, in CO-FREE, 4 promising alternative compounds were identified which can substantially contribute to copper reduction in grape and all measures will further positively impact the environment, when used in a refined strategy. Further development is however required before the alternative PPPs are available for market introduction. Overall, in grape, further research is required to investigate emergence of potential diseases when copper is omitted.
In conclusion, depending on the crop, a range of measures are ready to be implemented and used, while others need some more time. E.g. for several of the alternative compounds investigated in CO-FREE, completed registration is to be awaited earliest from 2022. Also, new varieties need time to be adopted by farmers and end-users. As shown in CO-FREE, communication and commitment along the whole value chain is essential here. Results indicated that strategies including the use of alternative compounds as one component together with DSS and further measures will be the way forward to further reduce/replace copper. The adoption of novel copper-reduced/free production systems by farmers depends directly on the economic feasibility and on the market demand for such commodities. The advanced production systems developed under WPs5-7 in CO-FREE have been submitted to a socio-economic assessment in order to determine profit margins, and it turned out that these are most likely to be more costly than copper. As discussed and stated in the final CO-FREE conference, the replacement and reduction of copper by other tools is currently slowed down beyond the technical potential by, for example, the legal frameworks, associated costs or lack of markets. Thus, exchange and discussions, including advisors, farmers, retailers and policy makers, needs to be continued and intensified to develop joint agreements and regulatory/subsidy schemes.
Altogether, the findings from CO-FREE have a high positive impact on the environment and support EU-policy to reduce chemical inputs, due to their current and future contribution to further reduce copper application in agriculture.

Meet the needs of the agricultural sector across different regions in Europe
The most important crops that currently depend on copper use in Europe are grapevine, pome and stone fruit, potato and tomato. Organic grapevine and field tomato production is mainly located in the Mediterranean countries and to some extent also in the moderate climates of Central Europe. Pome and stone fruit production is present in most European countries, with a focus on France, Italy, Germany, and the Netherlands. In contrast, potato production has a tradition in all European countries, including the Mediterranean, Atlantic, Central European and Northern countries. The market demand for products derived from organic and low input production systems is steadily increasing but production was limited so far, mostly due to limitations in disease control. CO-FREE focused on providing copper reduction/replacement strategies to be used in the current production areas. Moreover, the introduction of innovative production strategies that increase the resilience towards diseases and pests and thus reduce the overall dependency on pesticide impact will create the potential for substantial growth of organic and low input farming systems in regions where organic and low input farming was limited due to high disease-related production risks.
Technologies developed for replacement of copper will also benefit those regions where copper use is already banned, i.e. the Netherlands, Denmark, Sweden, Norway and Finland. Organic production is currently facing severe losses in these countries and growers are losing position on the competitive markets.
Therefore, CO-FREE has evaluated novel alternative products developed in WPs 2-3 in pome fruit, grape, potato and tomato in a wide range of pedo-climatic conditions covering Mediterranean, Atlantic, and Central European (moderate and continental) climates. Furthermore, refined production systems developed under WPs 5-7 have been tailored to the regional needs and the experiments have covered a wide range of climatic conditions, allowing for progressive uptake of innovations, taking into account regional and cultural differences as well as the economic realities and the local legal framework.

Provide results that are of interest and potential benefit to SMEs.
A total of ten SMEs have built an integral part of the CO-FREE consortium, each with a portfolio of at least one innovative pipeline product with proven efficacy and/or an innovative decision support tool. A cornerstone of the overall concept of CO-FREE was the development of novel copper-alternative compounds of plant and microbial origin under WP2-3. The involvement of industry partners is a prerequisite to achieve production, registration and marketing of plant protection products. Novel products of plant and microbial origin have in the past often been successfully developed in close cooperation between publicly funded research and industry partners. Therefore, the CO-FREE steering committee had identified the most promising and advanced candidate companies/products. In order to maximize benefit to SMEs and to achieve the overall aim of the project, each of the SMEs in the CO-FREE consortium involved in WP2-3 was partnered by an academic partner providing tailor-made complimentary research for the benefit of the product development of the SME. Thus, SMEs had privileged access to the knowledge of the academic partner while IP was protected from competitors. This enabled SMEs to make rapid progress in product development. Services provided included improvement of manufacturing processes, identification of active compounds, improved formulation, development of application strategies, identification of additional uses. Furthermore, in a workshop organized by CO-FREE at Annual Biocontrol Industry Meeting 2013, SMEs also had access to partners providing specific knowledge and technology and to policy makers involved in registration in order to optimize dossier preparation. A major breakthrough was the implementation of the guidance document for dossier preparation of botanicals that clarified some key uncertainties in product registration. CO-FREE also provided the SMEs with rapid access to potential end-users (farmers, advisors) in the main markets of Europe by integrating novel products into copper-reduced production systems in WPs 5-7 and by providing multi-criteria-assessments under WPs 8-9. SMEs took advantage of the complementary knowhow of scientific and field partner. In particular, efficacy evaluation in different pedo-climatic conditions and assessments of ecotoxicological and economic data was provided. SMEs had access to the expertise in organic crop protection and to testing facilities across Europe in the key crops. Thus, CO-FREE has facilitated the development and market introduction of novel plant protection products for use in the entire European agriculture. The overall market for biocontrol agents has duplicated every 5 years in the past, mainly due to plant protection innovations that are useful for both low input and intensive traditional agriculture. Efficient and economically feasible copper alternatives will find very large markets worldwide and thus strengthen the competitiveness of European biopesticides manufacturers.
Work in CO-FREE under WP4 has led to the development of a new DSS for grape downy mildew and a pilot model for grape black rot, which both are now offered by the respective SMEs to users.
Breeding of novel disease-resistant cultivars is currently mainly provided by publicly funded RTD and SMEs. A conceptual study in CO-FREE (WP 7) did not support the initial assumptions of markedly different traits being relevant for ideotype adaptation to different climatic environments in organic potato crops in Europe which is valuable information to breeders. How acceptance of novel varieties among farmers, advisors, retailers and consumers (WP 9) can be provided and fostered, has been shown in pilots in the project and will open new market opportunities for breeding companies. Possibilities of contacts to farmer´s breeders was provided to SMEs. All activities substantially contributed to the benefit of the industry.

Provide a beneficial economic impact to the sector concerned
As outlined above, immediate phasing out of copper is currently not feasible generally, since it would create unbearable risks and costs and would lead to rapid break-down of the organic sector. However, in the four systems under investigation in CO-FREE, further reduction was shown to be achievable and possibilities have been identified to reduce copper-dependency. This will provide the fundament to farmers for the economically feasible continuation of organic and low input farming in high value crops by a step by step continued reduction of copper to be further developed until copper can be phased out in the future. This helps to preserve the huge past investments by private (farmers, retailers) and public stakeholders (wider public, tax payers, policy makers). Further copper reduction/replacement is a declared goal of both, the EC and the organic sector. Thus, the possibilities of further copper reduction as shown in CO-FREE contribute not only to support EC policies but help to support existence and expansion of organic and low-input farming. It underlines and keeps the creditibility of organic farming in public recognition. The activities and outcomes of CO-FREE help to increase the productivity and yield security of high value crops and support the opening of markets for these products. This results in further potential to enlarge the proportion of organic/low input farming within the ‘traditional’ regions and to widen the potential production areas to regions with higher disease pressure.
CO-FREE has shown a medium term way to further copper reduction/replacement. However, refined strategies and especially novel compounds are most likely more expensive than copper. This is due to the fact that (i) the alternatives have shown in the best cases as high efficacy as copper and (ii) production costs for alternative compounds will in most cases be higher based on expenses for raw material and production.
Alltogether, CO-FREE contributed strongly to long-lasting credibility of organic farming by showing differentially in four of the most important copper-relevant crops that further reduction of copper use is possible.

Ensure a strong link between academia and end-users.
The ultimate purpose of CO-FREE was to achieve rapid development and adoption of innovations for copper-reduced/free plant production by European organic and low-input farmers, who are the main end-users profiting from RTD activities provided by academic partners. However, the SMEs included in the CO-FREE consortium also profited directly as end-users from CO-FREE results. The three key strategies of CO-FREE to achieve the RTD objectives and maximum use of new knowledge and technologies by end-users consisted in (i) the full integration of SMEs in the project consortium and RTD activities in WP 2-4, (ii) active (farmer, retailer) participation and on-farm research in WPs 5, 6, 7, 9, and (iii) dissemination and continuous dialogue with the main stakeholder groups and the wider public in WP 10.
In the consortium, partners FiBL, ITAB, LAI, ORC and LBI integrated RTD as well as advisory services. These partners have long-established contacts with (or are part of) (i) local and international organic and IPM farmers’ associations, (ii) inspection and certification bodies and (iii) the policy makers at EU and MS level involved in organic and low-input farming. Based on this interaction, only innovations that are potentially acceptable in the target farming systems have been developed. Partners JKI, DLO, INRA, FEM, LAI, BPI, FiBL, and IPP are closely involved in the regulation/registration of plant protection products including the development of the regulatory framework for biocontrol agents.
Academia and farmers have engaged in an intensive dialogue when CO-FREE results were presented at farmers reunions and in dissemination activities organized by local advisory services (WP 10). A very intensive dialogue between farmers (and advisors) and researchers has been established at ‘open field days’, when the field experiments conducted in WPs 5-7 in different countries were open for visits and active farmer participation was encouraged. Farmers and advisors were also directly involved in the activities in WP 9 dealing with the development of strategies to foster acceptance for novel varieties by consumers and retailers. The publication of articles in farmer´s magazines, lectures and flyers for consumers, the presentation of results to grower´s associations, and the production of a video that is available on the CO-FREE website further ensured dissemination from academia to end-users.

Dissemination activities
The rapid dissemination of experimental results to and the interaction with advisors, farmers, and other stakeholders is crucial for rapid implementation of techniques into farming practice. Results and outcomes of CO-FREE were actively communicated to stakeholders and policy makers throughout the project whenever results were sufficiently supported by data and as long as exploitation of IP was not infringed.
CO-FREE has disseminated information by (i) integration of leading partners within the consortium in the sector in the most important regions, (ii) by close interaction with and by feeding information into dissemination networks (i.e. established by ENDURE, continued by PURE, national organic advisory networks, TIPI - the Technology Innovation Platform of the International Federation of Organic Agriculture Movements (IFOAM)), and (iii) by intensifying the excellent direct contacts of the consortium partners with the main stakeholders of the sector (i.e. producers associations, retailers, advisory services, regulators and other policy makers). A project website was established, and newsletters were produced where events and results were made available to the public. Within CO-FREE, FiBL, ITAB, LAIM, ORC and LBI integrated RTD as well as advisory services and thus had direct contact to local and international organic and IPM farmers’ associations, inspection and certification bodies and policy makers at EU and MS level involved in organic and low-input farming.
CO-FREE was noticed beyond European boundaries e.g. by representatives of the Chinese Academy of Agricultural Sciences and during a visit of the Minister of Agriculture of Israel at BPI (2012). The Newsletter was subscribed by Europeans and by readers from Argentina and New Zealand, and CO-FREE was presented at EXPO Milan at the EU-pavillon in 2015 (live-streamed) for which a video was produced focussing on the issue of breeding and consumer acceptance of new cultivars (linked on the CO-FREE website). CO-FREE is one of 25 selected EU-funded projects presented in the EU-brochure “EU-funded Plant Health and Plant Protection Research (2007-2013)”.
In particular, CO-FREE partners published 18 peer reviewed articles so far and had more than 100 oral and poster presentations at conferences and stakeholder meetings. Field trials were presented to farmers on 13 open field days and in further events an exchange with farmers, breeders, advisors, consumers, industry and other stakeholdes was realized. Contacts and discussions with policy makers took place in different meetings. E.g. JKI was invited to give a lecture at the 8th IFOAM European Congress, which focussed on implementing innovative ecological solutions for farmers and rural communities and shared views on the European Innovation Partnership for Agricultural Productivity and Sustainability (EIP-AGRI), rural development and the organic regulation review (2014). CO-FREE was presented by JKI and FiBL in expert discussion on copper in Germany (2012, 2013, 2014). FiBL took part in DG SANTE’s "low risk substance" expert group that developed a guidance document for improved registration procedures for botanicals (2013-2015). The expert group comprised DG SANTE, EFSA, IBMA and several MS regulators. FiBL also contributed as to EGTOP (2014), and provided a key note address in the IFOAM EU Internal Meeting on future of Peronospora Control in Viticulture (2014). The final CO-FREE conference was organized during the world-leading trade fair for organic food (BIOFACH) in Nürnberg, Germany in February 2016 as part of the BIOFACH Congress.

Overall, the outcomes from CO-FREE will be further developed and continuously be made public also after the end of the project at conferences, stakeholder meetings etc., and thus will contribute to practical adoption of further copper-reduction both, for end-users and policy makers.

Exploitation of results
The partners involved in CO-FREE are committed to proceed with the development and market introduction of the copper alternatives and e.g. DSS tools. Our studies related to the market introduction of novel varieties will have a considerable impact on breeders, farmers and retailers, since we demonstrated that the close collaboration of the players along the value chain is essential for a successful shift towards copper-independent varieties. Results related to potential and limits of the experimental low copper production systems already find high interest among farmers. However, there are also obvious technical and economic limits and draw-backs that will prevent rapid wide-spread adoption. Here, continued research is clearly needed to further develop those systems into economically feasible and realistic options. However, the examples and outcomes do inspire farmers and the realistic assessment also paves the way towards higher acceptability among farmers as soon as the methodology is feasible.

List of Websites:
www.co-free.eu
Dr. Annegret Schmitt
Julius Kühn-Institut, Federal Research Centre for Cultivated Plants
Institute for Biological Control
Heinrichstr. 243
64287 Darmstadt, Germany

Related information

Contact

Knut Steffens, (Administrative officer)
Tel.: +49 531 299 3270
Fax: +49 531 299 3023
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Agriculture
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