Final Report Summary - DROPSA (Strategies to develop effective, innovative and practical approaches to protect major European fruit crops from pests and pathogens)
Executive Summary:
DROPSA's aims were to develop reliable, robust and cost-effective approaches to protect the major European fruit crops from Drosophila suzukii, and the quarantine pathogens Pseudomonas syringae pv. actinidiae (Psa), Xanthomonas fragariae (Xf) and X. arboricola pv. pruni (Xap) which are major phytosanitary risks and pose significant challenges to fruit production. It is estimated that pests and pathogens cause losses to the EU fruit industry of €10 billion and 3 million tonnes of produce.
Genetic studies and the spread models for D. suzukii, Psa and Xf provided information on the introduction and spread of new pests and diseases of fruit. Alert lists have been created for pests and pathogens that may be introduced into Europe with trade of Vaccinium berries, apples, table grapes and oranges/mandarins.
A comprehensive understanding of the biology of D. suzukii and the epidemiology of Psa, Xf and Xap provided key information for the development of prevention and control methods.
Extensive studies on non-crop hosts, spatial ecology, and interactions between crop and non-crop habitats of D. suzukii and the lack of effective native parasitoids in Europe indicate the need for an area wide control approach for this pest. A non-native Asian parasitoid has been selected to assess its suitability for classical biological control. The best commercial trap and attractant for mass trapping and monitoring were identified. A device to lure and kill D. suzukii was developed, as well as a lure and infect strategy using entomopathogenic fungi.
Field strategies for D. suzukii using conventional pesticides have been developed for crop protection with lower residues in harvested fruits. Some may be used in concert with biological control agents and in integrated pest management (IPM) with fruit removal, mass trapping. Insect proof nets effectively exclude D. suzukii, but costs are high, and nets impact on orchard management.
Future pest control solutions based on sterile insect techniques, modifying fly physiology and behaviour, and novel delivery mechanisms for biomolecules, have been developed in the laboratory.
Data on environmental requirements and disease epidemiology of the bacterial pathogens provided data for forecasting models and decision support systems. On-site detection tools and genotyping tools were developed for all three pathogens and can be used for phytosanitary inspection and monitoring, and to characterize pathogen population biodiversity.
Few existing products were effective for disease management confirming the need for new and alternative compounds. Novel antibacterial peptides and microbial biopesticides derived from bacteria were identified. Innovative formulations and stem injections were developed to apply antibacterial compounds into kiwi vines and peach trees.
Cultural management reduces pest and disease incidence. Psa contamination is associated with irrigation water, pruning instruments, grafting and pollen, the latter highlights the risk of commercial pollen production and assisted pollination in kiwifruit. The spread of pathogens by D. suzukii is of low risk. Orchard management practices (training, pruning, covers) facilitate spray treatment and limit disease incidence.
A Decision Support System, which integrates information on biology, ecology, and economic evaluation of management options revealed that strategies developed in DROPSA performed better than current pest management practices for Psa and D. suzukii.
Integrated pest management (IPM) strategies (orchard management, resistance inducers, biological control) for Psa significantly reduced bacterial canker incidence in plastic covered tunnels.
Economic analyses showed that the use of extra chemicals or netting with bait sprays to control D. suzukii leads to a higher marginal gross margin, but single tree netting is not economically sustainable. Covering kiwi plants can prevent yield reductions caused by Psa, but due to the relatively high investment costs this is not economically viable for green kiwifruit in Italy.
Project Context and Objectives:
Fruit crops account for around 17% of the value of the EU’s agricultural production and the fruit and vegetables supply chain has an estimated turnover of more than €120 billion with over 550,000 employees and around 1.4 million growers. Whilst reliable data on average yield and financial losses due to invasive pests and diseases are not currently available in Europe, it is estimated that insect pests destroy approximately 14% of all potential food production globally. Therefore, it is reasonable to estimate that fruit losses from pests and pathogens accounts for at least €10 billion in revenue and 3 million tonnes of produce to the EU fruit industry.
The European Fruit Sector delivers an essential and unique contribution to the economic, social, and environmental future of the EU; it is technologically advanced and includes a complex chain of production, storage, marketing, logistics, wholesaler and retailer input providers. This sector faces several challenges including financial competition, especially from developing countries, and the increased trade from outside Europe increases the risk of invasive pest species.
Modern fruit production must ensure consumer satisfaction and deliver high value quality fresh fruit that contributes to a healthy diet and overall wellbeing. It should also be free from contaminants such as pesticide residues which can be achieved through ensuring agricultural practices that reduce pesticide impacts on the environment and promote biodiversity.
This view has widespread public support and has been influential in scoping of EU regulations for pesticide use and plant protection strategies including the sustainable use directive (EU Directive 128/2009/EC) . This directive aims to achieve sustainable use of pesticides by minimising the risks and impact on human health and the environment, improving the quality and efficacy of pesticide application, and promoting the use of integrated pest management (IPM), and alternative approaches and techniques. Under article 14 of this directive EU member states are required to have a national action plan for the development and introduction of IPM. In addition, Regulation (EC) No1107/2009 , concerning the placing of plant protection products on the market, aims to ensure a high level of safeguarding human and animal health, and the environment, whilst protecting the competitiveness of EU agriculture, and at the same time, increasing the competitiveness of EU agriculture.
Delivering the DROPSA project objectives aimed to provide effective, sustainable, innovative and integrated management solutions to protect the major EU fruit crops, which addressed the above EU policies and regulations, the need for environmentally friendly control strategies, that are target specific to reduce the negative impact on beneficial organisms (e.g. bees), and the public concerns and desire for food free of pesticide residues.
New and emerging pests e.g. Drosophila suzukii, and quarantine pathogens such as Pseudomonas syringae pv. actinidiae (Psa), Xanthomonas fragariae (Xf) and X. arboricola pv. pruni (Xap) that have been introduced into Europe have been identified as major phytosanitary risks and pose a major challenge to fruit production.
Drosophila suzukii is an invasive pest native to Asia that was first reported in Europe in 2008, and has since rapidly spread across the continent. It is highly polyphagous and infests a wide variety of berry and stone fruits. The larvae feed and develop within ripening fruit causing it to soften and rapidly rot, resulting in significant reduction in crop yields and financial losses, and can be so devastating that 100% fruit losses on many crops (e.g. Cherries) have been reported. In Northern Italy, annual losses in fruit crops of over €8 million and over €4 million for cherries in France have been reported. A loss of just 10% (1.1 million tonnes) of the strawberry crop in the EU represents €50 million in lost income. In 2011, a pest risk assessment by the European and Mediterranean Plant Protection Organisation (EPPO) recognised the importance of D. suzukii as a serious pest of soft fruits, stone fruits and grapes in most parts of the EPPO region. It is also considered that an eradication programme and regulation would be unlikely to retard its spread, and that there was currently no effective control strategy against this pest. Hence, D. suzukii poses a serious challenge to fruit production in Europe, and the lack of knowledge on its basic biology has seriously hampered the development of rational and effective IPM strategies.
The production and quality of fruit in the EU is also severely threatened by bacterial diseases caused by Psa, Xap and Xf which represent the most recent and devastating outbreaks of disease to affect European fruit production. Control options are limited due to the virtual absence of, and restrictions on, effective bactericides, so there is an urgent need to develop effective and practical strategies for their management to protect the EU fruit sector. DROPSA has focussed on Psa on kiwifruit, Xap on stone fruits, and Xf on strawberry, but these pathogens affect all the main fruit types, so strategies developed for their control will be transferable to other fruit crops.
Italy are the second largest producer (after China) and second largest exporter (after New Zealand) of kiwifruit equating a value for agricultural of over €3 billion with a retail market value worth €10 billion, and is vital to the economy of the five main regions (Lazio, Emilia, Romagna, Piedmont, and Veneto) where 80% of Italy’s production is concentrated. Kiwi production has been compromised by Psa, which is currently the most virulent and devastating disease in fruit crops. Economic impacts for farmers have been estimated to be over €85000/hectare/year in production losses, orchard investment and plant destruction to stop the spread of infection, reducing production in orchards by 10-50%. Revenue losses of over €40 million have also been reported for Psa damage to kiwifruit in the Lazio region of Italy and in New Zealand Psa is estimated to cost the kiwifruit industry over €50 million per annum.
Among small fruits, strawberry is the most important berry crop in the EU, being produced in all member states, with a production exceeding 1.31 million tonnes per year. Strawberry production requires specialised knowledge, high energy costs (e.g. machinery fuel, irrigation), high external inputs (e.g. labour costs, fertilisers, water and pesticides). Thus, any development that may reduce the incidence of pests and pathogens in strawberry cultivation will be of high importance. Disease in strawberries due to Xf is predominantly destructive in nurseries, especially under protective cultivations with high humidity, and can cause up to 75% losses.
Present in central and southern Europe, but spreading, bacterial spot due to Xap has been reported on a wide range of Prunus species, and is particularly serious in peach and plum. Control relies primarily on copper compounds, but none are fully effective, hence reliable and durable control strategies for this disease are still needed.
Clearly control methods for both D. suzukii and microbial pathogens in Europe are severely inadequate. DROPSA has addressed these problems to advance options beyond those currently available to provide effective, sustainable and IPM strategies to protect and secure food production in the EU.
The primary research emphasis of the DROPSA project is on the development of effective, innovative and practical solutions for the control of D. suzukii, Psa, Xf and Xap on various fruit crops that are representative of the some of the major fruit sectors in the EU (namely berries, grapes, stone fruits and kiwifruit).
This is based on the following drivers:
• Pests and pathogens, particularly D. suzukii, Psa, Xf and Xap, are a major concern and challenge to fruit production and cause significant losses within and outside the EU region.
• Since its introduction into Europe D. suzukii continues to spread unabated. Eradication or containment is not possible, so there is a pressing need for effective, targeted, innovative, practical and integrated management solutions to minimise its economic impact.
• The pathogens Psa, Xf and Xap are currently emerging in the EU and Mediterranean region and there are no efficient methods of disease control available. Their spread from the initial outbreak to other fruit production areas is ever increasing.
• The invasion of, and damage by new exotic pests and pathogens is likely to increase with intensification of trade and climate change.
• The desire to develop IPM strategies able to guarantee high yields, high fruit quality and fruits free of pest, diseases and pesticides.
• Τhe translation of knowledge to practical solutions for the management of pests and pathogens, with the active engagement of SMEs and industry.
• To use DROPSA as an exemplar of how the investigation of the pathways of introduction of D. suzukii, Psa, Xf and Xap will provide recommendations and strategies against the invasion of other invasive pests and pathogens.
The principal objective of DROPSA was to develop reliable, robust and cost-effective approaches to protect the major European fruit crops from new and emerging threat dues to pests (D. suzukii) and pathogens (Psa and Xf and Xap) that could be widely implemented by the EU fruit industry.
DROPSA integrated leading expertise in fruit pests and pathogens, fruit production and orchard management from across Europe and more widely on a global basis, involving multidisciplinary, interrelated and complementary evaluations, methods and techniques. The main objectives were to:
• Determine the pathways of introduction and spread of D. suzukii and the above pathogens into the EU to help develop preventative strategies and recommendations against the introduction of other dangerous fruit pests/pathogens and diseases into Europe.
• Contribute towards minimising the risk of spreading the target pest and pathogens in other countries, thus securing the security of EU fruit exports.
• To identify the key mechanisms within the biology, epidemiology, ecology and interactions of these pests and diseases for the development of effective, innovative, practical and sustainable control methods. This includes a comprehensive evaluation of life cycles, host ranges, capacities to disperse, the identification of natural enemies, plant-pathogen interactions, and semiochemicals involved in D. suzukii behaviour.
• Develop control options including screening of existing compounds, semiochemical based technologies, novel antimicrobial compounds, biological control agents, as well as cultural practices, sterile insect techniques, and new mode of action compounds.
• Combine the most reliable and effective control options to develop and optimise an integrated pest management strategy to combat fruit pests and pathogens, utilising forecasting and decision support systems and risk mapping.
• Evaluate the economic viability of proposed strategies for fruit crop protection to support decision making in the development of practical solutions and integrated pest management strategies
DROPSA has included partners from non-EU countries in Oceania (New Zealand), North America (USA and Canada), and Asia (China and Japan) which have extensive expertise in the biology and control of D. suzukii, Psa, Xf and Xap. The exchange of information between countries in these regions (where the pests/pathogens either originated or show similar invasion characteristics) and EU partners, and the coordinated development of improved control methods, is recognised as key to the successful execution of the project.
Project Results:
Work package 1 – Pathways of introduction of fruit pests and pathogens
Aims and objectives
The aims and objectives of WP 1 were to determine the pathways of introduction and spread of Drosphila suzukii and pathogens into the EU and to develop preventative strategies and recommendations against the introduction of other dangerous fruit pests and pathogens.
Results and discussion
Pathways of introduction and spread of D. suzukii
The spread and potential distribution of D. suzukii was modelled with a dispersal kernel model based on a 2Dt-distribution developed in the former EU project PRATIQUE. The spread model predicted a rapid spread over the whole area of potential establishment (i.e. most of Europe) within seven years. D. suzukii is already widespread over a large area of the EU but has still the capacity to spread and establish in the northern EU countries (Ireland, Scandinavian states and Baltic States), though with limited damage potential.
It was found that modelling of D. suzukii with a dispersal kernel is possible. The dispersal kernel provides a quickly applicable tool with the possibility to adjust relevant parameters to new findings.
DROPSA partners participated in an international study to identify the routes of invasion of the European and North American invasive populations of D. suzukii, using molecular markers (microsatellites) directly with D. suzukii populations to perform a phylogeographic study on the roads of invasion of D. suzukii. In order to distinguish the different populations of D. suzukii, the presence and the phylogenetic relationship of the bacterial communities associated with the intestinal tract of natural populations of D. suzukii from different geographical areas were assessed, with the aim to study their phylogenetic relationship. The results indicate that habitat, food resources as well as the colonization phase of a new region contribute to shape the bacterial communities of the invasive species which, in turn, by evolving more quickly, could influence host adaptation in a new environment.
The study of the routes of invasion of D. suzukii worldwide revealed that Japan emerges as the most probable source of the earliest recorded invasion into Hawaii. Furthermore, Southeast China and Hawaii together are the most probable sources of populations in western North America, which then in turn served as sources for those in eastern North America. European populations are genetically more homogeneous than North American populations, and their most probable source is northeast China, with evidence of limited gene flow from the eastern US as well.
Pathways of introduction and spread of diseases
Spread models were also prepared for Pseudomonas syringae pv. actinidiae (Psa) and Xanthomonas fragariae (Xf) to evaluate the applicability of this model to bacterial pests. It was hitherto tested on insects, plants fungi and nematodes. It was not possible to model the spread of X. arboricola pv. pruni (Xap) due to the lack of key data. The area of potential establishment was plotted for all targeted bacteria. The modelling of bacterial spread with the dispersal kernel is in general possible. The model needs only a few key parameters as input and provides meaningful results. However, the modelling of bacterial spread is challenging due to the biological characteristics of bacteria and a lack and high uncertainty of data.
An alert list of pathways of introduction in Europe
A review on pests (including pathogens) that have fruit species as their host plants was conducted. The focus was on pests that have been introduced into Europe or were found in the fruit trade during the last 10–15 years. A total of 387 introduced pests were identified. The large number of pests intercepted on fruit, from different taxonomic groups and origins, shows that pests do move in trade on fruit. Conducting pathway analysis requires a considerable amount of time for each fruit species, therefore a priority list of fruit crops was established. Important factors for prioritizing the crops were for example trade volumes into the EU, the area of production in the EU, origins of trade (emerging markets) and the prior history of pest introduction. Furthermore, the overall selection aimed to provide a good geographical coverage of fruit production in the EU, to ensure a balance between fruit species (both regulated and non-regulated for fruit in the EU), and to cover crops from different groups (e.g. pome fruit, stone fruit, citrus, small fruit). After prioritization, Alert lists for Vaccinium berries, apples, table grapes and oranges/mandarins were prepared, as well as a description of the commodities traded for each species. Detailed information on the pest identification and rating system were provided. Pests from all parts of the world were reviewed and screened to assess the ones that were likely to be introduced with fruit trade. For the four selected pathways, 104 organisms likely to be introduced into Europe with foreseeable non-negligible economic impact were identified.
The pathway analysis provides the potential to identify pests that otherwise would have been unrecognised until they are detected in commodity inspections or in member states of the EU. These species represent a high risk for fruit production in Europe. It was shown that the risks of introducing new plant pests through the import of fruits have so far been underestimated. The Alert lists provided the basis for the identification of preventive strategies, which are based on recommendations for good professional practice for importers and producers as well as risk mitigation by legal regulation. These commodity assessments may be used to support regulation of high risk commodities at EU level (as defined in art 42 of EU Regulation 2016/2031). Pathway analyses provide the opportunity for horizon scanning and the identification of so far unnoticed risks and should be continued in the EU.
Methods for quantitative pathway risk analyses systems for fruit pests and pathogens
The quantitative pathway model, QPAFood, has been designed to support risk assessment for plant pest entry into EU territory on a range of edible plant commodities and for the pests’ transfer to a host, via trade flows. The model was developed originally for the European Food Safety Authority (EFSA) and has been extended in the context of DROPSA. It provides a stochastic description of the flow of infested commodity from one stage on the trade pathway to the next.
The practical modular approach applied here offers a high degree of potential application for fruits and grains such that different pests and commodities can be assessed by changing parameters on the spreadsheets indicated without the need to alter either model’s structure. A pathway in QPAFood concerns a particular pest on a particular commodity, but multiple commodity paths for a single pest or multiple pests for a single commodity can be considered by employing a series of parallel model calculations. The model enables the identification of high risk origins of the commodities and the assessment of the arrival risk on a monthly basis. The quantitative pathway model was applied to D. suzukii and the Apple fruit moth Argyresthia assimilis. The QPAFood model can be used to facilitate effective and efficient inspection methods of monitoring fruit imports; identify countries and products with a high risk profile and increase the awareness of the risk and the urgency for action to fruit industry. Within DROPSA the QPAFood pathway model was extended to cover multiple commodity pathways, reflecting the wide range of commercial hosts of D. suzukii.
Work package 2 – Biology and Ecology of Drosophila suzukii
Aims and Objectives
To identify the key mechanisms within the biology and ecology of D. suzukii for the development of sustainable control methods in WP 4 and WP 6 and to assess actual and potential host range and study spatial ecology in various environments.
Results and discussion
Life cycle and environmental requirements
The life cycle and environmental requirements of D. suzukii under various climatic conditions were assessed. These data have been used for the development of integrated control strategies in WP 4 and 6 and for risk mapping in WP 6.
Population dynamics of the fly in Europe and East Asia
For the first time, the population dynamics of D. suzukii was studied in east Asia, at various sites in Beijing (Temperate climate) and Kunming (Yunnan, subtropical climate), using traps. In the two regions the peak of the occurrence occurred in summer but dropped in late summer or early autumn. This is in contrast to what is observed in Europe and North America where, in general, populations are maintained or even dramatically increase in autumn. In Beijing, hardly any flies were caught from late November to late May. In Kunming, in contrast, flies were found in all seasons. However, in total, many more flies were caught in Beijing than in Kunming. An elevation gradient did not provide a clear pattern. Variations in natural control may explain the lower population levels in Yunnan compared to Beijing, where D. suzukii could possibly also be introduced. A subset of the females caught in the traps were dissected to assess their ovarian maturity, and dissection results were compared with similar studies in Europe. In Beijing and Europe, ovarian maturation stops rather early in autumn, and faster at the high elevation sites than at low elevation, suggesting that it may be temperature-driven, but maturation resumes earlier in Europe (April-May) than in Beijing (late May - early June). In the subtropical climate of Kunming, mature females are found throughout the year.
Degree-days and temperature requirements
Degree days and temperature thresholds were obtained based on developmental studies under constant temperatures. These were carried out in Switzerland and China on various fruits and diets. Data from the literature were also analysed. All data were compared and tested using field development data gathered in Switzerland and the Netherlands. Most data (from our own and others’ studies) were surprisingly similar, no matter the regional strain and the diet used. The values that suited best the field development data in Europe were used for the climate model and risk maps built in task 6.
Environmental requirement studies under natural conditions
As a fruit-feeder tracking the availability of wild fruits along latitudinal and elevational gradients, D. suzukii is expected to be rather flexible in relation to the environmental conditions, among which temperature is a major player. We sampled potential wild host fruits of D. suzukii along elevational gradients in a mountain area of northern Italy and tracked temperature conditions in the weeks before the sampling, in order to verify if data about temperature-related development available in the literature match with the exploitation of the hosts, and if there are hosts which cannot be exploited because of limiting temperature. In addition, we used a strong natural temperature gradient in a cave, in the lower range of performance known for D. suzukii, to mimic conditions existing along the elevational gradients. We used laboratory stock colonies of D. suzukii to assess insect performance under different naturally fluctuating temperatures in a cave and compared them with a reference stock kept under constant temperature in the laboratory. At least six species resulted to be suitable for the development of D. suzukii in mountain areas (Daphne mezereum, Lonicera alpigena, Lonicera nigra, Lonicera xylosteum, Sambucus nigra, and Sambucus racemosa). By comparing the data from the temperature experiment, the minimum threshold of average daily temperature under natural conditions for the complete development of D. suzukii corresponds to 11.6°C, a value slightly higher than that obtained by weather stations close to the collection site of wild fruits which yielded adults along the elevational gradient (11.2°C). These findings open the way to better understand the possibility of the fly to colonise mountain habitats, although in a temporary way because of limiting conditions during overwintering. They also indicate that D. suzukii shows a good performance at rather low temperatures, indicating that population build up may occur even those conditions and thus the species may recolonise the low elevation habitats at the end of the summer with abundant populations
Circadian behaviour of adults D. suzukii
Studies of the circadian cycle of D. suzukii were carried out, using simulated environmental conditions and field observations. Many results were gathered, which allowed us to make important observations to calibrate control methods. For example, we know now that, male and virgin female D. suzukii are active in the morning and early evening with a night time sleep period and afternoon siesta. The siesta in mated females is disrupted by elevated afternoon activity, a post-mating response probably triggered by the male sex peptide. The increased activity of mated females might explain the higher proportion of females migrating into fruit crops early in the growing season. When afternoon temperatures reach 30oC, both male and female D. suzukii increase activity, but this is a direct ‘escape’ response to dangerous temperatures. The timing of emergence is highest is early morning when light and temperature are starting to rise. Humidity does not greatly affect development of the pharate adult within the pupal case, but is critical for adult emergence with very few emerging at and below 50% RH. Mating behaviour of D. suzukii was compared with that of D. melanogaster. The courtship behaviour is rather similar in the two species. In contrast to D. melanogaster, 7-10 day old virgin D. suzukii are reluctant to mate in laboratory setting; e.g. males spend less time courting and they have a much longer latency before copulation. Once copulation takes place the mean copulation time is 25.73 min which is longer than that of D. melanogaster.
Host range and spatial ecology
• Host ranges of D. suzukii were assessed in various countries and continents, on wild and cultivated plants and well as on selected crop fruits.
• The spatial ecology of D. suzukii was investigated in a heavily infested region in NE Italy, with the aim of gathering new information on its distribution and the role played by the composition of the farmscape on its abundance.
Non-crop host plants
Field surveys were carried out during two years in Italy, the Netherlands and Switzerland. Fruits of 165 potential host plant species were collected, including mostly wild and ornamental plants. Over 24,000 D. suzukii adults emerged from 84 plant species belonging to 19 families, 38 of which being non-native. Forty-two plants were reported for the first time as hosts of D. suzukii. The highest infestations were found in fruits of the genera Cornus, Prunus, Rubus, Sambucus and Vaccinium as well as in Ficus carica, Frangula alnus, Phytolacca americana and Taxus baccata. Based on these data, management methods were suggested. Ornamental and hedge plants in the vicinity of fruit crops and orchards can be selected according to their susceptibility to D. suzukii. However, the widespread availability and abundance of non-crop hosts and the lack of efficient native parasitoids suggest the need for an area wide control approach.
The host range of D. suzukii was also investigated in its area of origin in China. Fruits of 104 plant species were collected in 2014 - 2016 in 10 Chinese provinces in different environments. Adults of D. suzukii were obtained from 29 plant species. Most of the recorded hosts are non-crop plants and several represent new host records. In general, the main host genera in Europe were also hosts in China. However, compared to our surveys in Europe, the percentage of fruit species attacked by D. suzukii was lower in China than in Europe (27.9% vs 50.9%). The level of attack on preferred hosts was also much lower than in Europe.
Cultivated fruits
Since many publications are already available on the susceptibility of cultivated fruits to D. suzukii, in DROPSA we decided to focus our work, in Italy, on the varietal susceptibility of grapes and strawberry. The grape study was particularly important because it is a well-known fact that grape is not the most suitable host for D. suzukii and that suitability varies between varieties. This was clearly shown in our study, which also demonstrated that the level of infestation in a variety depends on skin toughness, skin elasticity and pulp consistency. Even in heavily attacked varieties, only a small amount of the larvae developed to adult. Important differences in susceptibility were also found between strawberry cultivars. The number of eggs per strawberry and the infestation level were highly correlated with acidity but not with penetrating force and sugar level.
Correlation between catches in cherry orchards and damage on cherries
In Italy, we investigated the correlation between catches in cherry orchards and damage on cherries. The study showed that the relationship was very weak, most probably due to the fact that the best attractants available to date are not more attractive than cherries.
Influence of forest abundance on captures and damage on cherry
We selected 32 conventional cherry orchards in NE Italy surrounded by landscapes with different proportions of semi-natural habitats and quantified both pest density and crop damage using sentinel fruits. We observed a higher attack density in orchards surrounded by higher forest cover. The same trend was observed for female adult density. In forest habitats, the pest probably can find ideal microclimatic conditions and other biotic factors such as the presence of alternative host plants that can promote population growth. The effect size of forest cover on both adult density and crop damage, despite the very large tested forest gradient (0-60%), suggests that the removal of forest habitats does not seem to be a viable option for controlling D. suzukii. However, current integrated pest management should take into account landscape composition and interventions should be particularly timely in forested landscapes where D. suzukii can quickly attack the crop at higher density.
Spillover of Drosophila suzukii between non-crop and crop areas
Semi-natural areas may affect D. suzukii population dynamics by providing alternative host resources, overwintering habitats, or refuge areas when crops are sprayed with insecticides. We investigated the spillover of D. suzukii between non-crop and crop areas using sweet cherry as a model crop. We sampled the pest at different heights and at different distances from the forest from the stage of vegetative rest to the stage after crop harvest. Drosophila suzukii abundance in the orchards declined strongly with increasing distance from the forest margin and with increasing height from ground. The observed patterns varied across the crop phenological development stages indicating that the pest used multiple habitats across the seasons. When the host plant was not suitable for reproduction, D. suzukii preferred to fly closer to the forest margin and at lower heights. Differently, when the host plant was suitable (i.e. ripe cherries), D. suzukii colonized further the orchards both horizontally and vertically exploring more in depth the canopy volume.
Natural enemies and other mortality factors
Natural enemies and other mortality factors were identified to gather information for the development of biological control strategies against D. suzukii (WP 4). Two approaches were used: (1) Partial life tables to investigate the relative importance of natural mortality factors throughout the life cycle of the fly; (2) Study of the parasitoid complex of D. suzukii in Europe, Asia and North America, with the aim to identify potential candidates for biological control.
Partial life tables
This study aimed, by comparing mortality factors in Europe and China, at providing information on why D. suzukii populations are lower in China than in Europe. However, producing life tables for D. suzukii in the field was much more difficult than expected, for various reasons: overlapping generations, the cryptic life of immature stages and their very short duration, naturally occurring populations that disturb experiments with field-exposed stages, etc. Nevertheless, partial life tables were developed in Switzerland and China in 2015 and 2016, using various protocols. In China, no parasitism was observed whereas it was known to occur regularly in the area. Generational mortalities varied between 93.4% and 99.3% and the net reproductive rates (R0) varied between 7 in July and 0.7 in August. These numbers appear very close to what is observed in reality close to the reality. In Switzerland, the total generational mortality was 95.57%, which provided a realistic R0 of 4.7. Mortality was caused by two parameters: predation and abiotic mortality.
Parasitoid surveys
Classical biological control through the introduction of parasitoids from its region of origin could help reducing populations at landscape level and, thereby, decrease the need for management in cropping systems. However, little was known about the parasitoid complex of the fly in its region of origin, especially in China, which shares the largest part of its native distribution. Therefore, surveys for larval parasitoids of D. suzukii were carried out in 12 Chinese provinces and five Japanese prefectures between 2015 and 2017. Parasitoids of D. suzukii and other fruit-inhabiting drosophilids were found at 28 sites in four provinces in China and four prefectures in Japan. Larval parasitoids were obtained at most sites where D. suzukii was found, with parasitism varying from 0.0 to 75.6 %. At least eight parasitoid species were reared out. The most abundant and frequent parasitoids were the Figitidae Ganaspis cf. brasiliensis and Leptopilina japonica, but another Leptopilina species and at least five Braconidae species belonging to the genera Areotetes, Asobara and Tanycarpa were obtained in low numbers. In most samples, D. suzukii was accompanied by D. pulchrella or D. subpulchrella, two other Drosophilidae whose larvae live in fresh fruits in Asia and are probably attacked by the same parasitoid complex. Due to its abundance in Asia and its likely restricted host range, the most promising parasitoid for biological control is Ganaspis cf. brasiliensis. This and other parasitoids were extensively studied in Task 4.2
Semiochemicals of D. suzukii
Compounds attractive to adult flies were investigated using two approaches: (1) Identification of external compounds involved in the attraction of D. suzukii, particularly from fruits; (2) Identification of compounds emitted by D. suzukii, such as those involved in courtship and mating behaviour
Fruits and other compounds
The extraction and identification of fruit volatiles was carried out in China. Gas chromatograph- electroantennographic detection (GC-EAD), electrophysiological and behavioural responses of D. suzukii to plant volatiles at different ripening stages of fruits of bayberry (Myrica rubra) and cherry (Cerasus yunnanensis) were studied to identify volatile semiochemicals used in host finding behaviour. There were 11, 42, 23 and six main volatile compounds identified from cherries, ripe bayberry, half-ripe bayberry and green bayberry, respectively. GC-EAD results revealed the presence of six electrophysiological active compounds from ripe bayberry fruits. EAG responses of female and male D. suzukii to 12 compounds increased with increasing concentration (0.01 0.1 1, and 10μg/μl). Both cherry and bayberry fruits significantly attracted mated and virgin D. suzukii. Mated females were all attracted by the fruits of cherry and bayberry and showed no response to the control. Methyl 2-hexenoate and α-caryophyllene significantly attracted both virgin and mated female D. suzukii, whereas (E)-β-caryophyllene significantly attracted virgin females. Methyl 2-hexenoate could repel either virgin or mated female D. suzukii, and α-caryophyllene just repelled virgin D. suzukii. The results showed that methyl 2-hexenoate, α-humulene, (E)-β-caryophyllene could be used by D. suzukii to locate its host plant. In addition, we performed the identification and behavioural responses of D. suzukii to plant volatiles of the wild fruit Osyris wightiana. GC-EAD detection revealed the presence of 16 EAG active compounds. Novel attractant lures based on these volatile compounds have been field tested in WP4
Compounds emitted by D. suzukii
We investigated semiochemicals emitted by D. suzukii (e.g. aggregation pheromone) by profiling volatiles of both fly sexes. Given that cuticular hydrocarbons (CHCs) may be involved in courtship behaviour, the extraction of these compounds was also carried out. However, no compound involved in chemical communication was found. This confirms other studies carried out recently by other teams.
Natural dispersal capacities
The natural dispersion and spread capacity of adult flies was investigated using the mark-recapture technique.
Sterilisation and marking methods
In order to perform release-recapture experiments to study the natural dispersal capacities of D. suzukii, sterilization and marking methods for flies had to be defined and tested. For sterilisation, 40 Gy was selected as the irradiation dose as this dose ensures almost 0% egg hatching from the irradiated females and avoid complete development to pupa. For marking, in laboratory trials using fluorescein, flies were not effectively marked or not detectable after a short period. Thus, a non-specific protein was tested as insect marker, i.e. bovine serum albumin (BSA) which can be detected by ELISA technique. This method allowed a high sensitivity for BSA protein detection, even 7 days after marking.
Mark-recapture
In order to compare the dispersal capacity of D. suzukii with a well know fly, sterile marked Ceratitis capitata males were also released and recaptured. Sterilized and marked D. suzukii and C. capitata were released in a mixed orchard containing various citrus varieties. An array of 40 traps concentrically deployed at 10, 25, 50, 100 and 250 m from the releasing point was used to recapture the released flies. Flies were released six times, three during autumn and three during spring. The traps were inspected 3, 24, 48 h and one week after release.
In general, recapture rates for C. capitata were higher (7-69%) than for D. suzukii (2-14%), most probably due to the different attractant employed – trimedlure, a powerful parapheromone for C. capitata and the unspecific food-bait (wine + apple vinegar + sugar) for D. suzukii. However, the recapture rate of D. suzukii was fairly good compared to similar tests made with other Drosophila spp. For both flies, the majority of the captures were made at 10 m from the release point but a few were collected at all distances. These results suggest that D. suzukii has a relatively limited dispersal capacity compared to other fruit flies such as C. capitata, suggesting a shorter distance of influence. We can estimate a dispersal distance for D. suzukii between 200 and 300 m. This does not take into account potential occasional dispersal with strong winds. Given the low dispersal capacity of D. suzukii and the low efficacy of the current attractants, we suggest, as mass trapping strategy, a dense trap density to effectively trap the flies at short distances or a perimeter trapping design to prevent pest intrusion, especially when field margins contain alternative hosts.
Work package 3 – Biology and epidemiology of quarantine fruit crop diseases
Aims and objectives
Pseudomonas syringae pv. actinidiae (Psa), Xanthomonas arboricola pv. pruni (Xap), and X. fragarieae (Xf) are quarantine pathogens in the EU that seriously limit kiwifruit, peach-almond, cherry and strawberry production. Knowledge of pathogen biology and epidemiology is fundamental to develop effective sustainable control strategies. This information was however very scarce at the beginning of the DROPSA project. Thus DROPSA aimed to identify the principle elements in the life cycle and epidemiology of these quarantine pathogens in fruit crops, determine mechanisms involved with pathogen virulence and fitness level, host defence responses, and insect vectoring risks in order to develop effective and innovative control solutions and IPM.
Results and discussion
Fundamental insights on the environmental requirements and disease epidemiology were determined.
For Psa, temperature and humidity greatly influence growth and disease development whereas light amount and quality does not seem to be crucial. It does influence relevant functions for plant colonisation during in vitro tests. The bacterium appears to overwinter readily on host plants until budburst the following spring. Psa seems unable to survive in soil probably due to the high competition with the rich microbial community present in this ecosystem. Rain splashes, especially during spring and autumn, can spread Psa within the orchard. However mechanical pruning or ineffective disinfection of tools may be regarded as a major issue for the bacterial spread. Infection threshold was found to be directly correlated with cultivar susceptibility. Psa was occasionally found on non-host plants inside infected orchards, but could not multiply in any of the tested plants.
Concerning Xap, the basis for the development of the first predictive model of Xap growth and infections on peach were established and provide insights on fundamental climatic parameters.
The risk of Xf pathogen dispersal via aerosols and plant material when moving strawberry plants was evaluated. To reduce risk of Xf dissemination and subsequent plant infection, it is advised to mow during dry (sunny) weather conditions. Weather conditions have a minor influence on spread but a greater one on the risk of infection. Dissemination of Xf during mowing should be reduced by using physical barriers around the blades of the mower or by disinfecting (heat or biocides) the blades during mowing.
On-site detection tools and genotyping tools were developed.
Source tracking tools are available for all three pathogens. This was further developed for Xf using MLVA and the existence of four lineages was revealed. Genotyping with CRISPR revealed two main different lineages but four (sub)groups and this approach is complementary to MLVA as it allows retracing pathways of evolution by following newly acquired spacers. However, CRISPR sequence determination still requires whole genome sequencing of the pathogen. A quick CRISPR genotyping tool to distinguish the four main different lineages revealed in the frame of DROPSA has been developed in order to circumvent this problem.
The evolutionary risks of the pathogens were evaluated in silico
Several pathogen genomes were determined during the DROPSA project. In the case of Psa this revealed that large portions of specific DNA have been acquired by the Psa3 subpopulation through horizontal gene transfer. Mobile genetic elements (plasmids, ICEs, GIs, phages, prophages, etc.) seem to contribute greatly to the genetic diversity of Psa. Although some diversity was observed in Xap and Xf, the genomes of these pathogens seem more conserved. Some differences observed in the Xf genomes sequenced for the development of genotyping tools were further characterized and will be soon published.
Pathogen virulence factors and fitness determinants as well as their environmental modulators were determined
Pathogen transcriptomes under in planta (or mimicking) conditions were obtained for Psa and Xf and compared to in vitro condition transcriptomes. A list of up-regulated virulence factors and fitness determinants could be established for both pathogens. A prerequisite for optimal downstream analysis appeared to be the availability of a good reference genome for the phytopathogen. Indeed, in addition to specifying the location of the differentially expressed genes (i.e. chromosomal or on a plasmid), it can also help the identification of new genes and improvement of the genome annotation.
Host plant defence factors were identified using RNA-seq
The RNA-seq results highlighted a role of AtWRKY70 and actinidins in kiwifruit resistance against Psa. Therefore, they may be potential candidates for molecular markers for future breeding programs focused on obtaining novel kiwifruit cultivars resistant to Psa. Further experiments are however needed to prove their effectiveness. The lack of resistant genotypes of A. chinensis or A. deliciosa makes it very difficult to test these putative markers. To overcome this limitation, the expression of these two genes needs to be evaluated in a number of cultivars showing differential susceptibility to Psa and in Actinidia arguta, which is a species tolerant to the pathogen.
The first strawberry transcriptome in response to inoculation with Xf was established recently and revealed metabolic changes between short- and long-term symptoms. Results will be published shortly.
Insect vector interactions and critical factors to mitigate pollinator vector risks were characterised.
The role of pollen, bee pollinators, pathogen survival in beehives, role of phloem feeding insects (Metcalfa pruinosa) was investigated in the case of Psa. Pollen has a crucial role in vectoring the bacterium. Asymptomatic closed flowers may produce contaminated pollen, highlighting that Psa is adapted to the internal colonization of male flowers which increases the sexual transmission of the pathogen, providing an evolutionary advantage to the bacterium. Thus substantial attention should be paid to the commercial production of pollen and use of assisted pollination in kiwifruit. However it is unlikely that beehives provide a possible reservoir for the pathogen and transport of beehives from infected areas to Psa-free orchards present a very low risk for Psa spread. On the contrary, there is a risk of Psa spread via phloem feeding insects such as M. pruinosa.
The presence of Xf could not be confirmed using LAMP and qPCR on insects sampled on inoculated plants, suggesting that the spread of Xf to healthy plants by insect feeding on diseased plants is very low.
No Psa, Xap or Xf was identified in the gut microbiome of D. suzukii, thus the risk of spread of Psa and Xf by D. suzukii appears very low.
Work package 4. Effective and innovative solutions to control Drosophila suzukii
Aims and Objectives
To develop effective and innovative solutions to control Drosophila suzukii based on biological data produced in WP 2, transfer of best practices and adaptation of innovative technologies for inclusion in IPM strategies in WP6.
Monitoring, mass-trapping, lure & infect methods
The objective of this task was to develop monitoring, mass-trapping, lure and infect methods using available semiochemicals and new attractants identified in WP 2. Several volatile compounds extracted from fruits induced reaction and/or were found to be attractive to D. suzukii flies. Novel attractants based on these compounds have been tested in the laboratory and some have been selected to be further test in the fields, however none are yet commercially available at this time. Efficacy of several commercial trap designs and available liquid attractants (most wine-vinegar-sugar based) was also tested in several countries and agricultural environments.
The red Drosotrap® device was found to be the best trap and regarding liquid baits, Suzukii-trap® was the most suitable lure considering its high selectivity and persistence. Droskidrink® was the best lure in considering longevity and high attractiveness. Some of the liquid bait were not stable for long period (proliferation of fungi, gel formation, etc.) and needs a weekly replacement while others more stable needed periodic re-filling due to evaporation. To avoid some of these problems a more convenient attractant device was developed, the system includes a permeant plastic bag containing the liquid bait that are easier to handle and may be placed in commercial dispensers. This bait has been optimized for the attractant release and trapping success.
Mass trapping alone did not provide efficient practical solutions for D. suzukii during the fruit season, and it has been observed that fruit infestation increases while the season progressed, and plants with traps had greater fruit damage. Indeed, attractant traps could bring flies into an area, which may engage in feeding, mating and/or egg-laying before entering the trap. However, traps could be useful at the beginning of the D. suzukii season to decrease the first generation of reproductive females and to monitor the pest pressure to start and adjust other treatments in an IPM strategy.
Lure and Kill (L&K) and Lure and Infect (L&I) strategies could provide an alternative strategy as there is no need for a trap to physically capture the flies. Insects can be lured to baits or surfaces treated with the pest controlling agents, for instance insecticides or sterilizing agents (L&K) or entomopathogenic agents (L&I).
A L&K strategy was developed using the developed plastic bag dispenser device as attractant coated with insecticides and has shown its efficacy during field tests. A large array of sterilizing agents has also been tested (in relation with Task 4.4) to be included in this attractant method. However, D. suzukii proved to be quite resistant to these products and the dose necessary to induce sterility were too high and then excluded for economical and risks reasons.
For the L&I strategy different species and strains of entomopathogenic fungus have been tested for their effect on D. suzukii (in relation with Task 4.2). In laboratory Beauveria bassiana (Naturalis-L) causes 44% adult D. suzukii mortality after 7 days, whereas Lecanicilium muscarium (Mycotal) and B. bassiana solutions caused no significant decrease in fruit fly population. Three further fungi species (commercial formulations and different strains of Metarhizium anisopliae and Isaria fumosorosea of Beauveria) were also screened. Most of the fungi produced a high death of adult flies after exposition but in some case this death requires several days to week to happen and did not decrease the reproductive potential of females. The most efficient entomopathogenic fungi have been tested in the fields and PCR (TaqMan) test has been developed to follow horizontal and vertical transmission of entomopathogenic fungus spores. Preliminary results for both L&K and L&I were promising, but need to be confirmed in larger field trials.
Evaluation of natural enemies for the biological control of D. suzukii
Commercially available biological control agents (predatory bugs, nematodes, pathogens) have been tested in laboratory and field conditions using standard procedures. Predatory species screened were Orius majusculus and O. laevigatus (both efficient in thrips control); Atheta coriaria (a Staphylinid beetle); Hypoaspis miles (a soil dwelling predatory mite) and Anthocoris nemoralis (a generalist predatory bug). Orius spp. and A. coriaria did prey upon the larvae and pupae of D. suzukii but have a low impact, while H. miles, did not feed upon D. suzukii larvae or pupae. A. nemoralis displayed the best potential as a control agent; 45% of adult mortality was achieved over a five-day period under laboratory conditions. These predators may be potentially used in an augmentative biological control strategy. Different nematodes species (Steinernema kraussei, Heterorhabditis bacteriophora) that infect and kill arthropods were also tested. Both larvae and pupae of D. suzukii are susceptible to nematode infection. Larvae proved more susceptible than pupae under laboratory trials. S. kraussei caused the higher pupae mortality (55%) while H. bacteriophora produced the higher mortality in larvae (95%). The nematodes offer potential to be applied as soil drenches around infested fruit bushes for the suppression of D. suzukii populations.
Considering the fact that D. suzukii is multivoltine and highly polyphagous, attacking a wide range of fruits of wild and ornamental plants (see Task 2.2) it becomes more and more apparent that long term control solutions will have to involve both in cultura and area-wide control methods. Biological control using parasitoids is a promising strategy for D. suzukii because, in contrast to other methods, it has the potential to lower the population levels in all habitats, limiting the possibility for D. suzukii to reinvade crops generation after generation. Drosophila species are attacked by more than 40 different species of parasitoid wasps that locally may control up to 80% of the fly population. These wasps are laying their eggs in fly larvae (larval parasitoids) or in pupae (pupal parasitoids), but all emerge from pupae.
While generalist European pupal parasitoids (such as Trichopria drosophilae) can successfully develop on D. suzukii (albeit they are not able to control its population in the fields), European larval parasitoids do not naturally parasitize D. suzukii in the field. In lab studies they either do not show interest in D. suzukii larva or parasitize it but the eggs are destroyed by the fly larva immunity. Only one species, Leptopilina heterotoma, very occasionally succeeds to overcome the immune defence in laboratory experiments. An experiment was set up to assess whether the ability to attack and develop on D. suzukii was different between strains or could be increased and, thus, was probably genetically based. Seven strains of L. heterotoma were collected in Switzerland and compared in their ability to (1) attack and (2) successfully develop on D. suzukii. Significant differences were found in the attack rates and success rates. The most successful strains showed average success rates of 9% and the least successful 1%. The best strains were also reared alternatively on D. melanogaster and D. suzukii and the progenies tested for increase in parasitism success. Complementary studies, including semi-natural conditions tests, should tell us if European larval parasitoids of Drosophila spp. can become adapted to be efficient enough to control of D. suzukii.
Asian parasitoid species were collected in China and Japan in 2015-2017 (see Task 2.3) and are presently being reared in quarantine in Switzerland, France and China. Several strains of the braconid Asobara japonica, the figitid Leptopilina japonica and the figitid Ganaspis cf. brasiliensis are have been studied in various ways. The Asian surveys showed that G. cf. brasiliensis is the most frequent parasitoid of D. suzukii and is specialised in parasitizing drosophilids in fruits. This preference was retrieved in laboratory conditions since G. cf. brasiliensis oviposited in D. suzukii larvae almost exclusively in fruits. These parasitoids species and strains were also tested in a similar manner on five European Drosophila spp.: D. melanogaster, D. immigrans, D. subobscura, D. hydei and D. busckii, as well as on the tephritid fly Ceratitis capitata. Asobara japonica and L. japonica were tested with Drosophila spp. on regular Drosophila diet. However, since G. cf. brasiliensis does not successfully parasitize D. suzukii in the regular diet and the European Drosophila spp. cannot be reared on fresh fruit, a compromise had to be found. After various tests, it was found that both the parasitoid and the Drosophila spp. would accept a mixture of the diet with a high proportion of fresh fruits. Thus, the tests with G. cf. brasiliensis included a treatment on the fruit diet with all five Drosophila spp. and also a treatment with D. suzukii in fruits. The results of the specificity tests were as follows: Asobara japonica attacked and developed in all five Drosophila spp. Leptopilina japonica successfully and abundantly developed on D. suzukii and D. melanogaster, individuals emerged from D. immigrans and D. subobscura and none from D. busckii and D. hydei. Interestingly, one G. cf. brasiliensis strain from Japan showed a total specificity for D. suzukii on fruits whereas other strains successfully attacked D. melanogaster. Thus, so far, among Asian parasitoids, the specificity tests revealed polyphagous Asian species (A. japonica, L. japonica) and confirm the specificity of G. brasiliensis, with some variations in the specificity level between strains. Similar observations were obtained in olfactometer tests. These ecotypes of G. cf. brasiliensis with different behaviour may indicate sibling species since the molecular analyses also showed important genetic variations between individuals. Ganaspis cf. brasiliensis is thus the most promising biological control agent of D. suzukii but the following aspects still needs to be further investigated to fulfil the regulatory requirements before a possible release in the fields: completion of the specificity tests; climatic requirements; existence of sibling species or ecotypes varying in their specificity and climatic suitability; mechanisms inducing specificity, e.g. chemical attraction.
Chemical control
In this task, a range of compounds appropriate for use on fruit in Europe at the beginning of the program were assessed for efficacy against D. suzukii including already commercialized insecticides as well as novel materials. Insecticides with different modes of action and routes to target were examined including those with direct contact toxicity towards adults and larvae. Considering that the use of insecticides for the control of SWD can be critical since it damages ripening fruits, only those with a favorable toxicological profile were considered, and analysis on treated commodity were performed. First of all, standard laboratory test procedures were used for a large group of products, after screening, products with the best performance were tested in open field conditions in different countries.
The results strongly support the earlier findings that pyrethroids, organophosphates and spinosyns provide controls from 5 to 14 days after treatment (DAT), showing an excellent activity concerning adult mortality, eggs laying and hatching, female fecundity, larvae and pupae development and adult emerging. Neonicotinoids caused somewhat moderate impact for managing D. suzukii providing 1 to 3 days of control. Natural pyrethrum exhibited a moderate mortality especially at 2 and 3 DAT.
Results from open field trials carried out on Cherry, Grapevine and Strawberry in Italy and in France suggest:
• on Cherry, to maintain a protection throughout the harvest period, four applications with insecticides belonging different mode-of-action groups are strongly recommended, provided that the first application should be applied when monitoring traps indicate the presence of D. suzukii populations.
• on Grape 1 to 2 applications are necessary depending upon the penetration resistance of the berries.
• on Strawberry 3 to 6 applications are necessary every 3 to 7 days between the fruits collection
Due to the fact that the applications should be made near the harvest date, residues on fruits may be present even if they are below the MRL (maximum residue level admitted), representing a problem for export. Rotation and diversity in applying of insecticides with different and unique modes of action are greatly necessary to combat or at least delay widespread insecticide resistance.
Sterile insect techniques to reduce D. suzukii populations
The release of insects carrying ‘self-limiting’ genetic traits that induce mortality to some or all progeny at an immature life-stage, has been demonstrated as an effective management tool for specific insect pests across both agricultural and public health sectors. This female-specific self-limiting approach allows for a male-only release cohort, that has been previously shown to increase the productivity of mating-based insect pest management programs and is not detrimental to agriculture. Mating between self-limiting males and wild females produces no viable female offspring, thereby decreasing the reproductive potential of the wild population. If sufficient numbers of wild females’ mate with self-limiting males over time, then the pest population will decline. Oxitec has developed and optimized a conditional self-limiting technology in D. suzukii that enables genetic sexing of genetically engineered insects based on the tetracycline-off system and the tetracycline transactivator (tTAV) gene. A fluorescent protein marker gene is linked to the self-limiting trait and serves as a tool for visual monitoring of the genetically engineered flies. A number of strains displaying the correct expression of the self-limiting trait have been obtained; i.e. absence of females when larvae were reared without the addition of the antidote in the larval medium (off-tet) and a 1:1 (female: male) ratio on larvae reared in larval medium with tetracycline (on-tet). However, none of these strains could be made homozygous may be because that basal tTAV levels cause an increased mortality in larvae that contain two copies of the transgene. In order to develop a homozygous male-selecting strain, redesign of the tetracycline repressible self-limiting trait has been carried out and new transgenic lines with different construct configuration to minimize larval mortality are being produced and will be tested under controlled condition for their ability to decrease the reproductive potential of D. suzukii.
Potential of new mode of action compounds as alternative solutions in D. suzukii control
Two approaches were investigated as alternatives solutions for D. suzukii control which targeted essential components of the fly’s physiology and/or behaviour. The expression of specific genes was down-regulated by RNA interference using double-stranded RNA (dsRNA) and G protein-coupled receptors (GPCRs) were characterised to develop screening assays for pesticide discovery. New microencapsulation methods for stabilising and delivering dsRNA and receptor agonists/antagonists were investigated.
As a first step in identifying insecticidal targets, the neuropeptide and GPCR precursors from the D. suzukii genome database and the corresponding peptides present in the central nervous system of the fly were identified. Selected targets included the hormones and receptors involved in osmoregulation (diuretic hormone) feeding and gut motility (myosuppressin) and reproductive behaviour (sex peptide).
A range of compounds were tested on functional receptor assays revealing receptor specificity in that they were activated by the native ligands and some synthetic analogues, but not by mammalian peptide orthologues. This work has provided an understanding of the ligand requirements for each of the D. suzukii receptors investigated, enhancing the prospects of developing novel insecticidal molecules for use against D. suzukii.
Microcapsules, which provide protection upon application and ingestion, have been developed which selectively release insecticidal bio-molecules in response to the alkali pH of the insect. Furthermore, dsRNA, targeting V-ATPases in the fly gut, have been complexed with polymers and shown to stabilise the dsRNA. Significant pupal mortality occurred when D. suzukii larvae were fed with complexed dsRNA, whereas naked dsRNA was ineffective. The complexed dsRNA had no significant effect D. melanogaster, demonstrating that this method could provide species specific control.
Work package 5 – Effective and innovative solutions for quarantine fruit crop disease control
Aims and objectives
P. syringae pv. actinidiae (Psa), X. arboricola pv. pruni (Xap), and X. fragariae (Xf) are quarantine pathogens in the EU that seriously limit kiwifruit, peach-almond, cherry and strawberry production. There are no existing authorized chemical compounds, nor a suitable strategy for efficient disease control in the EU. Thus, novel formulations and delivery technologies are greatly needed. Project DROPSA deals with confirming the efficacy of existing commercial products, develop novel biopesticides and physical or plant-defence stimulation approaches for disease control.
Results and discussion
Existing compounds and products
Existing compounds and products were evaluated under controlled environment greenhouse conditions and potted host plants (12 products, Xap on peach), and in the field in four regions where kiwifruit is cultivated (30 products, Psa on kiwifruit). Only a few existing compounds were sufficiently effective to manage the three diseases, and this effect was greatly dependent on the pathogen-host system. Thus, it was evident that there is a need of research on new compounds.
Novel antimicrobial compounds
Novel antimicrobial compounds were identified and tested. Among 38 synthetic antimicrobial peptides studied, the linear undecapeptides BP77 and BP100, the D-amino acid derivative of BP100 BP157, the magainin-melittin enlarged BP100 derivatives BP208, BP211 and BP178, and the BPC192 and BPC194 cyclic decapeptides, were the most active against Psa, Xap and Xf. Peptide BP100 (H-KKLFKKILKYL-NH2), was the best lead because it is small size peptide with linear structure and contains proteinogenous aminoacids, with a good antibacterial activity, low toxicity and a relatively low effort of synthesis. BP100 performed well in the in planta assays at doses between 100-200 microM (0,225 g/L) for controlling kiwifruit, peach and strawberry pathogen infections. The effective dose of BP100 was similar to the recommended for streptomycin, kasugamycin or tetracycline in the countries where antibiotics are authorized for plant bacterial disease control. However, the cost of synthesis is too high for field use and BP100 will need additional efforts to decrease costs of production (not a DROPSA objective). The peptide fulfils the recent regulation on low risk plant protection products in the EU.
Novel biological control agents
Leaf- and the flower-associated bacterial biota of two kiwifruit species were analyzed in healthy and Psa-infected plants using a metagenomic approach. Some non-pathogenic species with a potential interest as biocontrol agents were detected. In non-diseased plants Pseudomonas spp. were the most abundant, and Lactobacillus was detectable in all samples. A total of around 700 isolates from a comprehensive bacteria collection from a wide range of plants including Bacillus spp. and lactic acid bacteria were characterized in relation to antimicrobial peptide (AMP) biosynthetic genes and multilocus sequence typing (MLST) analysis. Antibacterial activity of the putative BCAs was determined against the plant pathogenic bacteria Psa, Xap and Xf. Polyphasic and phylogenetic analysis permitted the selection of the most adequate BCAs. Selection of putative BCAs lead to B. amyloliquefaciens strains 2017, 2059, 2128, 2130 and 2151, and Lactobacillus plantarum TC92, PM411 and CC100, and Leuconostoc mesenteroides CM160 and CM209. Strains EPS2017, TC92 and PM411 were selected for further work. Fitness of BCAs was dependent on environmental conditions and host plant. The survival of B. amyloliquefaciens 2017 and L. plantarum PM411 and TC92 onto leaves of GF677 Prunus plants in the greenhouse was strongly dependent on relative humidity conditions. In in vivo trials, BX44 tagged with gfp reporter was able to colonize flowers of both A. deliciosa and A. chinensis leaves better than any other commercial BCAs. BX44 is able to compete with Psa for the same substrates. A combination of methods based on qPCR (total cells), and dead and alive cells (PMAX-qPCR) have been developed for quantitative molecular monitoring of L. plantarum PM411. These approaches provide with robust tools for monitoring viability of the strains in the field to study the best formulations and conditions of application, host and pathogen range. Greenhouse and semi-field experiments for disease control concluded that the strains Lactobacillus plantarum TC92/PM411 and Bacillus amyloliquefaciens EPS2017 were gobally effective (40-70% efficacy) for control of Psa in kiwifruit plants (cv. Hayward), Xap in Prunus GF677 plants and Xf in strawberry cv. Darselect, and did not differ significantly from reference products BlossomProtect and Serenade. Overall, two novel strains of L. plantarum (TC92 and PM411), one strain of B. amyloliquefaciens EPS2017, and a strain of Pantoea vagans BX44 were candidates for use in field programs for disease control of Xap, Xf and Psa, and to develop novel microbial pesticides.
Manipulation of plant metabolism and physical methods for disease control
Azybenzolar-S-methyl (ASM) resulted to be the most effective and reliable resistance inducer, by both root and leaf treatment, and with a prolonged effect over time, but may cause phytotoxicity at high treatment rates. Infrared radiation system does work in reducing the bacterial population, although possibly not in eradicating it, but the general health status of the plant may influence its tolerance to the heat treatment. Ozone has an antimicrobial effect on the survival of Psa in kiwifruit pollen, but unfortunately, the treatments significantly reduced also pollen viability.
Innovative formulations
Innovative formulations were developed by improving the fitness of the BCAs, testing suitable growth media for scale-up production and dehydration processes for formulation. Physiological improvement and nutritional enhancement of strains and formulations was successful. In Bacillus, maltose used by Bacillus EPS2017 and not by the plant pathogenic bacteria, and the best combination to adapt cells to hyperosmotic conditions is PM medium amended with NaCl and with several osmolytes. In L. plantarum PM411 and TC92 acidic and hyperosmotic pre-treatments during growth lead to a better stress tolerance, and induced stress and defence related genes. Thus, it is possible to increase the fitness of the BCA strains by using fermentation conditions including stress stages, with a better consistency of disease control.
Growth medium for scale-up processes and mass production was improved in Bacillus by different combinations of PM with molasses and soybean meal, including bioreactor performance providing optimum amounts of metabolites with antibacterial properties. In L. plantarum the medium MRS was also improved, but in a less extent. The shelf life of different Bacillus products obtained by lyophilization, from a set of mixtures was maximum with the combination of osmoadaptation with several osmoprotectants. Several formulations of L. plantarum strains TC92 and PM411 were prepared based on freeze drying, spray drying, and encapsulation. The combination of antimicrobial peptides and L. plantarum strains, and of copper bactericides with plant defence elicitors were compatible to increase the range of effective strategies for disease control. Finally, a novel approach to efficiently deliver the antimicrobial peptide BP100 to plants was evaluated with success by endotherapy in kiwifruit against Psa, and in peach against Xap, which decreases the need of high costly doses of the expensive compound, and reduces environmental impact.
Work Package 6 – Practical solutions for control
Aims and objectives
To develop innovative, practical, preventive and integrated control solutions for quarantine fruit pests and diseases, utilising current control strategies and those developed in WP 2, 3, 4 and 5, with an emphasis on the prevention of D. suzukii and diseases that infect crops. Integrated strategies (IPM) for the major economic fruit crops in Europe will be combined, concentrating on kiwifruit, strawberry, peach, cherry and grapes. This will involve ex-ante economic assessments from WP 7 and the pathways and impact analyses developed in WP 1.
Risk mapping for D. suzukii
Risk maps built in DROPSA were based on climatic requirements of D. suzukii as determined in Task 2.1 and in the previous literature on climatic requirements. Due to its broad host range (revealed by Task 2.2) the risk and impact of D. suzukii is probably more related to climatic suitability than to host distribution. Therefore, the study focused on climatic parameters only.
Due to its short development time, D. suzukii can build very high densities, even when winter mortality is close to 100%. As a result, in spite of the narrow climatic range, the pest is found in most of Europe. For these reasons, the model was based largely on pest occurrence and damage data (Deliverables D2.1 and D2.2 and literature search). The model was validated by comparing model output to reported distributions, seasonal phenology and tested for consistency with empirical data.
Damage prediction values for Europe correspond well to the present situation, with heavy damage in Western/Central Europe, and lower risk associated to hotter/drier areas or the occurrence of cold stress. The model will be improved to better fit with data from Asia and North America, further considering climate change (particularly for water availability) and including field observation in addition to lab data.
Role of cultural management
Cultural practices (e.g. clean harvest, wild fruit removal, netting) were developed in field conditions to limit abundance of, and damage by D. suzukii (WP 2).
The effect of pollination, irrigation, fertilization, use of bio-regulators and pruning on the incidence and epidemiology of Psa (WP 3) was evaluated in small-scale and field experiments.
The use of insect-proof nets covering fruit bearing trees or shrubs is an effective method to physically exclude D. suzukii from cherry, grapevine and blueberry plants, preventing pest damage. Downsides are the high initial costs and difficulty of orchard management. Reducing cool-humid microhabitats (by pruning, mulching, mowing) and removing unharvested fruit further limit the pest population.
As for Psa, risks of contamination are associated to irrigation water, pruning instruments, grafting and pollen. Water stresses and auxin treatments may increase disease severity. Orchard management practices (training, pruning, covers) can be implemented to facilitate spray treatment efficacy and limit leaf wetness, which is one of the main factors promoting the establishment of the pathogen. Cytokinin treatments may induce a moderate resistance. Traits of resistance to P. syringae pv. actinidiae are explored in the Actinida genus (notably in A. arguta) for the development of rootstocks. Fruit from diseased plants was smaller and more perishable.
Development and validation of decision support and forecasting systems
The knowledge acquired about biology and ecology of the pests and pathogens (WP2 and WP3), the management options identified in WPs 4 and 5, and the economic aspects of plant protection (WP7) were integrated in a decision support system (DSS). This tool, by the specification of indicators and the opinion of experts, encourages complete and careful consideration of individual measures.
A DSS evaluation tool was developed to assess the performance of current (“Baseline”) pest management practice against the packages proposed and tested in DROPSA field trials. For Psa, the DROPSA package is predicted to be more favourable than the current management system, with an estimated overall benefit of €27.75 per hectare when using the DROPSA package compared with the Baseline package.
By reducing the loss of production, the DROPSA package is also strongly favoured in case of high D. suzukii pressure sites, while a lower utility is generated by lower infestation levels, because of the conspicuous costs and management efforts inherent to nettings.
Integrated pest management strategies
The results obtained in WP4 and 5 and in Tasks 6.2 and 6.3 have been integrated to develop orchard IPM strategies based on the most reliable and effective combinations of synthetic and natural pesticides, biological control agents, semiochemical-based techniques and cultural methods.
IPM strategies for Psa were implemented in a demonstration orchard, incorporating copper application and treatment with resistance inducer (ASM). In addition, biological control agents (BCA) selected in WP 5 were applied at blooming, and part of the experimental plot was under plastic cover. The IPM strategies significantly reduced bacterial canker incidence, particularly in the plastic-covered parcel. Overall, the combined use of proper management, resistance inducers and BCAs, on one hand, can provide an effective protection against Psa, on the other hand, allow a substantial limitation of copper (thus meeting the current EU regulations) and other chemical pesticides, particularly in phenological stages, such as flowering or pre-harvest, in which copper application may result in phytotoxic effects or residues on fruit. However, further testing is required to ascertain the effects of weather and local climate conditions on the IPM strategies.
IPM strategies for D. suzukii were investigated in cherry, blueberry and raspberry orchards. The control methods derived from WP 4 and included pruning, fruit removal and mass trapping, along with a rational use of chemicals of synthetic and natural origin. The impact on non-target organisms (bees, butterflies, snails and earthworms) was also monitored. Preventing a wet-cool microclimate in the canopy (by pruning or mulching, which also enhanced the penetration of spray treatments), and removing non-harvested fruit was useful to control pest infestation. Larvae surviving within the removed fruit were effectively killed by solarisation. In addition, traps were designed for a Lure & Kill control strategy with an extended lifespan (6 weeks), to minimise servicing by the grower. However, the control efficacy depended on the trap positioning, decreasing at the borders of the plot, as a result of pest immigration from outside. Overall, the use of IPM strategies for the control of D. suzukii is envisaged to limit the use of insecticides, which may have adverse effects on the environment and crop quality, may lead to the selection of resistant insects, and do not prevent pest infestation, due to its wide host range, rapid development rate and high dispersal ability. Lure & Kill devices can be useful in combination with other measures to reduce the initial population of D. suzukii and to retard its infestation peak.
Results have been, and will continue to be, made available to growers via informative websites, advisory services and field demonstration.
Work package 7 – Economic analysis
Aims and objectives
The aim was to provide an economic analysis of the prevention and control strategies developed in the WP 2, 3, 4 and 5, and their implementation in practical solutions and IPM strategies (WP 6). This was achieved by determining the economic viability of proposed preventive and control strategies using ex-ante analyses of critical success factors with regards to costs and returns, and evaluating the economic opportunities for fruit growers of the innovative solutions and practical strategies developed by DROPSA as alternative measures of IPM prevention and crop protection using ex-post analysis.
Economic viability: ex-ante analysis of proposed alternatives
Ex-ante analyses of critical success factors with regards to costs and returns to determine the economic viability of proposed preventive and control strategies. The conditions for the improvement and implementation of the alternatives were identified and used as research guide in the other work packages (WP 4, 5 and 6) for the improvement and development of successful alternative strategies.
This task was carried out at the beginning of the project as research guide for the other work packages in order to develop management strategies that are also sustainable in an economic view.
At the start of the project, the following ex-ante calculations were made in order to provide critical factors for the further development of the proposed strategies:
• Sterile insects against D. suzukii in Cherries (one country)
• Netting against D. suzukii in Cherries (one country)
• Use of pesticides against D. Suzuki in Strawberry (four countries)
• Covers against Psa in Kiwi (one country)
As these ex-ante calculations are preliminary, many data (costs and effects) of proposed techniques were estimations. However, these calculations give critical factors for the further development of the innovations. More specific were the concluding remarks about the critical success factors:
• The use of sterile insects seems a possible solution, but the final economic outcome depends on the area to be treated, the period of treatment, the amount of sterile insects needed and of course the reduction of the damage by D. suzukii. It is clear that a percentage loss in yield has more (negative) impact on the marginal gross margin than a percentage loss in picking and grading speed.
• The ex-ante analysis for sterile insects is done for Cherries. The work in the DROPSA project should make clear what the real data are for Cherries, as well as for other crops. In other crops like Raspberry, the period during which fruits are attractive for D. suzukii is (much) longer. Perhaps in the ex-post economic calculations several measurements need to be combined.
• For the calculations of the use of netting, trials need to make clear which systems are most effective, which amount of labour is required, and how it is working in practice as the nets have to be opened regularly for people and machinery.
• The life-span of the netting is not known yet, but firmly affects the costs of this measure. The life-span of the nets is something to be varied in the ex-post calculations as the period of the DROPSA project will presumably be shorter than the expected life span of the nets.
• Spraying pesticides: economic viability seems positive, but due to differences in growing systems of Strawberry, varies much between countries.
• Plastic covers against bacterial infections: trials, like for netting, need to make clear which systems are best working, which amount of labour is required, and how it is working in practice at which effect on yield and quality.
Economic viability: ex-post analysis of new developed strategies
For the ex-post calculations relevant topics and crops were selected, based on the ex-ante calculations but also on the developments in the different work packages. For the data collection of these topics and crops, two templates are made:
• The A-form is meant for the initial information about the relevant crops. This information is required for the calculations of the costs and returns of the crops in a situation without infestations of D. suzukii or diseases of pathogens. It concerns economic and technical data of the fruit crops as yield, labour (costs), prices etc.
• The B-form is meant for the specific data about the consequences of D. suzukii or pathogen in the crops. Two situations must be described, completed by data:
o The first situation shows the effects with infestations of D. suzukii or diseases of pathogens, but without new DROPSA-strategies, on economic and technical data, as yield, labour, investments, use of materials etc.
o The second situation shows the effect of the new strategy on economic and technical data.
For both forms a filled example is made to show the technical researchers what is needed. The forms as well as the first data are discussed with individual researchers of other WPs in order to provide the best situation for data collection during the coming season.
New calculations were made based on the achieved results and the collected data of the work in the WPs 2-6. Concluding remarks of these calculations:
The invasive pest D. suzukii causes a lot of economic damage to individual growers, several strategies were tested to diminish the damage.
• The strategy of the use of Attract and Kill D. suzukii showed an effective population reduction but not enough to avoid damage when populations are already established.
• The strategy to use extra chemical control, specific for D. suzukii, showed in The Netherlands in sweet cherry an average yield reduction of 10-15%, compared to the situation before D. suzukii and therefore no damage or loss of production. The decrease in damage, due to the chemical control, leads to a higher marginal gross margin (mgm) despite the extra costs for the chemical control.
• The strategy of clean harvesting in combination with other measurements is effective, but the data for an economic evaluation are not sufficient at the moment.
• The combination of side netting (in the case rain covers are standard) with bait sprays diminished the damage to nearly zero in a pilot in The Netherlands. The costs for the investment are between 11,000 and 17,000 €/ha. The year costs for this investment are lower than the increase in mgm.
• Netting of sweet cherry plantings in Italy might reduce the damage to zero. The planting system and the percentage damage before netting determine if the return on investment of the netting is high enough. However, single tree netting, is due to the high investment costs not economically sustainable.
• The effect of mass trapping in Italian sweet cherry plantings is not clear, but at the moment not economically sustainable.
• The measurements against D. suzukii in Blueberries in The Netherlands are complicated due to the long production season with different varieties. The use of extra chemical control as only measurement has only limited effects: on average still 30% yield reduction.
The pathogen Psa is negatively affecting the European kiwi fruit production. Several strategies were tested to diminish the damage.
• The yield reduction due to Psa can be decreased to zero by the use of covering/nets.
• Due to the relatively high investment costs this is at the moment not economic sustainable for green kiwifruit in Italy.
• For yellow kiwifruit in Italy this may be positive, but further work is needed to confirm this.
Potential Impact:
The joint dissemination, exploitation of project results and management of intellectual property is covered in DROPSA project consortium agreement, agreed by each partner organisation and the project management committee.
A work package was dedicated to dissemination with the main objective:
To undertake dissemination actions that enables the partners to maximise the commercialisation of the project results and to plan for post project exploitation. During the project lifetime this will include the knowledge transfer of results to all interested stakeholders (industry and governmental) through the website, e-Newsletters, publications and presentations at scientific meetings, project workshops and other events, and the exploitation of the results and outcomes of the project.
This WP also aimed to maximise the project’s visibility, create a distinctive project identity, spread information on its objectives, activities and achievements, and facilitate commercial exploitation of the research output.
All partners contributed additional resource to ensure maximum impact and members of the DROPSA consortium facilitated dissemination to all stakeholders. The latest guidance from the EC was used on how to maximise the impact of research, development and demonstration, to ensure that the results are effectively used, through their exploitation and dissemination.
Project website
1. A project website was created (http://dropsaproject.eu/) which is being used to inform the public of progress and activities in the project. Links to documents and other relevant materials are available on the website.
2. A database was created of >1600 stakeholders from industry and end-users from European countries. A link on the website also allows the public, companies and others to register their interest in DROPSA and to receive publicity materials. A mailing list of these stakeholders has also been created for electronic distribution of publicity material.
Dissemination of project results
Scientific and practical dissemination has been achieved through publications and DROPSA partners’ attendance at conferences and workshops. The consortium partners have been a very proactive group and have attended and participated in a wide range of conferences, workshops and promotional activities.
The main dissemination activities were:
• Project brochure and four annual newsletters to introduce and give updates on the project, which were distributed electronically to registered stakeholders and available via the website
• Conferences - overviews of the DROPSA-project and scientific results were presented, either as a poster or oral presentation at:
o International conferences and symposia (> 60 oral and poster presentations).
o National conferences and meetings (approximately 45 oral and poster presentations).
• Peer-reviewed Scientific publications. A variety of publications (approximately 60 publications to date) have been produced during the time frame of DROPSA (listed above in the publications section). It is anticipated that further publications will include data from this project. These will be listed on the DROPSA website or made available if open access.
• Future funding opportunities: continued funding for research undertaken in DROPSA will be sought by individual partners. Potential opportunities would be via Euphresco or Eranet funding, as well as from individual partner governments.
• Lectures, post-graduate courses, workshops, training and knowledge exchange (approximately 42 meetings).
• Publications in national (growers) magazines and websites (>4 0 publications and 26 websites) to inform growers, plant health inspectors and NPPOs.
• Interaction COST FA1104, Sustainable Production of High-Quality Cherries for the European Market (3 interactions).
• Stakeholder workshops for training and dissemination of project results and outcomes. At each stakeholder event local interested parties were invited to attend and were able to the network with the project partners. Experts from the field of industry/growers/plant protection officers, were also in attendance to provide feedback from an industry perspective.
o DROPSA Stakeholder Workshop 2016: Strategies to develop effective, innovative and practical approaches to protect major European fruit crops from pests and pathogens, Girona, Spain 10th March 2016.
o Stakeholder (technical) meetings and field visits involving growers and technicians (approximately 70 meetings and field visits).
• Collaboration with external projects/organisations (2).
• Scientific and public stakeholder meetings (approximately 11 meetings).
A final DROPSA dissemination conference was organised. At the annual International Soft Fruit Conference (ISFC) in Den Bosch (The Netherlands), the European members of the Dropsa project presented their findings to stakeholders. In total 760 people from 32 nationalities, 20 presenters and 98 companies from 13 countries were present at the ISFC.
International Soft Fruit Conference – Thursday 11 January 2018 (http://www.softfruitconference.com/).
DROPSA participated with 8 oral presentations and 10 poster presentations:
1. Oral presentations
• Introduction to DROPSA: Strategies to develop effective, innovative and practical approaches to protect major European fruit crops from pests and pathogens.
Neil Audsley, Fera, UK.
• Integrated pest management of Drosophila suzukii.
Nicola Mori, University of Padova, Italy.
• Biocontrol of Drosophila suzukii.
Marc Kenis, CABI, Switzerland.
• Spotless strawberry plants: how to keep them free from Xanthomonas?
Jan M. van der Wolf, Wageningen University & Research, The Netherlands.
• Managing pathways of pest and disease introduction.
John Mumford, Imperial College London, UK.
• Toxic baits for the control of Drosophila suzukii.
Herman Helsen, Wageningen University and Research, The Netherlands.
• Pesticide-free crop protection with self-limiting fruit flies.
Carlos Pedraz, Oxitec Ltd, UK.
• Comparison of traps and attractants for Drosophila suzukii monitoring in North-East Italy. Lorenzo Tonina, University of Padua, Italy.
2. Poster presentations
• Strategies to develop effective, innovative and practical approaches to protect major European fruit crops from pests and pathogens. DROPSA - Partners.
• Comparison of two integrated management plans for kiwi crops against Pseudomonas syringae pv. actinidiae using a Decision Support System. Olliff L, Humphreys A, Leach A, Holt J, Mumford J. Imperial College London, UK.
• Assessment of IPM packages for Drosophila suzukii – A Decision Support System. Humphreys A, Olliff L, Leach A, Holt J, Mumford J. Imperial College London, UK.
• Identification of pests likely to be imported with fruit to Europe. Suffert M, European and Mediterranean Plant Protection Organization, France.
• Insect netting as a vital component of a Drosophila suzukii control strategy. Helsen H, Van der Sluis B, Wageningen University & Research, The Netherlands.
• A diagnostic tool for improved detection of Xanthomonas fragariae using a rapid and highly specific LAMP assay designed with comparative genomics. Gétaz M, Bühlmann A, Schneeberger PHH, Van Malderghem C, Duffy B, Maes M, Pothier JF, Cottyn B. Zürich University of Applied Sciences, Switzerland.
• Lure and kill devices against Drosophila suzukii: first field trials. Navarro-Llopis V, Primo J, Sanchis J, Barroso JM, Riba JM, Navarro I, Fratantuono M, Alvarado P, Vacas S. Universidad Politècnica de València and Endoterapia Vegetal, Spain.
• Identification of pests likely to be imported with fruit to Europe. Suffert M, Steffen K, Wilstermann A, Schrader G, Grousset F. EPPO, France and Julius Kühn-Institut, Germany.
• New mode of action compounds for the control of Drosophila suzukii. Down R, Matthews J, Audsley N. Fera, UK.
• Drosophila suzukii spillover from natural habitats to vineyards and cherry orchards. Tonina L, Marini L, Sancassani M, Marchesini E, Mori N. University of Padova, Italy.
Dr. Francesco Spinelli (Department of Agricultural and Food Sciences - University of Bologna) organized a presentation during the kiwifruit day for stakeholders (growers, advisors, industry) by the Italian Society for Horticultural Sciences (1st February 2018). The presentation focussed on bacterial diseases (especially Pseudomonas syringae pv. actinidiae), which were not represented at the meeting in Den Bosch.
The presentations are available on the DROPSA-website: http://dropsaproject.eu/
Potential impacts and wider societal implications
Estimates of potential pest impacts and routes of introduction and spread add to the body of work on impact of invasive pests and diseases species and support EU phytosanitary regulations and EPPO alert lists so as to reduce the likelihood and frequency of such invasions occurring in future.
Fruit crops account for around 17% of the value of the EU’s agricultural production and the fruit and vegetables supply chain has an estimated turnover of more than €120 billion with over 550,000 employees and around 1.4 million growers. Losses from pests and pathogens are estimated to account for at least €10 billion in revenue and 3 million tonnes of produce to the EU fruit industry. Despite measures to control D. suzukii and bacterial pathogens (Psa, Xf and Xap) they continue to spread. Implementing new detection and surveillance, control measures and integrated pest management strategies could save tens of millions of Euros and protect jobs and growers. The outcomes and impacts of DROPSA are described below:
WP1 - Pathways of introduction of fruit pests and pathogens
Genetic studies of D. suzukii and the spread models of Drosophila suzukii, Xanthomonas fragariae and Pseudomonas syringae provided valuable information on the introduction and spread of new pests of fruits in Europe. The commodity based risk analysis allows the identification many high-risk pests at an early stage by means of horizon scanning and to develop preventive measures before their introduction. A high number likely to be introduced into Europe with a high risk were identified. The prevention of their introduction will help to secure fruit production in Europe. The QPAFood model enables the identification of particularly risky countries of origin and import periods of the goods. Inspections and measures can be focused and therefore will efficiently and effectively reduce the risk to an acceptable level.
The process used to carry out the pathway risk analyses has been used as a basis to develop EPPO Standard PM 5/9 (EPPO, 2017).
DROPSA alert lists have been reviewed and discussed by experts of the NPPOs in the framework of EPPO (Panel on Phytosanitary Measures 2017-03 and 2017-10). The Datasheets of all alert list species are already available on the EPPO Global Database and some additional species will be added to the EPPO Alert List in 2017-2018 to raise awareness.
The identification of the pests may result in the adaptation of legislation such as for Thaumatotibia leucotreta which was added to Section I of part A Annex I to Directive 2000/29/EC within period of the DROPSA project.
WP2 Biology and Ecology of Drosophila suzukii
• A set of climatic requirement data and development parameters, which can support the building up of climate models and risk maps
• The first data on the biology and ecology of D. suzukii in China, e.g. on its population dynamics and reproductive diapause, which can be compared to those observed in Europe.
• A better understanding of the capacity of the fly to colonise mountainous areas.
• A set of unique data on the circadian cycle of adults D. suzukii fly, which allow to make important observations to calibrate control methods.
• First detailed information on non-crop hosts in China and the most extensive study on non-crop hosts in Europe. Based on these data, management methods can be suggested. E.g. ornamental and hedge plants in the vicinity of fruit crops and orchards can be selected according to their susceptibility to D. suzukii. However, the widespread availability and abundance of non-crop hosts and the lack of efficient native parasitoids suggest the need for an area wide control approach.
• Better information on variations in susceptibilities between varieties and cultivars of grapes and strawberries, two important fruit crops that are only partially susceptible to D. suzukii.
• The most extensive studies on spatial ecology of D. suzukii and interactions between crop and non-crop habitats. These studies are very valuable for developing management methods. For example, the spillover study described in the project results can contribute to help developing physical or mechanical control measures such as the modification of the spatial configuration of the orchards and surrounding native vegetation or the possibility of application of insect-proof nets.
• For the first time, a large survey for parasitoids of D. suzukii was carried out in China. They revealed the same main parasitoids as those previously collected in Japan by our DROPSA collaborator at University of Hokkaido, i.e. Leptopilina japonica and Ganaspis cf. brasiliensis. The latter species is now being studied under quarantine conditions, to assess its suitability for classical biological control.
• Several volatile compounds extracted from fruits induced reaction and/or were found to be attractive for D. suzukii flies.
• The observation that, without strong winds, D. suzukii probably does not spread more than a few hundred meters, which has implications for the development of control strategies
WP3.- Biology and epidemiology of quarantine fruit crop diseases
• Diagnostic tools based on LAMP technology for the bacterial pathogens have been developed. There were initially plans to commercialize these assays as a ready to use kit with the non-DROPSA partner SME OptiGene Ltd. (Horsham, UK) but those were not pursued mainly due to the need to disclose the primer sequences for publications. If required these assays could however be easily developed into ready to use commercial kits.
• Future plans to exploit the CRISPR typing data concerning X. fragariae with the SME Beamedex SAS (Orsay, France) in order to develop a spoligotyping tool for plant pathogenic bacteria.
• The knowledge developed concerning RNA-seq of Actinidia-Psa interaction will be applied in the Italian project “Biotech” (2018-2021) with the aim to use key genes involved in the interaction for genome editing in Actinidia in order to obtain tolerant genotypes.
WP4.- Effective and innovative solutions to control Drosophila suzukii
• Determination of the best commercial trap device and attractant lure for mass trapping and monitoring. Indication for the best use of this type of traps both temporally and specially.
• Development of a new type of device for lure and kill strategies that could decrease the need for conventional broad-spectrum foliar pesticides, reduce costs and environmental impacts.
• The demonstration of the efficiency of different commercial biocontrol agents (entomopathogenic fungi and nematodes) and arthropods and the development of a lure and infect strategy based on entomopathogenic fungi that can be included in IPM strategies to help to control the pest at low levels of infestation.
• Selection among the Asian parasitoids of Ganaspis sp brasiliensis as the best candidate for introduction in Europe that could control the pest both in the cultivated and wild areas. Future work will continue on risk assessment and licence application for the introduction of the best candidate exotic parastioid.
• Assessment of the effectiveness of different commercial pesticides and development of field strategies for the best crop protection with lower pesticide residues in harvested fruits. Some of these pesticides may be used in concert with BCA (such as fungi) while other may have side effects particularly on parasitoids.
• Oxitiec Ltd has developed and optimized of conditional self-limiting technology in D. suzukii for the production of sterile males which may be a solution to control this pest in the future. Further product development for commercial registration, including scale-up production will continue.
• Receptor screening assays have been developed for pesticide discovery, further funding will be sought to continue this work, which is transferable to other pests.
• Novel delivery mechanisms for insecticidal biomolecules and dsRNA have been developed which can be developed further for other applications.
WP5. Effective and innovative solutions for quarantine fruit crop disease control
• Three strains of microorganisms have been developed by UdG as microbial biopesticides against Psa, Xap and Xf. Strains PM411 and TC92 of Lactobacillus plantarum were the object of an agreement between, Anti Microbial Peptide Biotechnologies S.L. (receptor of a license from the owner of the technology, the University of Girona) and Agrifutur s.r.l. for further development and commercial exploitation. A further agreement has been signed between Agrifutur s.r.l. and the University of Bologna to perform field testing with the LAB strains.
• A patent extension from the original application, prior to DROPSA, has been extended to EU, USA and Brasil (EP14798394.4).
• The strain EPS2017 of Bacillus amyloliquefaciens, that was developed by the University of Girona (inventors) for Industrias Químicas del Vallés S.A. (IQV Agro; owners) has resulted in an EU patent (EP16711642.5).
• The antimicrobial peptide BP100 (patent EP2017285A1) is being licensed to companies interested in their exploitation, through Anti Microbial Peptide Biotechnologies S.L. and the University of Girona.
• The strategy for Psa control in kiwifruit based on existing compounds (antibacterials and plant-defence elicitors) has been transferred to the kiwifruit growers in Italy, and will be extended to other growing areas.
• Endoterapia Vegetal have patented an improved injection device to apply microvolumes of antibacterial compounds into plants to protect against PSA and Xap.
WP 6 Practical solutions for control
• Development of new policy to prevent introduction of D. suzukii in new uninvaded area.
• Tailoring monitoring inputs according to the possible paths of diffusion and risk map
• Development of innovative and sustainable cultural management tools creating a preventive control of D. suzukii and P. syringae pv. actinidiae maintaining, at the same time, high production efficiency and economic turnover of the fruit industry
• Reduce pesticide inputs by green, ready to use, green-technologies such as netting, curative pruning and physical methods
• Increase orchard gate revenues: the new cultural management tools generally have higher costs and need an increase in labour. However, the higher costs are outclassed by the reduction of fruit losses and the increase in fruit marketable quality
• Development of DSS system to predict the sustainability of the new control strategies for D. suzukii and P. syringae pv. actinidiae
• Adaptation of the DSS system to other invasive pests and pathogen
• Estimate the long-term sustainability of control strategies for D. suzukii and P. syringae pv. actinidiae
• Achievement of a long term environmental, social and economic sustainable control strategies of the major invasive pests and pathogen threatening European horticulture
• Provide information to develop new GAP policy
• Provide scientifically-based advices to growers and stakeholders of the horticultural industry
• Produce fruit with excellent organoleptic and nutraceutical characteristics and zero chemical contaminant
• Increase the consumption of safe fruit with potential benefits on consumers’ health
WP7 – Economic analysis
• Evaluation of the economic opportunities for fruit growers of the management strategies developed by DROPSA as alternative measures of IPM prevention and crop protection, which can be transferred to other pest and disease scenarios.
• The conditions required to improve and implement alternative strategies that can be used as a guide for the improvement and development of future successful alternative strategies.
List of Websites:
www.dropsaproject.eu
DROPSA's aims were to develop reliable, robust and cost-effective approaches to protect the major European fruit crops from Drosophila suzukii, and the quarantine pathogens Pseudomonas syringae pv. actinidiae (Psa), Xanthomonas fragariae (Xf) and X. arboricola pv. pruni (Xap) which are major phytosanitary risks and pose significant challenges to fruit production. It is estimated that pests and pathogens cause losses to the EU fruit industry of €10 billion and 3 million tonnes of produce.
Genetic studies and the spread models for D. suzukii, Psa and Xf provided information on the introduction and spread of new pests and diseases of fruit. Alert lists have been created for pests and pathogens that may be introduced into Europe with trade of Vaccinium berries, apples, table grapes and oranges/mandarins.
A comprehensive understanding of the biology of D. suzukii and the epidemiology of Psa, Xf and Xap provided key information for the development of prevention and control methods.
Extensive studies on non-crop hosts, spatial ecology, and interactions between crop and non-crop habitats of D. suzukii and the lack of effective native parasitoids in Europe indicate the need for an area wide control approach for this pest. A non-native Asian parasitoid has been selected to assess its suitability for classical biological control. The best commercial trap and attractant for mass trapping and monitoring were identified. A device to lure and kill D. suzukii was developed, as well as a lure and infect strategy using entomopathogenic fungi.
Field strategies for D. suzukii using conventional pesticides have been developed for crop protection with lower residues in harvested fruits. Some may be used in concert with biological control agents and in integrated pest management (IPM) with fruit removal, mass trapping. Insect proof nets effectively exclude D. suzukii, but costs are high, and nets impact on orchard management.
Future pest control solutions based on sterile insect techniques, modifying fly physiology and behaviour, and novel delivery mechanisms for biomolecules, have been developed in the laboratory.
Data on environmental requirements and disease epidemiology of the bacterial pathogens provided data for forecasting models and decision support systems. On-site detection tools and genotyping tools were developed for all three pathogens and can be used for phytosanitary inspection and monitoring, and to characterize pathogen population biodiversity.
Few existing products were effective for disease management confirming the need for new and alternative compounds. Novel antibacterial peptides and microbial biopesticides derived from bacteria were identified. Innovative formulations and stem injections were developed to apply antibacterial compounds into kiwi vines and peach trees.
Cultural management reduces pest and disease incidence. Psa contamination is associated with irrigation water, pruning instruments, grafting and pollen, the latter highlights the risk of commercial pollen production and assisted pollination in kiwifruit. The spread of pathogens by D. suzukii is of low risk. Orchard management practices (training, pruning, covers) facilitate spray treatment and limit disease incidence.
A Decision Support System, which integrates information on biology, ecology, and economic evaluation of management options revealed that strategies developed in DROPSA performed better than current pest management practices for Psa and D. suzukii.
Integrated pest management (IPM) strategies (orchard management, resistance inducers, biological control) for Psa significantly reduced bacterial canker incidence in plastic covered tunnels.
Economic analyses showed that the use of extra chemicals or netting with bait sprays to control D. suzukii leads to a higher marginal gross margin, but single tree netting is not economically sustainable. Covering kiwi plants can prevent yield reductions caused by Psa, but due to the relatively high investment costs this is not economically viable for green kiwifruit in Italy.
Project Context and Objectives:
Fruit crops account for around 17% of the value of the EU’s agricultural production and the fruit and vegetables supply chain has an estimated turnover of more than €120 billion with over 550,000 employees and around 1.4 million growers. Whilst reliable data on average yield and financial losses due to invasive pests and diseases are not currently available in Europe, it is estimated that insect pests destroy approximately 14% of all potential food production globally. Therefore, it is reasonable to estimate that fruit losses from pests and pathogens accounts for at least €10 billion in revenue and 3 million tonnes of produce to the EU fruit industry.
The European Fruit Sector delivers an essential and unique contribution to the economic, social, and environmental future of the EU; it is technologically advanced and includes a complex chain of production, storage, marketing, logistics, wholesaler and retailer input providers. This sector faces several challenges including financial competition, especially from developing countries, and the increased trade from outside Europe increases the risk of invasive pest species.
Modern fruit production must ensure consumer satisfaction and deliver high value quality fresh fruit that contributes to a healthy diet and overall wellbeing. It should also be free from contaminants such as pesticide residues which can be achieved through ensuring agricultural practices that reduce pesticide impacts on the environment and promote biodiversity.
This view has widespread public support and has been influential in scoping of EU regulations for pesticide use and plant protection strategies including the sustainable use directive (EU Directive 128/2009/EC) . This directive aims to achieve sustainable use of pesticides by minimising the risks and impact on human health and the environment, improving the quality and efficacy of pesticide application, and promoting the use of integrated pest management (IPM), and alternative approaches and techniques. Under article 14 of this directive EU member states are required to have a national action plan for the development and introduction of IPM. In addition, Regulation (EC) No1107/2009 , concerning the placing of plant protection products on the market, aims to ensure a high level of safeguarding human and animal health, and the environment, whilst protecting the competitiveness of EU agriculture, and at the same time, increasing the competitiveness of EU agriculture.
Delivering the DROPSA project objectives aimed to provide effective, sustainable, innovative and integrated management solutions to protect the major EU fruit crops, which addressed the above EU policies and regulations, the need for environmentally friendly control strategies, that are target specific to reduce the negative impact on beneficial organisms (e.g. bees), and the public concerns and desire for food free of pesticide residues.
New and emerging pests e.g. Drosophila suzukii, and quarantine pathogens such as Pseudomonas syringae pv. actinidiae (Psa), Xanthomonas fragariae (Xf) and X. arboricola pv. pruni (Xap) that have been introduced into Europe have been identified as major phytosanitary risks and pose a major challenge to fruit production.
Drosophila suzukii is an invasive pest native to Asia that was first reported in Europe in 2008, and has since rapidly spread across the continent. It is highly polyphagous and infests a wide variety of berry and stone fruits. The larvae feed and develop within ripening fruit causing it to soften and rapidly rot, resulting in significant reduction in crop yields and financial losses, and can be so devastating that 100% fruit losses on many crops (e.g. Cherries) have been reported. In Northern Italy, annual losses in fruit crops of over €8 million and over €4 million for cherries in France have been reported. A loss of just 10% (1.1 million tonnes) of the strawberry crop in the EU represents €50 million in lost income. In 2011, a pest risk assessment by the European and Mediterranean Plant Protection Organisation (EPPO) recognised the importance of D. suzukii as a serious pest of soft fruits, stone fruits and grapes in most parts of the EPPO region. It is also considered that an eradication programme and regulation would be unlikely to retard its spread, and that there was currently no effective control strategy against this pest. Hence, D. suzukii poses a serious challenge to fruit production in Europe, and the lack of knowledge on its basic biology has seriously hampered the development of rational and effective IPM strategies.
The production and quality of fruit in the EU is also severely threatened by bacterial diseases caused by Psa, Xap and Xf which represent the most recent and devastating outbreaks of disease to affect European fruit production. Control options are limited due to the virtual absence of, and restrictions on, effective bactericides, so there is an urgent need to develop effective and practical strategies for their management to protect the EU fruit sector. DROPSA has focussed on Psa on kiwifruit, Xap on stone fruits, and Xf on strawberry, but these pathogens affect all the main fruit types, so strategies developed for their control will be transferable to other fruit crops.
Italy are the second largest producer (after China) and second largest exporter (after New Zealand) of kiwifruit equating a value for agricultural of over €3 billion with a retail market value worth €10 billion, and is vital to the economy of the five main regions (Lazio, Emilia, Romagna, Piedmont, and Veneto) where 80% of Italy’s production is concentrated. Kiwi production has been compromised by Psa, which is currently the most virulent and devastating disease in fruit crops. Economic impacts for farmers have been estimated to be over €85000/hectare/year in production losses, orchard investment and plant destruction to stop the spread of infection, reducing production in orchards by 10-50%. Revenue losses of over €40 million have also been reported for Psa damage to kiwifruit in the Lazio region of Italy and in New Zealand Psa is estimated to cost the kiwifruit industry over €50 million per annum.
Among small fruits, strawberry is the most important berry crop in the EU, being produced in all member states, with a production exceeding 1.31 million tonnes per year. Strawberry production requires specialised knowledge, high energy costs (e.g. machinery fuel, irrigation), high external inputs (e.g. labour costs, fertilisers, water and pesticides). Thus, any development that may reduce the incidence of pests and pathogens in strawberry cultivation will be of high importance. Disease in strawberries due to Xf is predominantly destructive in nurseries, especially under protective cultivations with high humidity, and can cause up to 75% losses.
Present in central and southern Europe, but spreading, bacterial spot due to Xap has been reported on a wide range of Prunus species, and is particularly serious in peach and plum. Control relies primarily on copper compounds, but none are fully effective, hence reliable and durable control strategies for this disease are still needed.
Clearly control methods for both D. suzukii and microbial pathogens in Europe are severely inadequate. DROPSA has addressed these problems to advance options beyond those currently available to provide effective, sustainable and IPM strategies to protect and secure food production in the EU.
The primary research emphasis of the DROPSA project is on the development of effective, innovative and practical solutions for the control of D. suzukii, Psa, Xf and Xap on various fruit crops that are representative of the some of the major fruit sectors in the EU (namely berries, grapes, stone fruits and kiwifruit).
This is based on the following drivers:
• Pests and pathogens, particularly D. suzukii, Psa, Xf and Xap, are a major concern and challenge to fruit production and cause significant losses within and outside the EU region.
• Since its introduction into Europe D. suzukii continues to spread unabated. Eradication or containment is not possible, so there is a pressing need for effective, targeted, innovative, practical and integrated management solutions to minimise its economic impact.
• The pathogens Psa, Xf and Xap are currently emerging in the EU and Mediterranean region and there are no efficient methods of disease control available. Their spread from the initial outbreak to other fruit production areas is ever increasing.
• The invasion of, and damage by new exotic pests and pathogens is likely to increase with intensification of trade and climate change.
• The desire to develop IPM strategies able to guarantee high yields, high fruit quality and fruits free of pest, diseases and pesticides.
• Τhe translation of knowledge to practical solutions for the management of pests and pathogens, with the active engagement of SMEs and industry.
• To use DROPSA as an exemplar of how the investigation of the pathways of introduction of D. suzukii, Psa, Xf and Xap will provide recommendations and strategies against the invasion of other invasive pests and pathogens.
The principal objective of DROPSA was to develop reliable, robust and cost-effective approaches to protect the major European fruit crops from new and emerging threat dues to pests (D. suzukii) and pathogens (Psa and Xf and Xap) that could be widely implemented by the EU fruit industry.
DROPSA integrated leading expertise in fruit pests and pathogens, fruit production and orchard management from across Europe and more widely on a global basis, involving multidisciplinary, interrelated and complementary evaluations, methods and techniques. The main objectives were to:
• Determine the pathways of introduction and spread of D. suzukii and the above pathogens into the EU to help develop preventative strategies and recommendations against the introduction of other dangerous fruit pests/pathogens and diseases into Europe.
• Contribute towards minimising the risk of spreading the target pest and pathogens in other countries, thus securing the security of EU fruit exports.
• To identify the key mechanisms within the biology, epidemiology, ecology and interactions of these pests and diseases for the development of effective, innovative, practical and sustainable control methods. This includes a comprehensive evaluation of life cycles, host ranges, capacities to disperse, the identification of natural enemies, plant-pathogen interactions, and semiochemicals involved in D. suzukii behaviour.
• Develop control options including screening of existing compounds, semiochemical based technologies, novel antimicrobial compounds, biological control agents, as well as cultural practices, sterile insect techniques, and new mode of action compounds.
• Combine the most reliable and effective control options to develop and optimise an integrated pest management strategy to combat fruit pests and pathogens, utilising forecasting and decision support systems and risk mapping.
• Evaluate the economic viability of proposed strategies for fruit crop protection to support decision making in the development of practical solutions and integrated pest management strategies
DROPSA has included partners from non-EU countries in Oceania (New Zealand), North America (USA and Canada), and Asia (China and Japan) which have extensive expertise in the biology and control of D. suzukii, Psa, Xf and Xap. The exchange of information between countries in these regions (where the pests/pathogens either originated or show similar invasion characteristics) and EU partners, and the coordinated development of improved control methods, is recognised as key to the successful execution of the project.
Project Results:
Work package 1 – Pathways of introduction of fruit pests and pathogens
Aims and objectives
The aims and objectives of WP 1 were to determine the pathways of introduction and spread of Drosphila suzukii and pathogens into the EU and to develop preventative strategies and recommendations against the introduction of other dangerous fruit pests and pathogens.
Results and discussion
Pathways of introduction and spread of D. suzukii
The spread and potential distribution of D. suzukii was modelled with a dispersal kernel model based on a 2Dt-distribution developed in the former EU project PRATIQUE. The spread model predicted a rapid spread over the whole area of potential establishment (i.e. most of Europe) within seven years. D. suzukii is already widespread over a large area of the EU but has still the capacity to spread and establish in the northern EU countries (Ireland, Scandinavian states and Baltic States), though with limited damage potential.
It was found that modelling of D. suzukii with a dispersal kernel is possible. The dispersal kernel provides a quickly applicable tool with the possibility to adjust relevant parameters to new findings.
DROPSA partners participated in an international study to identify the routes of invasion of the European and North American invasive populations of D. suzukii, using molecular markers (microsatellites) directly with D. suzukii populations to perform a phylogeographic study on the roads of invasion of D. suzukii. In order to distinguish the different populations of D. suzukii, the presence and the phylogenetic relationship of the bacterial communities associated with the intestinal tract of natural populations of D. suzukii from different geographical areas were assessed, with the aim to study their phylogenetic relationship. The results indicate that habitat, food resources as well as the colonization phase of a new region contribute to shape the bacterial communities of the invasive species which, in turn, by evolving more quickly, could influence host adaptation in a new environment.
The study of the routes of invasion of D. suzukii worldwide revealed that Japan emerges as the most probable source of the earliest recorded invasion into Hawaii. Furthermore, Southeast China and Hawaii together are the most probable sources of populations in western North America, which then in turn served as sources for those in eastern North America. European populations are genetically more homogeneous than North American populations, and their most probable source is northeast China, with evidence of limited gene flow from the eastern US as well.
Pathways of introduction and spread of diseases
Spread models were also prepared for Pseudomonas syringae pv. actinidiae (Psa) and Xanthomonas fragariae (Xf) to evaluate the applicability of this model to bacterial pests. It was hitherto tested on insects, plants fungi and nematodes. It was not possible to model the spread of X. arboricola pv. pruni (Xap) due to the lack of key data. The area of potential establishment was plotted for all targeted bacteria. The modelling of bacterial spread with the dispersal kernel is in general possible. The model needs only a few key parameters as input and provides meaningful results. However, the modelling of bacterial spread is challenging due to the biological characteristics of bacteria and a lack and high uncertainty of data.
An alert list of pathways of introduction in Europe
A review on pests (including pathogens) that have fruit species as their host plants was conducted. The focus was on pests that have been introduced into Europe or were found in the fruit trade during the last 10–15 years. A total of 387 introduced pests were identified. The large number of pests intercepted on fruit, from different taxonomic groups and origins, shows that pests do move in trade on fruit. Conducting pathway analysis requires a considerable amount of time for each fruit species, therefore a priority list of fruit crops was established. Important factors for prioritizing the crops were for example trade volumes into the EU, the area of production in the EU, origins of trade (emerging markets) and the prior history of pest introduction. Furthermore, the overall selection aimed to provide a good geographical coverage of fruit production in the EU, to ensure a balance between fruit species (both regulated and non-regulated for fruit in the EU), and to cover crops from different groups (e.g. pome fruit, stone fruit, citrus, small fruit). After prioritization, Alert lists for Vaccinium berries, apples, table grapes and oranges/mandarins were prepared, as well as a description of the commodities traded for each species. Detailed information on the pest identification and rating system were provided. Pests from all parts of the world were reviewed and screened to assess the ones that were likely to be introduced with fruit trade. For the four selected pathways, 104 organisms likely to be introduced into Europe with foreseeable non-negligible economic impact were identified.
The pathway analysis provides the potential to identify pests that otherwise would have been unrecognised until they are detected in commodity inspections or in member states of the EU. These species represent a high risk for fruit production in Europe. It was shown that the risks of introducing new plant pests through the import of fruits have so far been underestimated. The Alert lists provided the basis for the identification of preventive strategies, which are based on recommendations for good professional practice for importers and producers as well as risk mitigation by legal regulation. These commodity assessments may be used to support regulation of high risk commodities at EU level (as defined in art 42 of EU Regulation 2016/2031). Pathway analyses provide the opportunity for horizon scanning and the identification of so far unnoticed risks and should be continued in the EU.
Methods for quantitative pathway risk analyses systems for fruit pests and pathogens
The quantitative pathway model, QPAFood, has been designed to support risk assessment for plant pest entry into EU territory on a range of edible plant commodities and for the pests’ transfer to a host, via trade flows. The model was developed originally for the European Food Safety Authority (EFSA) and has been extended in the context of DROPSA. It provides a stochastic description of the flow of infested commodity from one stage on the trade pathway to the next.
The practical modular approach applied here offers a high degree of potential application for fruits and grains such that different pests and commodities can be assessed by changing parameters on the spreadsheets indicated without the need to alter either model’s structure. A pathway in QPAFood concerns a particular pest on a particular commodity, but multiple commodity paths for a single pest or multiple pests for a single commodity can be considered by employing a series of parallel model calculations. The model enables the identification of high risk origins of the commodities and the assessment of the arrival risk on a monthly basis. The quantitative pathway model was applied to D. suzukii and the Apple fruit moth Argyresthia assimilis. The QPAFood model can be used to facilitate effective and efficient inspection methods of monitoring fruit imports; identify countries and products with a high risk profile and increase the awareness of the risk and the urgency for action to fruit industry. Within DROPSA the QPAFood pathway model was extended to cover multiple commodity pathways, reflecting the wide range of commercial hosts of D. suzukii.
Work package 2 – Biology and Ecology of Drosophila suzukii
Aims and Objectives
To identify the key mechanisms within the biology and ecology of D. suzukii for the development of sustainable control methods in WP 4 and WP 6 and to assess actual and potential host range and study spatial ecology in various environments.
Results and discussion
Life cycle and environmental requirements
The life cycle and environmental requirements of D. suzukii under various climatic conditions were assessed. These data have been used for the development of integrated control strategies in WP 4 and 6 and for risk mapping in WP 6.
Population dynamics of the fly in Europe and East Asia
For the first time, the population dynamics of D. suzukii was studied in east Asia, at various sites in Beijing (Temperate climate) and Kunming (Yunnan, subtropical climate), using traps. In the two regions the peak of the occurrence occurred in summer but dropped in late summer or early autumn. This is in contrast to what is observed in Europe and North America where, in general, populations are maintained or even dramatically increase in autumn. In Beijing, hardly any flies were caught from late November to late May. In Kunming, in contrast, flies were found in all seasons. However, in total, many more flies were caught in Beijing than in Kunming. An elevation gradient did not provide a clear pattern. Variations in natural control may explain the lower population levels in Yunnan compared to Beijing, where D. suzukii could possibly also be introduced. A subset of the females caught in the traps were dissected to assess their ovarian maturity, and dissection results were compared with similar studies in Europe. In Beijing and Europe, ovarian maturation stops rather early in autumn, and faster at the high elevation sites than at low elevation, suggesting that it may be temperature-driven, but maturation resumes earlier in Europe (April-May) than in Beijing (late May - early June). In the subtropical climate of Kunming, mature females are found throughout the year.
Degree-days and temperature requirements
Degree days and temperature thresholds were obtained based on developmental studies under constant temperatures. These were carried out in Switzerland and China on various fruits and diets. Data from the literature were also analysed. All data were compared and tested using field development data gathered in Switzerland and the Netherlands. Most data (from our own and others’ studies) were surprisingly similar, no matter the regional strain and the diet used. The values that suited best the field development data in Europe were used for the climate model and risk maps built in task 6.
Environmental requirement studies under natural conditions
As a fruit-feeder tracking the availability of wild fruits along latitudinal and elevational gradients, D. suzukii is expected to be rather flexible in relation to the environmental conditions, among which temperature is a major player. We sampled potential wild host fruits of D. suzukii along elevational gradients in a mountain area of northern Italy and tracked temperature conditions in the weeks before the sampling, in order to verify if data about temperature-related development available in the literature match with the exploitation of the hosts, and if there are hosts which cannot be exploited because of limiting temperature. In addition, we used a strong natural temperature gradient in a cave, in the lower range of performance known for D. suzukii, to mimic conditions existing along the elevational gradients. We used laboratory stock colonies of D. suzukii to assess insect performance under different naturally fluctuating temperatures in a cave and compared them with a reference stock kept under constant temperature in the laboratory. At least six species resulted to be suitable for the development of D. suzukii in mountain areas (Daphne mezereum, Lonicera alpigena, Lonicera nigra, Lonicera xylosteum, Sambucus nigra, and Sambucus racemosa). By comparing the data from the temperature experiment, the minimum threshold of average daily temperature under natural conditions for the complete development of D. suzukii corresponds to 11.6°C, a value slightly higher than that obtained by weather stations close to the collection site of wild fruits which yielded adults along the elevational gradient (11.2°C). These findings open the way to better understand the possibility of the fly to colonise mountain habitats, although in a temporary way because of limiting conditions during overwintering. They also indicate that D. suzukii shows a good performance at rather low temperatures, indicating that population build up may occur even those conditions and thus the species may recolonise the low elevation habitats at the end of the summer with abundant populations
Circadian behaviour of adults D. suzukii
Studies of the circadian cycle of D. suzukii were carried out, using simulated environmental conditions and field observations. Many results were gathered, which allowed us to make important observations to calibrate control methods. For example, we know now that, male and virgin female D. suzukii are active in the morning and early evening with a night time sleep period and afternoon siesta. The siesta in mated females is disrupted by elevated afternoon activity, a post-mating response probably triggered by the male sex peptide. The increased activity of mated females might explain the higher proportion of females migrating into fruit crops early in the growing season. When afternoon temperatures reach 30oC, both male and female D. suzukii increase activity, but this is a direct ‘escape’ response to dangerous temperatures. The timing of emergence is highest is early morning when light and temperature are starting to rise. Humidity does not greatly affect development of the pharate adult within the pupal case, but is critical for adult emergence with very few emerging at and below 50% RH. Mating behaviour of D. suzukii was compared with that of D. melanogaster. The courtship behaviour is rather similar in the two species. In contrast to D. melanogaster, 7-10 day old virgin D. suzukii are reluctant to mate in laboratory setting; e.g. males spend less time courting and they have a much longer latency before copulation. Once copulation takes place the mean copulation time is 25.73 min which is longer than that of D. melanogaster.
Host range and spatial ecology
• Host ranges of D. suzukii were assessed in various countries and continents, on wild and cultivated plants and well as on selected crop fruits.
• The spatial ecology of D. suzukii was investigated in a heavily infested region in NE Italy, with the aim of gathering new information on its distribution and the role played by the composition of the farmscape on its abundance.
Non-crop host plants
Field surveys were carried out during two years in Italy, the Netherlands and Switzerland. Fruits of 165 potential host plant species were collected, including mostly wild and ornamental plants. Over 24,000 D. suzukii adults emerged from 84 plant species belonging to 19 families, 38 of which being non-native. Forty-two plants were reported for the first time as hosts of D. suzukii. The highest infestations were found in fruits of the genera Cornus, Prunus, Rubus, Sambucus and Vaccinium as well as in Ficus carica, Frangula alnus, Phytolacca americana and Taxus baccata. Based on these data, management methods were suggested. Ornamental and hedge plants in the vicinity of fruit crops and orchards can be selected according to their susceptibility to D. suzukii. However, the widespread availability and abundance of non-crop hosts and the lack of efficient native parasitoids suggest the need for an area wide control approach.
The host range of D. suzukii was also investigated in its area of origin in China. Fruits of 104 plant species were collected in 2014 - 2016 in 10 Chinese provinces in different environments. Adults of D. suzukii were obtained from 29 plant species. Most of the recorded hosts are non-crop plants and several represent new host records. In general, the main host genera in Europe were also hosts in China. However, compared to our surveys in Europe, the percentage of fruit species attacked by D. suzukii was lower in China than in Europe (27.9% vs 50.9%). The level of attack on preferred hosts was also much lower than in Europe.
Cultivated fruits
Since many publications are already available on the susceptibility of cultivated fruits to D. suzukii, in DROPSA we decided to focus our work, in Italy, on the varietal susceptibility of grapes and strawberry. The grape study was particularly important because it is a well-known fact that grape is not the most suitable host for D. suzukii and that suitability varies between varieties. This was clearly shown in our study, which also demonstrated that the level of infestation in a variety depends on skin toughness, skin elasticity and pulp consistency. Even in heavily attacked varieties, only a small amount of the larvae developed to adult. Important differences in susceptibility were also found between strawberry cultivars. The number of eggs per strawberry and the infestation level were highly correlated with acidity but not with penetrating force and sugar level.
Correlation between catches in cherry orchards and damage on cherries
In Italy, we investigated the correlation between catches in cherry orchards and damage on cherries. The study showed that the relationship was very weak, most probably due to the fact that the best attractants available to date are not more attractive than cherries.
Influence of forest abundance on captures and damage on cherry
We selected 32 conventional cherry orchards in NE Italy surrounded by landscapes with different proportions of semi-natural habitats and quantified both pest density and crop damage using sentinel fruits. We observed a higher attack density in orchards surrounded by higher forest cover. The same trend was observed for female adult density. In forest habitats, the pest probably can find ideal microclimatic conditions and other biotic factors such as the presence of alternative host plants that can promote population growth. The effect size of forest cover on both adult density and crop damage, despite the very large tested forest gradient (0-60%), suggests that the removal of forest habitats does not seem to be a viable option for controlling D. suzukii. However, current integrated pest management should take into account landscape composition and interventions should be particularly timely in forested landscapes where D. suzukii can quickly attack the crop at higher density.
Spillover of Drosophila suzukii between non-crop and crop areas
Semi-natural areas may affect D. suzukii population dynamics by providing alternative host resources, overwintering habitats, or refuge areas when crops are sprayed with insecticides. We investigated the spillover of D. suzukii between non-crop and crop areas using sweet cherry as a model crop. We sampled the pest at different heights and at different distances from the forest from the stage of vegetative rest to the stage after crop harvest. Drosophila suzukii abundance in the orchards declined strongly with increasing distance from the forest margin and with increasing height from ground. The observed patterns varied across the crop phenological development stages indicating that the pest used multiple habitats across the seasons. When the host plant was not suitable for reproduction, D. suzukii preferred to fly closer to the forest margin and at lower heights. Differently, when the host plant was suitable (i.e. ripe cherries), D. suzukii colonized further the orchards both horizontally and vertically exploring more in depth the canopy volume.
Natural enemies and other mortality factors
Natural enemies and other mortality factors were identified to gather information for the development of biological control strategies against D. suzukii (WP 4). Two approaches were used: (1) Partial life tables to investigate the relative importance of natural mortality factors throughout the life cycle of the fly; (2) Study of the parasitoid complex of D. suzukii in Europe, Asia and North America, with the aim to identify potential candidates for biological control.
Partial life tables
This study aimed, by comparing mortality factors in Europe and China, at providing information on why D. suzukii populations are lower in China than in Europe. However, producing life tables for D. suzukii in the field was much more difficult than expected, for various reasons: overlapping generations, the cryptic life of immature stages and their very short duration, naturally occurring populations that disturb experiments with field-exposed stages, etc. Nevertheless, partial life tables were developed in Switzerland and China in 2015 and 2016, using various protocols. In China, no parasitism was observed whereas it was known to occur regularly in the area. Generational mortalities varied between 93.4% and 99.3% and the net reproductive rates (R0) varied between 7 in July and 0.7 in August. These numbers appear very close to what is observed in reality close to the reality. In Switzerland, the total generational mortality was 95.57%, which provided a realistic R0 of 4.7. Mortality was caused by two parameters: predation and abiotic mortality.
Parasitoid surveys
Classical biological control through the introduction of parasitoids from its region of origin could help reducing populations at landscape level and, thereby, decrease the need for management in cropping systems. However, little was known about the parasitoid complex of the fly in its region of origin, especially in China, which shares the largest part of its native distribution. Therefore, surveys for larval parasitoids of D. suzukii were carried out in 12 Chinese provinces and five Japanese prefectures between 2015 and 2017. Parasitoids of D. suzukii and other fruit-inhabiting drosophilids were found at 28 sites in four provinces in China and four prefectures in Japan. Larval parasitoids were obtained at most sites where D. suzukii was found, with parasitism varying from 0.0 to 75.6 %. At least eight parasitoid species were reared out. The most abundant and frequent parasitoids were the Figitidae Ganaspis cf. brasiliensis and Leptopilina japonica, but another Leptopilina species and at least five Braconidae species belonging to the genera Areotetes, Asobara and Tanycarpa were obtained in low numbers. In most samples, D. suzukii was accompanied by D. pulchrella or D. subpulchrella, two other Drosophilidae whose larvae live in fresh fruits in Asia and are probably attacked by the same parasitoid complex. Due to its abundance in Asia and its likely restricted host range, the most promising parasitoid for biological control is Ganaspis cf. brasiliensis. This and other parasitoids were extensively studied in Task 4.2
Semiochemicals of D. suzukii
Compounds attractive to adult flies were investigated using two approaches: (1) Identification of external compounds involved in the attraction of D. suzukii, particularly from fruits; (2) Identification of compounds emitted by D. suzukii, such as those involved in courtship and mating behaviour
Fruits and other compounds
The extraction and identification of fruit volatiles was carried out in China. Gas chromatograph- electroantennographic detection (GC-EAD), electrophysiological and behavioural responses of D. suzukii to plant volatiles at different ripening stages of fruits of bayberry (Myrica rubra) and cherry (Cerasus yunnanensis) were studied to identify volatile semiochemicals used in host finding behaviour. There were 11, 42, 23 and six main volatile compounds identified from cherries, ripe bayberry, half-ripe bayberry and green bayberry, respectively. GC-EAD results revealed the presence of six electrophysiological active compounds from ripe bayberry fruits. EAG responses of female and male D. suzukii to 12 compounds increased with increasing concentration (0.01 0.1 1, and 10μg/μl). Both cherry and bayberry fruits significantly attracted mated and virgin D. suzukii. Mated females were all attracted by the fruits of cherry and bayberry and showed no response to the control. Methyl 2-hexenoate and α-caryophyllene significantly attracted both virgin and mated female D. suzukii, whereas (E)-β-caryophyllene significantly attracted virgin females. Methyl 2-hexenoate could repel either virgin or mated female D. suzukii, and α-caryophyllene just repelled virgin D. suzukii. The results showed that methyl 2-hexenoate, α-humulene, (E)-β-caryophyllene could be used by D. suzukii to locate its host plant. In addition, we performed the identification and behavioural responses of D. suzukii to plant volatiles of the wild fruit Osyris wightiana. GC-EAD detection revealed the presence of 16 EAG active compounds. Novel attractant lures based on these volatile compounds have been field tested in WP4
Compounds emitted by D. suzukii
We investigated semiochemicals emitted by D. suzukii (e.g. aggregation pheromone) by profiling volatiles of both fly sexes. Given that cuticular hydrocarbons (CHCs) may be involved in courtship behaviour, the extraction of these compounds was also carried out. However, no compound involved in chemical communication was found. This confirms other studies carried out recently by other teams.
Natural dispersal capacities
The natural dispersion and spread capacity of adult flies was investigated using the mark-recapture technique.
Sterilisation and marking methods
In order to perform release-recapture experiments to study the natural dispersal capacities of D. suzukii, sterilization and marking methods for flies had to be defined and tested. For sterilisation, 40 Gy was selected as the irradiation dose as this dose ensures almost 0% egg hatching from the irradiated females and avoid complete development to pupa. For marking, in laboratory trials using fluorescein, flies were not effectively marked or not detectable after a short period. Thus, a non-specific protein was tested as insect marker, i.e. bovine serum albumin (BSA) which can be detected by ELISA technique. This method allowed a high sensitivity for BSA protein detection, even 7 days after marking.
Mark-recapture
In order to compare the dispersal capacity of D. suzukii with a well know fly, sterile marked Ceratitis capitata males were also released and recaptured. Sterilized and marked D. suzukii and C. capitata were released in a mixed orchard containing various citrus varieties. An array of 40 traps concentrically deployed at 10, 25, 50, 100 and 250 m from the releasing point was used to recapture the released flies. Flies were released six times, three during autumn and three during spring. The traps were inspected 3, 24, 48 h and one week after release.
In general, recapture rates for C. capitata were higher (7-69%) than for D. suzukii (2-14%), most probably due to the different attractant employed – trimedlure, a powerful parapheromone for C. capitata and the unspecific food-bait (wine + apple vinegar + sugar) for D. suzukii. However, the recapture rate of D. suzukii was fairly good compared to similar tests made with other Drosophila spp. For both flies, the majority of the captures were made at 10 m from the release point but a few were collected at all distances. These results suggest that D. suzukii has a relatively limited dispersal capacity compared to other fruit flies such as C. capitata, suggesting a shorter distance of influence. We can estimate a dispersal distance for D. suzukii between 200 and 300 m. This does not take into account potential occasional dispersal with strong winds. Given the low dispersal capacity of D. suzukii and the low efficacy of the current attractants, we suggest, as mass trapping strategy, a dense trap density to effectively trap the flies at short distances or a perimeter trapping design to prevent pest intrusion, especially when field margins contain alternative hosts.
Work package 3 – Biology and epidemiology of quarantine fruit crop diseases
Aims and objectives
Pseudomonas syringae pv. actinidiae (Psa), Xanthomonas arboricola pv. pruni (Xap), and X. fragarieae (Xf) are quarantine pathogens in the EU that seriously limit kiwifruit, peach-almond, cherry and strawberry production. Knowledge of pathogen biology and epidemiology is fundamental to develop effective sustainable control strategies. This information was however very scarce at the beginning of the DROPSA project. Thus DROPSA aimed to identify the principle elements in the life cycle and epidemiology of these quarantine pathogens in fruit crops, determine mechanisms involved with pathogen virulence and fitness level, host defence responses, and insect vectoring risks in order to develop effective and innovative control solutions and IPM.
Results and discussion
Fundamental insights on the environmental requirements and disease epidemiology were determined.
For Psa, temperature and humidity greatly influence growth and disease development whereas light amount and quality does not seem to be crucial. It does influence relevant functions for plant colonisation during in vitro tests. The bacterium appears to overwinter readily on host plants until budburst the following spring. Psa seems unable to survive in soil probably due to the high competition with the rich microbial community present in this ecosystem. Rain splashes, especially during spring and autumn, can spread Psa within the orchard. However mechanical pruning or ineffective disinfection of tools may be regarded as a major issue for the bacterial spread. Infection threshold was found to be directly correlated with cultivar susceptibility. Psa was occasionally found on non-host plants inside infected orchards, but could not multiply in any of the tested plants.
Concerning Xap, the basis for the development of the first predictive model of Xap growth and infections on peach were established and provide insights on fundamental climatic parameters.
The risk of Xf pathogen dispersal via aerosols and plant material when moving strawberry plants was evaluated. To reduce risk of Xf dissemination and subsequent plant infection, it is advised to mow during dry (sunny) weather conditions. Weather conditions have a minor influence on spread but a greater one on the risk of infection. Dissemination of Xf during mowing should be reduced by using physical barriers around the blades of the mower or by disinfecting (heat or biocides) the blades during mowing.
On-site detection tools and genotyping tools were developed.
Source tracking tools are available for all three pathogens. This was further developed for Xf using MLVA and the existence of four lineages was revealed. Genotyping with CRISPR revealed two main different lineages but four (sub)groups and this approach is complementary to MLVA as it allows retracing pathways of evolution by following newly acquired spacers. However, CRISPR sequence determination still requires whole genome sequencing of the pathogen. A quick CRISPR genotyping tool to distinguish the four main different lineages revealed in the frame of DROPSA has been developed in order to circumvent this problem.
The evolutionary risks of the pathogens were evaluated in silico
Several pathogen genomes were determined during the DROPSA project. In the case of Psa this revealed that large portions of specific DNA have been acquired by the Psa3 subpopulation through horizontal gene transfer. Mobile genetic elements (plasmids, ICEs, GIs, phages, prophages, etc.) seem to contribute greatly to the genetic diversity of Psa. Although some diversity was observed in Xap and Xf, the genomes of these pathogens seem more conserved. Some differences observed in the Xf genomes sequenced for the development of genotyping tools were further characterized and will be soon published.
Pathogen virulence factors and fitness determinants as well as their environmental modulators were determined
Pathogen transcriptomes under in planta (or mimicking) conditions were obtained for Psa and Xf and compared to in vitro condition transcriptomes. A list of up-regulated virulence factors and fitness determinants could be established for both pathogens. A prerequisite for optimal downstream analysis appeared to be the availability of a good reference genome for the phytopathogen. Indeed, in addition to specifying the location of the differentially expressed genes (i.e. chromosomal or on a plasmid), it can also help the identification of new genes and improvement of the genome annotation.
Host plant defence factors were identified using RNA-seq
The RNA-seq results highlighted a role of AtWRKY70 and actinidins in kiwifruit resistance against Psa. Therefore, they may be potential candidates for molecular markers for future breeding programs focused on obtaining novel kiwifruit cultivars resistant to Psa. Further experiments are however needed to prove their effectiveness. The lack of resistant genotypes of A. chinensis or A. deliciosa makes it very difficult to test these putative markers. To overcome this limitation, the expression of these two genes needs to be evaluated in a number of cultivars showing differential susceptibility to Psa and in Actinidia arguta, which is a species tolerant to the pathogen.
The first strawberry transcriptome in response to inoculation with Xf was established recently and revealed metabolic changes between short- and long-term symptoms. Results will be published shortly.
Insect vector interactions and critical factors to mitigate pollinator vector risks were characterised.
The role of pollen, bee pollinators, pathogen survival in beehives, role of phloem feeding insects (Metcalfa pruinosa) was investigated in the case of Psa. Pollen has a crucial role in vectoring the bacterium. Asymptomatic closed flowers may produce contaminated pollen, highlighting that Psa is adapted to the internal colonization of male flowers which increases the sexual transmission of the pathogen, providing an evolutionary advantage to the bacterium. Thus substantial attention should be paid to the commercial production of pollen and use of assisted pollination in kiwifruit. However it is unlikely that beehives provide a possible reservoir for the pathogen and transport of beehives from infected areas to Psa-free orchards present a very low risk for Psa spread. On the contrary, there is a risk of Psa spread via phloem feeding insects such as M. pruinosa.
The presence of Xf could not be confirmed using LAMP and qPCR on insects sampled on inoculated plants, suggesting that the spread of Xf to healthy plants by insect feeding on diseased plants is very low.
No Psa, Xap or Xf was identified in the gut microbiome of D. suzukii, thus the risk of spread of Psa and Xf by D. suzukii appears very low.
Work package 4. Effective and innovative solutions to control Drosophila suzukii
Aims and Objectives
To develop effective and innovative solutions to control Drosophila suzukii based on biological data produced in WP 2, transfer of best practices and adaptation of innovative technologies for inclusion in IPM strategies in WP6.
Monitoring, mass-trapping, lure & infect methods
The objective of this task was to develop monitoring, mass-trapping, lure and infect methods using available semiochemicals and new attractants identified in WP 2. Several volatile compounds extracted from fruits induced reaction and/or were found to be attractive to D. suzukii flies. Novel attractants based on these compounds have been tested in the laboratory and some have been selected to be further test in the fields, however none are yet commercially available at this time. Efficacy of several commercial trap designs and available liquid attractants (most wine-vinegar-sugar based) was also tested in several countries and agricultural environments.
The red Drosotrap® device was found to be the best trap and regarding liquid baits, Suzukii-trap® was the most suitable lure considering its high selectivity and persistence. Droskidrink® was the best lure in considering longevity and high attractiveness. Some of the liquid bait were not stable for long period (proliferation of fungi, gel formation, etc.) and needs a weekly replacement while others more stable needed periodic re-filling due to evaporation. To avoid some of these problems a more convenient attractant device was developed, the system includes a permeant plastic bag containing the liquid bait that are easier to handle and may be placed in commercial dispensers. This bait has been optimized for the attractant release and trapping success.
Mass trapping alone did not provide efficient practical solutions for D. suzukii during the fruit season, and it has been observed that fruit infestation increases while the season progressed, and plants with traps had greater fruit damage. Indeed, attractant traps could bring flies into an area, which may engage in feeding, mating and/or egg-laying before entering the trap. However, traps could be useful at the beginning of the D. suzukii season to decrease the first generation of reproductive females and to monitor the pest pressure to start and adjust other treatments in an IPM strategy.
Lure and Kill (L&K) and Lure and Infect (L&I) strategies could provide an alternative strategy as there is no need for a trap to physically capture the flies. Insects can be lured to baits or surfaces treated with the pest controlling agents, for instance insecticides or sterilizing agents (L&K) or entomopathogenic agents (L&I).
A L&K strategy was developed using the developed plastic bag dispenser device as attractant coated with insecticides and has shown its efficacy during field tests. A large array of sterilizing agents has also been tested (in relation with Task 4.4) to be included in this attractant method. However, D. suzukii proved to be quite resistant to these products and the dose necessary to induce sterility were too high and then excluded for economical and risks reasons.
For the L&I strategy different species and strains of entomopathogenic fungus have been tested for their effect on D. suzukii (in relation with Task 4.2). In laboratory Beauveria bassiana (Naturalis-L) causes 44% adult D. suzukii mortality after 7 days, whereas Lecanicilium muscarium (Mycotal) and B. bassiana solutions caused no significant decrease in fruit fly population. Three further fungi species (commercial formulations and different strains of Metarhizium anisopliae and Isaria fumosorosea of Beauveria) were also screened. Most of the fungi produced a high death of adult flies after exposition but in some case this death requires several days to week to happen and did not decrease the reproductive potential of females. The most efficient entomopathogenic fungi have been tested in the fields and PCR (TaqMan) test has been developed to follow horizontal and vertical transmission of entomopathogenic fungus spores. Preliminary results for both L&K and L&I were promising, but need to be confirmed in larger field trials.
Evaluation of natural enemies for the biological control of D. suzukii
Commercially available biological control agents (predatory bugs, nematodes, pathogens) have been tested in laboratory and field conditions using standard procedures. Predatory species screened were Orius majusculus and O. laevigatus (both efficient in thrips control); Atheta coriaria (a Staphylinid beetle); Hypoaspis miles (a soil dwelling predatory mite) and Anthocoris nemoralis (a generalist predatory bug). Orius spp. and A. coriaria did prey upon the larvae and pupae of D. suzukii but have a low impact, while H. miles, did not feed upon D. suzukii larvae or pupae. A. nemoralis displayed the best potential as a control agent; 45% of adult mortality was achieved over a five-day period under laboratory conditions. These predators may be potentially used in an augmentative biological control strategy. Different nematodes species (Steinernema kraussei, Heterorhabditis bacteriophora) that infect and kill arthropods were also tested. Both larvae and pupae of D. suzukii are susceptible to nematode infection. Larvae proved more susceptible than pupae under laboratory trials. S. kraussei caused the higher pupae mortality (55%) while H. bacteriophora produced the higher mortality in larvae (95%). The nematodes offer potential to be applied as soil drenches around infested fruit bushes for the suppression of D. suzukii populations.
Considering the fact that D. suzukii is multivoltine and highly polyphagous, attacking a wide range of fruits of wild and ornamental plants (see Task 2.2) it becomes more and more apparent that long term control solutions will have to involve both in cultura and area-wide control methods. Biological control using parasitoids is a promising strategy for D. suzukii because, in contrast to other methods, it has the potential to lower the population levels in all habitats, limiting the possibility for D. suzukii to reinvade crops generation after generation. Drosophila species are attacked by more than 40 different species of parasitoid wasps that locally may control up to 80% of the fly population. These wasps are laying their eggs in fly larvae (larval parasitoids) or in pupae (pupal parasitoids), but all emerge from pupae.
While generalist European pupal parasitoids (such as Trichopria drosophilae) can successfully develop on D. suzukii (albeit they are not able to control its population in the fields), European larval parasitoids do not naturally parasitize D. suzukii in the field. In lab studies they either do not show interest in D. suzukii larva or parasitize it but the eggs are destroyed by the fly larva immunity. Only one species, Leptopilina heterotoma, very occasionally succeeds to overcome the immune defence in laboratory experiments. An experiment was set up to assess whether the ability to attack and develop on D. suzukii was different between strains or could be increased and, thus, was probably genetically based. Seven strains of L. heterotoma were collected in Switzerland and compared in their ability to (1) attack and (2) successfully develop on D. suzukii. Significant differences were found in the attack rates and success rates. The most successful strains showed average success rates of 9% and the least successful 1%. The best strains were also reared alternatively on D. melanogaster and D. suzukii and the progenies tested for increase in parasitism success. Complementary studies, including semi-natural conditions tests, should tell us if European larval parasitoids of Drosophila spp. can become adapted to be efficient enough to control of D. suzukii.
Asian parasitoid species were collected in China and Japan in 2015-2017 (see Task 2.3) and are presently being reared in quarantine in Switzerland, France and China. Several strains of the braconid Asobara japonica, the figitid Leptopilina japonica and the figitid Ganaspis cf. brasiliensis are have been studied in various ways. The Asian surveys showed that G. cf. brasiliensis is the most frequent parasitoid of D. suzukii and is specialised in parasitizing drosophilids in fruits. This preference was retrieved in laboratory conditions since G. cf. brasiliensis oviposited in D. suzukii larvae almost exclusively in fruits. These parasitoids species and strains were also tested in a similar manner on five European Drosophila spp.: D. melanogaster, D. immigrans, D. subobscura, D. hydei and D. busckii, as well as on the tephritid fly Ceratitis capitata. Asobara japonica and L. japonica were tested with Drosophila spp. on regular Drosophila diet. However, since G. cf. brasiliensis does not successfully parasitize D. suzukii in the regular diet and the European Drosophila spp. cannot be reared on fresh fruit, a compromise had to be found. After various tests, it was found that both the parasitoid and the Drosophila spp. would accept a mixture of the diet with a high proportion of fresh fruits. Thus, the tests with G. cf. brasiliensis included a treatment on the fruit diet with all five Drosophila spp. and also a treatment with D. suzukii in fruits. The results of the specificity tests were as follows: Asobara japonica attacked and developed in all five Drosophila spp. Leptopilina japonica successfully and abundantly developed on D. suzukii and D. melanogaster, individuals emerged from D. immigrans and D. subobscura and none from D. busckii and D. hydei. Interestingly, one G. cf. brasiliensis strain from Japan showed a total specificity for D. suzukii on fruits whereas other strains successfully attacked D. melanogaster. Thus, so far, among Asian parasitoids, the specificity tests revealed polyphagous Asian species (A. japonica, L. japonica) and confirm the specificity of G. brasiliensis, with some variations in the specificity level between strains. Similar observations were obtained in olfactometer tests. These ecotypes of G. cf. brasiliensis with different behaviour may indicate sibling species since the molecular analyses also showed important genetic variations between individuals. Ganaspis cf. brasiliensis is thus the most promising biological control agent of D. suzukii but the following aspects still needs to be further investigated to fulfil the regulatory requirements before a possible release in the fields: completion of the specificity tests; climatic requirements; existence of sibling species or ecotypes varying in their specificity and climatic suitability; mechanisms inducing specificity, e.g. chemical attraction.
Chemical control
In this task, a range of compounds appropriate for use on fruit in Europe at the beginning of the program were assessed for efficacy against D. suzukii including already commercialized insecticides as well as novel materials. Insecticides with different modes of action and routes to target were examined including those with direct contact toxicity towards adults and larvae. Considering that the use of insecticides for the control of SWD can be critical since it damages ripening fruits, only those with a favorable toxicological profile were considered, and analysis on treated commodity were performed. First of all, standard laboratory test procedures were used for a large group of products, after screening, products with the best performance were tested in open field conditions in different countries.
The results strongly support the earlier findings that pyrethroids, organophosphates and spinosyns provide controls from 5 to 14 days after treatment (DAT), showing an excellent activity concerning adult mortality, eggs laying and hatching, female fecundity, larvae and pupae development and adult emerging. Neonicotinoids caused somewhat moderate impact for managing D. suzukii providing 1 to 3 days of control. Natural pyrethrum exhibited a moderate mortality especially at 2 and 3 DAT.
Results from open field trials carried out on Cherry, Grapevine and Strawberry in Italy and in France suggest:
• on Cherry, to maintain a protection throughout the harvest period, four applications with insecticides belonging different mode-of-action groups are strongly recommended, provided that the first application should be applied when monitoring traps indicate the presence of D. suzukii populations.
• on Grape 1 to 2 applications are necessary depending upon the penetration resistance of the berries.
• on Strawberry 3 to 6 applications are necessary every 3 to 7 days between the fruits collection
Due to the fact that the applications should be made near the harvest date, residues on fruits may be present even if they are below the MRL (maximum residue level admitted), representing a problem for export. Rotation and diversity in applying of insecticides with different and unique modes of action are greatly necessary to combat or at least delay widespread insecticide resistance.
Sterile insect techniques to reduce D. suzukii populations
The release of insects carrying ‘self-limiting’ genetic traits that induce mortality to some or all progeny at an immature life-stage, has been demonstrated as an effective management tool for specific insect pests across both agricultural and public health sectors. This female-specific self-limiting approach allows for a male-only release cohort, that has been previously shown to increase the productivity of mating-based insect pest management programs and is not detrimental to agriculture. Mating between self-limiting males and wild females produces no viable female offspring, thereby decreasing the reproductive potential of the wild population. If sufficient numbers of wild females’ mate with self-limiting males over time, then the pest population will decline. Oxitec has developed and optimized a conditional self-limiting technology in D. suzukii that enables genetic sexing of genetically engineered insects based on the tetracycline-off system and the tetracycline transactivator (tTAV) gene. A fluorescent protein marker gene is linked to the self-limiting trait and serves as a tool for visual monitoring of the genetically engineered flies. A number of strains displaying the correct expression of the self-limiting trait have been obtained; i.e. absence of females when larvae were reared without the addition of the antidote in the larval medium (off-tet) and a 1:1 (female: male) ratio on larvae reared in larval medium with tetracycline (on-tet). However, none of these strains could be made homozygous may be because that basal tTAV levels cause an increased mortality in larvae that contain two copies of the transgene. In order to develop a homozygous male-selecting strain, redesign of the tetracycline repressible self-limiting trait has been carried out and new transgenic lines with different construct configuration to minimize larval mortality are being produced and will be tested under controlled condition for their ability to decrease the reproductive potential of D. suzukii.
Potential of new mode of action compounds as alternative solutions in D. suzukii control
Two approaches were investigated as alternatives solutions for D. suzukii control which targeted essential components of the fly’s physiology and/or behaviour. The expression of specific genes was down-regulated by RNA interference using double-stranded RNA (dsRNA) and G protein-coupled receptors (GPCRs) were characterised to develop screening assays for pesticide discovery. New microencapsulation methods for stabilising and delivering dsRNA and receptor agonists/antagonists were investigated.
As a first step in identifying insecticidal targets, the neuropeptide and GPCR precursors from the D. suzukii genome database and the corresponding peptides present in the central nervous system of the fly were identified. Selected targets included the hormones and receptors involved in osmoregulation (diuretic hormone) feeding and gut motility (myosuppressin) and reproductive behaviour (sex peptide).
A range of compounds were tested on functional receptor assays revealing receptor specificity in that they were activated by the native ligands and some synthetic analogues, but not by mammalian peptide orthologues. This work has provided an understanding of the ligand requirements for each of the D. suzukii receptors investigated, enhancing the prospects of developing novel insecticidal molecules for use against D. suzukii.
Microcapsules, which provide protection upon application and ingestion, have been developed which selectively release insecticidal bio-molecules in response to the alkali pH of the insect. Furthermore, dsRNA, targeting V-ATPases in the fly gut, have been complexed with polymers and shown to stabilise the dsRNA. Significant pupal mortality occurred when D. suzukii larvae were fed with complexed dsRNA, whereas naked dsRNA was ineffective. The complexed dsRNA had no significant effect D. melanogaster, demonstrating that this method could provide species specific control.
Work package 5 – Effective and innovative solutions for quarantine fruit crop disease control
Aims and objectives
P. syringae pv. actinidiae (Psa), X. arboricola pv. pruni (Xap), and X. fragariae (Xf) are quarantine pathogens in the EU that seriously limit kiwifruit, peach-almond, cherry and strawberry production. There are no existing authorized chemical compounds, nor a suitable strategy for efficient disease control in the EU. Thus, novel formulations and delivery technologies are greatly needed. Project DROPSA deals with confirming the efficacy of existing commercial products, develop novel biopesticides and physical or plant-defence stimulation approaches for disease control.
Results and discussion
Existing compounds and products
Existing compounds and products were evaluated under controlled environment greenhouse conditions and potted host plants (12 products, Xap on peach), and in the field in four regions where kiwifruit is cultivated (30 products, Psa on kiwifruit). Only a few existing compounds were sufficiently effective to manage the three diseases, and this effect was greatly dependent on the pathogen-host system. Thus, it was evident that there is a need of research on new compounds.
Novel antimicrobial compounds
Novel antimicrobial compounds were identified and tested. Among 38 synthetic antimicrobial peptides studied, the linear undecapeptides BP77 and BP100, the D-amino acid derivative of BP100 BP157, the magainin-melittin enlarged BP100 derivatives BP208, BP211 and BP178, and the BPC192 and BPC194 cyclic decapeptides, were the most active against Psa, Xap and Xf. Peptide BP100 (H-KKLFKKILKYL-NH2), was the best lead because it is small size peptide with linear structure and contains proteinogenous aminoacids, with a good antibacterial activity, low toxicity and a relatively low effort of synthesis. BP100 performed well in the in planta assays at doses between 100-200 microM (0,225 g/L) for controlling kiwifruit, peach and strawberry pathogen infections. The effective dose of BP100 was similar to the recommended for streptomycin, kasugamycin or tetracycline in the countries where antibiotics are authorized for plant bacterial disease control. However, the cost of synthesis is too high for field use and BP100 will need additional efforts to decrease costs of production (not a DROPSA objective). The peptide fulfils the recent regulation on low risk plant protection products in the EU.
Novel biological control agents
Leaf- and the flower-associated bacterial biota of two kiwifruit species were analyzed in healthy and Psa-infected plants using a metagenomic approach. Some non-pathogenic species with a potential interest as biocontrol agents were detected. In non-diseased plants Pseudomonas spp. were the most abundant, and Lactobacillus was detectable in all samples. A total of around 700 isolates from a comprehensive bacteria collection from a wide range of plants including Bacillus spp. and lactic acid bacteria were characterized in relation to antimicrobial peptide (AMP) biosynthetic genes and multilocus sequence typing (MLST) analysis. Antibacterial activity of the putative BCAs was determined against the plant pathogenic bacteria Psa, Xap and Xf. Polyphasic and phylogenetic analysis permitted the selection of the most adequate BCAs. Selection of putative BCAs lead to B. amyloliquefaciens strains 2017, 2059, 2128, 2130 and 2151, and Lactobacillus plantarum TC92, PM411 and CC100, and Leuconostoc mesenteroides CM160 and CM209. Strains EPS2017, TC92 and PM411 were selected for further work. Fitness of BCAs was dependent on environmental conditions and host plant. The survival of B. amyloliquefaciens 2017 and L. plantarum PM411 and TC92 onto leaves of GF677 Prunus plants in the greenhouse was strongly dependent on relative humidity conditions. In in vivo trials, BX44 tagged with gfp reporter was able to colonize flowers of both A. deliciosa and A. chinensis leaves better than any other commercial BCAs. BX44 is able to compete with Psa for the same substrates. A combination of methods based on qPCR (total cells), and dead and alive cells (PMAX-qPCR) have been developed for quantitative molecular monitoring of L. plantarum PM411. These approaches provide with robust tools for monitoring viability of the strains in the field to study the best formulations and conditions of application, host and pathogen range. Greenhouse and semi-field experiments for disease control concluded that the strains Lactobacillus plantarum TC92/PM411 and Bacillus amyloliquefaciens EPS2017 were gobally effective (40-70% efficacy) for control of Psa in kiwifruit plants (cv. Hayward), Xap in Prunus GF677 plants and Xf in strawberry cv. Darselect, and did not differ significantly from reference products BlossomProtect and Serenade. Overall, two novel strains of L. plantarum (TC92 and PM411), one strain of B. amyloliquefaciens EPS2017, and a strain of Pantoea vagans BX44 were candidates for use in field programs for disease control of Xap, Xf and Psa, and to develop novel microbial pesticides.
Manipulation of plant metabolism and physical methods for disease control
Azybenzolar-S-methyl (ASM) resulted to be the most effective and reliable resistance inducer, by both root and leaf treatment, and with a prolonged effect over time, but may cause phytotoxicity at high treatment rates. Infrared radiation system does work in reducing the bacterial population, although possibly not in eradicating it, but the general health status of the plant may influence its tolerance to the heat treatment. Ozone has an antimicrobial effect on the survival of Psa in kiwifruit pollen, but unfortunately, the treatments significantly reduced also pollen viability.
Innovative formulations
Innovative formulations were developed by improving the fitness of the BCAs, testing suitable growth media for scale-up production and dehydration processes for formulation. Physiological improvement and nutritional enhancement of strains and formulations was successful. In Bacillus, maltose used by Bacillus EPS2017 and not by the plant pathogenic bacteria, and the best combination to adapt cells to hyperosmotic conditions is PM medium amended with NaCl and with several osmolytes. In L. plantarum PM411 and TC92 acidic and hyperosmotic pre-treatments during growth lead to a better stress tolerance, and induced stress and defence related genes. Thus, it is possible to increase the fitness of the BCA strains by using fermentation conditions including stress stages, with a better consistency of disease control.
Growth medium for scale-up processes and mass production was improved in Bacillus by different combinations of PM with molasses and soybean meal, including bioreactor performance providing optimum amounts of metabolites with antibacterial properties. In L. plantarum the medium MRS was also improved, but in a less extent. The shelf life of different Bacillus products obtained by lyophilization, from a set of mixtures was maximum with the combination of osmoadaptation with several osmoprotectants. Several formulations of L. plantarum strains TC92 and PM411 were prepared based on freeze drying, spray drying, and encapsulation. The combination of antimicrobial peptides and L. plantarum strains, and of copper bactericides with plant defence elicitors were compatible to increase the range of effective strategies for disease control. Finally, a novel approach to efficiently deliver the antimicrobial peptide BP100 to plants was evaluated with success by endotherapy in kiwifruit against Psa, and in peach against Xap, which decreases the need of high costly doses of the expensive compound, and reduces environmental impact.
Work Package 6 – Practical solutions for control
Aims and objectives
To develop innovative, practical, preventive and integrated control solutions for quarantine fruit pests and diseases, utilising current control strategies and those developed in WP 2, 3, 4 and 5, with an emphasis on the prevention of D. suzukii and diseases that infect crops. Integrated strategies (IPM) for the major economic fruit crops in Europe will be combined, concentrating on kiwifruit, strawberry, peach, cherry and grapes. This will involve ex-ante economic assessments from WP 7 and the pathways and impact analyses developed in WP 1.
Risk mapping for D. suzukii
Risk maps built in DROPSA were based on climatic requirements of D. suzukii as determined in Task 2.1 and in the previous literature on climatic requirements. Due to its broad host range (revealed by Task 2.2) the risk and impact of D. suzukii is probably more related to climatic suitability than to host distribution. Therefore, the study focused on climatic parameters only.
Due to its short development time, D. suzukii can build very high densities, even when winter mortality is close to 100%. As a result, in spite of the narrow climatic range, the pest is found in most of Europe. For these reasons, the model was based largely on pest occurrence and damage data (Deliverables D2.1 and D2.2 and literature search). The model was validated by comparing model output to reported distributions, seasonal phenology and tested for consistency with empirical data.
Damage prediction values for Europe correspond well to the present situation, with heavy damage in Western/Central Europe, and lower risk associated to hotter/drier areas or the occurrence of cold stress. The model will be improved to better fit with data from Asia and North America, further considering climate change (particularly for water availability) and including field observation in addition to lab data.
Role of cultural management
Cultural practices (e.g. clean harvest, wild fruit removal, netting) were developed in field conditions to limit abundance of, and damage by D. suzukii (WP 2).
The effect of pollination, irrigation, fertilization, use of bio-regulators and pruning on the incidence and epidemiology of Psa (WP 3) was evaluated in small-scale and field experiments.
The use of insect-proof nets covering fruit bearing trees or shrubs is an effective method to physically exclude D. suzukii from cherry, grapevine and blueberry plants, preventing pest damage. Downsides are the high initial costs and difficulty of orchard management. Reducing cool-humid microhabitats (by pruning, mulching, mowing) and removing unharvested fruit further limit the pest population.
As for Psa, risks of contamination are associated to irrigation water, pruning instruments, grafting and pollen. Water stresses and auxin treatments may increase disease severity. Orchard management practices (training, pruning, covers) can be implemented to facilitate spray treatment efficacy and limit leaf wetness, which is one of the main factors promoting the establishment of the pathogen. Cytokinin treatments may induce a moderate resistance. Traits of resistance to P. syringae pv. actinidiae are explored in the Actinida genus (notably in A. arguta) for the development of rootstocks. Fruit from diseased plants was smaller and more perishable.
Development and validation of decision support and forecasting systems
The knowledge acquired about biology and ecology of the pests and pathogens (WP2 and WP3), the management options identified in WPs 4 and 5, and the economic aspects of plant protection (WP7) were integrated in a decision support system (DSS). This tool, by the specification of indicators and the opinion of experts, encourages complete and careful consideration of individual measures.
A DSS evaluation tool was developed to assess the performance of current (“Baseline”) pest management practice against the packages proposed and tested in DROPSA field trials. For Psa, the DROPSA package is predicted to be more favourable than the current management system, with an estimated overall benefit of €27.75 per hectare when using the DROPSA package compared with the Baseline package.
By reducing the loss of production, the DROPSA package is also strongly favoured in case of high D. suzukii pressure sites, while a lower utility is generated by lower infestation levels, because of the conspicuous costs and management efforts inherent to nettings.
Integrated pest management strategies
The results obtained in WP4 and 5 and in Tasks 6.2 and 6.3 have been integrated to develop orchard IPM strategies based on the most reliable and effective combinations of synthetic and natural pesticides, biological control agents, semiochemical-based techniques and cultural methods.
IPM strategies for Psa were implemented in a demonstration orchard, incorporating copper application and treatment with resistance inducer (ASM). In addition, biological control agents (BCA) selected in WP 5 were applied at blooming, and part of the experimental plot was under plastic cover. The IPM strategies significantly reduced bacterial canker incidence, particularly in the plastic-covered parcel. Overall, the combined use of proper management, resistance inducers and BCAs, on one hand, can provide an effective protection against Psa, on the other hand, allow a substantial limitation of copper (thus meeting the current EU regulations) and other chemical pesticides, particularly in phenological stages, such as flowering or pre-harvest, in which copper application may result in phytotoxic effects or residues on fruit. However, further testing is required to ascertain the effects of weather and local climate conditions on the IPM strategies.
IPM strategies for D. suzukii were investigated in cherry, blueberry and raspberry orchards. The control methods derived from WP 4 and included pruning, fruit removal and mass trapping, along with a rational use of chemicals of synthetic and natural origin. The impact on non-target organisms (bees, butterflies, snails and earthworms) was also monitored. Preventing a wet-cool microclimate in the canopy (by pruning or mulching, which also enhanced the penetration of spray treatments), and removing non-harvested fruit was useful to control pest infestation. Larvae surviving within the removed fruit were effectively killed by solarisation. In addition, traps were designed for a Lure & Kill control strategy with an extended lifespan (6 weeks), to minimise servicing by the grower. However, the control efficacy depended on the trap positioning, decreasing at the borders of the plot, as a result of pest immigration from outside. Overall, the use of IPM strategies for the control of D. suzukii is envisaged to limit the use of insecticides, which may have adverse effects on the environment and crop quality, may lead to the selection of resistant insects, and do not prevent pest infestation, due to its wide host range, rapid development rate and high dispersal ability. Lure & Kill devices can be useful in combination with other measures to reduce the initial population of D. suzukii and to retard its infestation peak.
Results have been, and will continue to be, made available to growers via informative websites, advisory services and field demonstration.
Work package 7 – Economic analysis
Aims and objectives
The aim was to provide an economic analysis of the prevention and control strategies developed in the WP 2, 3, 4 and 5, and their implementation in practical solutions and IPM strategies (WP 6). This was achieved by determining the economic viability of proposed preventive and control strategies using ex-ante analyses of critical success factors with regards to costs and returns, and evaluating the economic opportunities for fruit growers of the innovative solutions and practical strategies developed by DROPSA as alternative measures of IPM prevention and crop protection using ex-post analysis.
Economic viability: ex-ante analysis of proposed alternatives
Ex-ante analyses of critical success factors with regards to costs and returns to determine the economic viability of proposed preventive and control strategies. The conditions for the improvement and implementation of the alternatives were identified and used as research guide in the other work packages (WP 4, 5 and 6) for the improvement and development of successful alternative strategies.
This task was carried out at the beginning of the project as research guide for the other work packages in order to develop management strategies that are also sustainable in an economic view.
At the start of the project, the following ex-ante calculations were made in order to provide critical factors for the further development of the proposed strategies:
• Sterile insects against D. suzukii in Cherries (one country)
• Netting against D. suzukii in Cherries (one country)
• Use of pesticides against D. Suzuki in Strawberry (four countries)
• Covers against Psa in Kiwi (one country)
As these ex-ante calculations are preliminary, many data (costs and effects) of proposed techniques were estimations. However, these calculations give critical factors for the further development of the innovations. More specific were the concluding remarks about the critical success factors:
• The use of sterile insects seems a possible solution, but the final economic outcome depends on the area to be treated, the period of treatment, the amount of sterile insects needed and of course the reduction of the damage by D. suzukii. It is clear that a percentage loss in yield has more (negative) impact on the marginal gross margin than a percentage loss in picking and grading speed.
• The ex-ante analysis for sterile insects is done for Cherries. The work in the DROPSA project should make clear what the real data are for Cherries, as well as for other crops. In other crops like Raspberry, the period during which fruits are attractive for D. suzukii is (much) longer. Perhaps in the ex-post economic calculations several measurements need to be combined.
• For the calculations of the use of netting, trials need to make clear which systems are most effective, which amount of labour is required, and how it is working in practice as the nets have to be opened regularly for people and machinery.
• The life-span of the netting is not known yet, but firmly affects the costs of this measure. The life-span of the nets is something to be varied in the ex-post calculations as the period of the DROPSA project will presumably be shorter than the expected life span of the nets.
• Spraying pesticides: economic viability seems positive, but due to differences in growing systems of Strawberry, varies much between countries.
• Plastic covers against bacterial infections: trials, like for netting, need to make clear which systems are best working, which amount of labour is required, and how it is working in practice at which effect on yield and quality.
Economic viability: ex-post analysis of new developed strategies
For the ex-post calculations relevant topics and crops were selected, based on the ex-ante calculations but also on the developments in the different work packages. For the data collection of these topics and crops, two templates are made:
• The A-form is meant for the initial information about the relevant crops. This information is required for the calculations of the costs and returns of the crops in a situation without infestations of D. suzukii or diseases of pathogens. It concerns economic and technical data of the fruit crops as yield, labour (costs), prices etc.
• The B-form is meant for the specific data about the consequences of D. suzukii or pathogen in the crops. Two situations must be described, completed by data:
o The first situation shows the effects with infestations of D. suzukii or diseases of pathogens, but without new DROPSA-strategies, on economic and technical data, as yield, labour, investments, use of materials etc.
o The second situation shows the effect of the new strategy on economic and technical data.
For both forms a filled example is made to show the technical researchers what is needed. The forms as well as the first data are discussed with individual researchers of other WPs in order to provide the best situation for data collection during the coming season.
New calculations were made based on the achieved results and the collected data of the work in the WPs 2-6. Concluding remarks of these calculations:
The invasive pest D. suzukii causes a lot of economic damage to individual growers, several strategies were tested to diminish the damage.
• The strategy of the use of Attract and Kill D. suzukii showed an effective population reduction but not enough to avoid damage when populations are already established.
• The strategy to use extra chemical control, specific for D. suzukii, showed in The Netherlands in sweet cherry an average yield reduction of 10-15%, compared to the situation before D. suzukii and therefore no damage or loss of production. The decrease in damage, due to the chemical control, leads to a higher marginal gross margin (mgm) despite the extra costs for the chemical control.
• The strategy of clean harvesting in combination with other measurements is effective, but the data for an economic evaluation are not sufficient at the moment.
• The combination of side netting (in the case rain covers are standard) with bait sprays diminished the damage to nearly zero in a pilot in The Netherlands. The costs for the investment are between 11,000 and 17,000 €/ha. The year costs for this investment are lower than the increase in mgm.
• Netting of sweet cherry plantings in Italy might reduce the damage to zero. The planting system and the percentage damage before netting determine if the return on investment of the netting is high enough. However, single tree netting, is due to the high investment costs not economically sustainable.
• The effect of mass trapping in Italian sweet cherry plantings is not clear, but at the moment not economically sustainable.
• The measurements against D. suzukii in Blueberries in The Netherlands are complicated due to the long production season with different varieties. The use of extra chemical control as only measurement has only limited effects: on average still 30% yield reduction.
The pathogen Psa is negatively affecting the European kiwi fruit production. Several strategies were tested to diminish the damage.
• The yield reduction due to Psa can be decreased to zero by the use of covering/nets.
• Due to the relatively high investment costs this is at the moment not economic sustainable for green kiwifruit in Italy.
• For yellow kiwifruit in Italy this may be positive, but further work is needed to confirm this.
Potential Impact:
The joint dissemination, exploitation of project results and management of intellectual property is covered in DROPSA project consortium agreement, agreed by each partner organisation and the project management committee.
A work package was dedicated to dissemination with the main objective:
To undertake dissemination actions that enables the partners to maximise the commercialisation of the project results and to plan for post project exploitation. During the project lifetime this will include the knowledge transfer of results to all interested stakeholders (industry and governmental) through the website, e-Newsletters, publications and presentations at scientific meetings, project workshops and other events, and the exploitation of the results and outcomes of the project.
This WP also aimed to maximise the project’s visibility, create a distinctive project identity, spread information on its objectives, activities and achievements, and facilitate commercial exploitation of the research output.
All partners contributed additional resource to ensure maximum impact and members of the DROPSA consortium facilitated dissemination to all stakeholders. The latest guidance from the EC was used on how to maximise the impact of research, development and demonstration, to ensure that the results are effectively used, through their exploitation and dissemination.
Project website
1. A project website was created (http://dropsaproject.eu/) which is being used to inform the public of progress and activities in the project. Links to documents and other relevant materials are available on the website.
2. A database was created of >1600 stakeholders from industry and end-users from European countries. A link on the website also allows the public, companies and others to register their interest in DROPSA and to receive publicity materials. A mailing list of these stakeholders has also been created for electronic distribution of publicity material.
Dissemination of project results
Scientific and practical dissemination has been achieved through publications and DROPSA partners’ attendance at conferences and workshops. The consortium partners have been a very proactive group and have attended and participated in a wide range of conferences, workshops and promotional activities.
The main dissemination activities were:
• Project brochure and four annual newsletters to introduce and give updates on the project, which were distributed electronically to registered stakeholders and available via the website
• Conferences - overviews of the DROPSA-project and scientific results were presented, either as a poster or oral presentation at:
o International conferences and symposia (> 60 oral and poster presentations).
o National conferences and meetings (approximately 45 oral and poster presentations).
• Peer-reviewed Scientific publications. A variety of publications (approximately 60 publications to date) have been produced during the time frame of DROPSA (listed above in the publications section). It is anticipated that further publications will include data from this project. These will be listed on the DROPSA website or made available if open access.
• Future funding opportunities: continued funding for research undertaken in DROPSA will be sought by individual partners. Potential opportunities would be via Euphresco or Eranet funding, as well as from individual partner governments.
• Lectures, post-graduate courses, workshops, training and knowledge exchange (approximately 42 meetings).
• Publications in national (growers) magazines and websites (>4 0 publications and 26 websites) to inform growers, plant health inspectors and NPPOs.
• Interaction COST FA1104, Sustainable Production of High-Quality Cherries for the European Market (3 interactions).
• Stakeholder workshops for training and dissemination of project results and outcomes. At each stakeholder event local interested parties were invited to attend and were able to the network with the project partners. Experts from the field of industry/growers/plant protection officers, were also in attendance to provide feedback from an industry perspective.
o DROPSA Stakeholder Workshop 2016: Strategies to develop effective, innovative and practical approaches to protect major European fruit crops from pests and pathogens, Girona, Spain 10th March 2016.
o Stakeholder (technical) meetings and field visits involving growers and technicians (approximately 70 meetings and field visits).
• Collaboration with external projects/organisations (2).
• Scientific and public stakeholder meetings (approximately 11 meetings).
A final DROPSA dissemination conference was organised. At the annual International Soft Fruit Conference (ISFC) in Den Bosch (The Netherlands), the European members of the Dropsa project presented their findings to stakeholders. In total 760 people from 32 nationalities, 20 presenters and 98 companies from 13 countries were present at the ISFC.
International Soft Fruit Conference – Thursday 11 January 2018 (http://www.softfruitconference.com/).
DROPSA participated with 8 oral presentations and 10 poster presentations:
1. Oral presentations
• Introduction to DROPSA: Strategies to develop effective, innovative and practical approaches to protect major European fruit crops from pests and pathogens.
Neil Audsley, Fera, UK.
• Integrated pest management of Drosophila suzukii.
Nicola Mori, University of Padova, Italy.
• Biocontrol of Drosophila suzukii.
Marc Kenis, CABI, Switzerland.
• Spotless strawberry plants: how to keep them free from Xanthomonas?
Jan M. van der Wolf, Wageningen University & Research, The Netherlands.
• Managing pathways of pest and disease introduction.
John Mumford, Imperial College London, UK.
• Toxic baits for the control of Drosophila suzukii.
Herman Helsen, Wageningen University and Research, The Netherlands.
• Pesticide-free crop protection with self-limiting fruit flies.
Carlos Pedraz, Oxitec Ltd, UK.
• Comparison of traps and attractants for Drosophila suzukii monitoring in North-East Italy. Lorenzo Tonina, University of Padua, Italy.
2. Poster presentations
• Strategies to develop effective, innovative and practical approaches to protect major European fruit crops from pests and pathogens. DROPSA - Partners.
• Comparison of two integrated management plans for kiwi crops against Pseudomonas syringae pv. actinidiae using a Decision Support System. Olliff L, Humphreys A, Leach A, Holt J, Mumford J. Imperial College London, UK.
• Assessment of IPM packages for Drosophila suzukii – A Decision Support System. Humphreys A, Olliff L, Leach A, Holt J, Mumford J. Imperial College London, UK.
• Identification of pests likely to be imported with fruit to Europe. Suffert M, European and Mediterranean Plant Protection Organization, France.
• Insect netting as a vital component of a Drosophila suzukii control strategy. Helsen H, Van der Sluis B, Wageningen University & Research, The Netherlands.
• A diagnostic tool for improved detection of Xanthomonas fragariae using a rapid and highly specific LAMP assay designed with comparative genomics. Gétaz M, Bühlmann A, Schneeberger PHH, Van Malderghem C, Duffy B, Maes M, Pothier JF, Cottyn B. Zürich University of Applied Sciences, Switzerland.
• Lure and kill devices against Drosophila suzukii: first field trials. Navarro-Llopis V, Primo J, Sanchis J, Barroso JM, Riba JM, Navarro I, Fratantuono M, Alvarado P, Vacas S. Universidad Politècnica de València and Endoterapia Vegetal, Spain.
• Identification of pests likely to be imported with fruit to Europe. Suffert M, Steffen K, Wilstermann A, Schrader G, Grousset F. EPPO, France and Julius Kühn-Institut, Germany.
• New mode of action compounds for the control of Drosophila suzukii. Down R, Matthews J, Audsley N. Fera, UK.
• Drosophila suzukii spillover from natural habitats to vineyards and cherry orchards. Tonina L, Marini L, Sancassani M, Marchesini E, Mori N. University of Padova, Italy.
Dr. Francesco Spinelli (Department of Agricultural and Food Sciences - University of Bologna) organized a presentation during the kiwifruit day for stakeholders (growers, advisors, industry) by the Italian Society for Horticultural Sciences (1st February 2018). The presentation focussed on bacterial diseases (especially Pseudomonas syringae pv. actinidiae), which were not represented at the meeting in Den Bosch.
The presentations are available on the DROPSA-website: http://dropsaproject.eu/
Potential impacts and wider societal implications
Estimates of potential pest impacts and routes of introduction and spread add to the body of work on impact of invasive pests and diseases species and support EU phytosanitary regulations and EPPO alert lists so as to reduce the likelihood and frequency of such invasions occurring in future.
Fruit crops account for around 17% of the value of the EU’s agricultural production and the fruit and vegetables supply chain has an estimated turnover of more than €120 billion with over 550,000 employees and around 1.4 million growers. Losses from pests and pathogens are estimated to account for at least €10 billion in revenue and 3 million tonnes of produce to the EU fruit industry. Despite measures to control D. suzukii and bacterial pathogens (Psa, Xf and Xap) they continue to spread. Implementing new detection and surveillance, control measures and integrated pest management strategies could save tens of millions of Euros and protect jobs and growers. The outcomes and impacts of DROPSA are described below:
WP1 - Pathways of introduction of fruit pests and pathogens
Genetic studies of D. suzukii and the spread models of Drosophila suzukii, Xanthomonas fragariae and Pseudomonas syringae provided valuable information on the introduction and spread of new pests of fruits in Europe. The commodity based risk analysis allows the identification many high-risk pests at an early stage by means of horizon scanning and to develop preventive measures before their introduction. A high number likely to be introduced into Europe with a high risk were identified. The prevention of their introduction will help to secure fruit production in Europe. The QPAFood model enables the identification of particularly risky countries of origin and import periods of the goods. Inspections and measures can be focused and therefore will efficiently and effectively reduce the risk to an acceptable level.
The process used to carry out the pathway risk analyses has been used as a basis to develop EPPO Standard PM 5/9 (EPPO, 2017).
DROPSA alert lists have been reviewed and discussed by experts of the NPPOs in the framework of EPPO (Panel on Phytosanitary Measures 2017-03 and 2017-10). The Datasheets of all alert list species are already available on the EPPO Global Database and some additional species will be added to the EPPO Alert List in 2017-2018 to raise awareness.
The identification of the pests may result in the adaptation of legislation such as for Thaumatotibia leucotreta which was added to Section I of part A Annex I to Directive 2000/29/EC within period of the DROPSA project.
WP2 Biology and Ecology of Drosophila suzukii
• A set of climatic requirement data and development parameters, which can support the building up of climate models and risk maps
• The first data on the biology and ecology of D. suzukii in China, e.g. on its population dynamics and reproductive diapause, which can be compared to those observed in Europe.
• A better understanding of the capacity of the fly to colonise mountainous areas.
• A set of unique data on the circadian cycle of adults D. suzukii fly, which allow to make important observations to calibrate control methods.
• First detailed information on non-crop hosts in China and the most extensive study on non-crop hosts in Europe. Based on these data, management methods can be suggested. E.g. ornamental and hedge plants in the vicinity of fruit crops and orchards can be selected according to their susceptibility to D. suzukii. However, the widespread availability and abundance of non-crop hosts and the lack of efficient native parasitoids suggest the need for an area wide control approach.
• Better information on variations in susceptibilities between varieties and cultivars of grapes and strawberries, two important fruit crops that are only partially susceptible to D. suzukii.
• The most extensive studies on spatial ecology of D. suzukii and interactions between crop and non-crop habitats. These studies are very valuable for developing management methods. For example, the spillover study described in the project results can contribute to help developing physical or mechanical control measures such as the modification of the spatial configuration of the orchards and surrounding native vegetation or the possibility of application of insect-proof nets.
• For the first time, a large survey for parasitoids of D. suzukii was carried out in China. They revealed the same main parasitoids as those previously collected in Japan by our DROPSA collaborator at University of Hokkaido, i.e. Leptopilina japonica and Ganaspis cf. brasiliensis. The latter species is now being studied under quarantine conditions, to assess its suitability for classical biological control.
• Several volatile compounds extracted from fruits induced reaction and/or were found to be attractive for D. suzukii flies.
• The observation that, without strong winds, D. suzukii probably does not spread more than a few hundred meters, which has implications for the development of control strategies
WP3.- Biology and epidemiology of quarantine fruit crop diseases
• Diagnostic tools based on LAMP technology for the bacterial pathogens have been developed. There were initially plans to commercialize these assays as a ready to use kit with the non-DROPSA partner SME OptiGene Ltd. (Horsham, UK) but those were not pursued mainly due to the need to disclose the primer sequences for publications. If required these assays could however be easily developed into ready to use commercial kits.
• Future plans to exploit the CRISPR typing data concerning X. fragariae with the SME Beamedex SAS (Orsay, France) in order to develop a spoligotyping tool for plant pathogenic bacteria.
• The knowledge developed concerning RNA-seq of Actinidia-Psa interaction will be applied in the Italian project “Biotech” (2018-2021) with the aim to use key genes involved in the interaction for genome editing in Actinidia in order to obtain tolerant genotypes.
WP4.- Effective and innovative solutions to control Drosophila suzukii
• Determination of the best commercial trap device and attractant lure for mass trapping and monitoring. Indication for the best use of this type of traps both temporally and specially.
• Development of a new type of device for lure and kill strategies that could decrease the need for conventional broad-spectrum foliar pesticides, reduce costs and environmental impacts.
• The demonstration of the efficiency of different commercial biocontrol agents (entomopathogenic fungi and nematodes) and arthropods and the development of a lure and infect strategy based on entomopathogenic fungi that can be included in IPM strategies to help to control the pest at low levels of infestation.
• Selection among the Asian parasitoids of Ganaspis sp brasiliensis as the best candidate for introduction in Europe that could control the pest both in the cultivated and wild areas. Future work will continue on risk assessment and licence application for the introduction of the best candidate exotic parastioid.
• Assessment of the effectiveness of different commercial pesticides and development of field strategies for the best crop protection with lower pesticide residues in harvested fruits. Some of these pesticides may be used in concert with BCA (such as fungi) while other may have side effects particularly on parasitoids.
• Oxitiec Ltd has developed and optimized of conditional self-limiting technology in D. suzukii for the production of sterile males which may be a solution to control this pest in the future. Further product development for commercial registration, including scale-up production will continue.
• Receptor screening assays have been developed for pesticide discovery, further funding will be sought to continue this work, which is transferable to other pests.
• Novel delivery mechanisms for insecticidal biomolecules and dsRNA have been developed which can be developed further for other applications.
WP5. Effective and innovative solutions for quarantine fruit crop disease control
• Three strains of microorganisms have been developed by UdG as microbial biopesticides against Psa, Xap and Xf. Strains PM411 and TC92 of Lactobacillus plantarum were the object of an agreement between, Anti Microbial Peptide Biotechnologies S.L. (receptor of a license from the owner of the technology, the University of Girona) and Agrifutur s.r.l. for further development and commercial exploitation. A further agreement has been signed between Agrifutur s.r.l. and the University of Bologna to perform field testing with the LAB strains.
• A patent extension from the original application, prior to DROPSA, has been extended to EU, USA and Brasil (EP14798394.4).
• The strain EPS2017 of Bacillus amyloliquefaciens, that was developed by the University of Girona (inventors) for Industrias Químicas del Vallés S.A. (IQV Agro; owners) has resulted in an EU patent (EP16711642.5).
• The antimicrobial peptide BP100 (patent EP2017285A1) is being licensed to companies interested in their exploitation, through Anti Microbial Peptide Biotechnologies S.L. and the University of Girona.
• The strategy for Psa control in kiwifruit based on existing compounds (antibacterials and plant-defence elicitors) has been transferred to the kiwifruit growers in Italy, and will be extended to other growing areas.
• Endoterapia Vegetal have patented an improved injection device to apply microvolumes of antibacterial compounds into plants to protect against PSA and Xap.
WP 6 Practical solutions for control
• Development of new policy to prevent introduction of D. suzukii in new uninvaded area.
• Tailoring monitoring inputs according to the possible paths of diffusion and risk map
• Development of innovative and sustainable cultural management tools creating a preventive control of D. suzukii and P. syringae pv. actinidiae maintaining, at the same time, high production efficiency and economic turnover of the fruit industry
• Reduce pesticide inputs by green, ready to use, green-technologies such as netting, curative pruning and physical methods
• Increase orchard gate revenues: the new cultural management tools generally have higher costs and need an increase in labour. However, the higher costs are outclassed by the reduction of fruit losses and the increase in fruit marketable quality
• Development of DSS system to predict the sustainability of the new control strategies for D. suzukii and P. syringae pv. actinidiae
• Adaptation of the DSS system to other invasive pests and pathogen
• Estimate the long-term sustainability of control strategies for D. suzukii and P. syringae pv. actinidiae
• Achievement of a long term environmental, social and economic sustainable control strategies of the major invasive pests and pathogen threatening European horticulture
• Provide information to develop new GAP policy
• Provide scientifically-based advices to growers and stakeholders of the horticultural industry
• Produce fruit with excellent organoleptic and nutraceutical characteristics and zero chemical contaminant
• Increase the consumption of safe fruit with potential benefits on consumers’ health
WP7 – Economic analysis
• Evaluation of the economic opportunities for fruit growers of the management strategies developed by DROPSA as alternative measures of IPM prevention and crop protection, which can be transferred to other pest and disease scenarios.
• The conditions required to improve and implement alternative strategies that can be used as a guide for the improvement and development of future successful alternative strategies.
List of Websites:
www.dropsaproject.eu
