The immune barriers of host insects represent an important strategy for defending themselves against parasite/predator and pathogen attack. However, many parasitoids and predators have evolved effective virulence strategies and host-regulation mechanisms to circumvent their hosts’ immune defence system e.g. some species produce potent toxins with strong and selective insecticidal activity. EcoStack has provided greater insights into the molecular mechanisms underlying these antagonistic interactions and provides a platform for the development of new bioinspired tools for pest control, mimicking insect suppression strategies existing in nature. The EcoStack project has identified new bioactive molecules, which are currently being used to develop new bioinsecticides. One such example is a venom peptide (from the predatory spider Segestria florentina; SFl6), which is a potent neurotoxin modulating the activity of voltage-gated channels, disrupting essential neuronal messages. Delivery of this neurotoxin to the site of action uses Bovine Serum Albumin (BSA) as a carrier molecule. BSA is known to enter the plant vascular system via roots and also to cross the insect gut thus delivering the SFl6 neurotoxin to the insect haemocoel and central nervous system. Production of this novel biopesticide has been scaled up by our industry partner, CPI, freeze-dried and shipped to UNEW where its efficacy against two target insect pests, the aphids Myzus persicae and Sitobion avenae has been confirmed. This material has been despatched to UNA for further toxicity studies (against lepidopteran sp) prior to biosafety studies against beneficial non-target organisms. The outcome will be the production of safe and effective biopesticides.

ALMaSS is a simulation system comprising a large number of fully interconnected sub-models. It has two key components, a model of the environment and models of people and organisms. The environment component is represented by a detailed map of a real area, selected from a number of countries in Europe where ALMaSS capability is established. This ‘Landscape Model’ is dynamic and of a high resolution, including aspects such as vegetation growth of crops and non-crop areas, field margins and other small habitats. The key feature of the landscape model is the farming model. This works by modelling each farm unit. Each model farmer carries out daily management of crops on all individual fields, affecting crop growth and also providing events that impact the organisms, including modelling pesticides and their environmental fate.

The organism and people models interact with the landscape and each other to create realistic patterns of behaviour and population changes. This provides a research/policy tool to evaluate the impact of changes in farm/crop management on the range of species modelled. Here we have a particular focus on ecosystem service providers of pollination and pest control.

Using the model to assess new ways to combine environmentally beneficial management without compromising productivity will provide evaluations of how to achieve targets (e.g. 50% pesticide reduction under the Green Deal), at the same time protecting or boosting, ecosystem service providers. As part of multi-criteria decision making process the simulation modelling will allow the testing of a range of ideas in silico, which would otherwise not be possible or practical in real life.

We have tested various plant species as candidates for sown flower margins specifically designed to enhance biological control and pollination ecosystems services in oilseed rape. We conducted this study in four different regional settings (UK, Serbia, Bulgaria & Spain) in order to tailor region-specific flower margins. Each team selected 20-25 local flowering plant species and established an experiment with 1 m2 flower plots; the abundance of pests, natural enemies and pollinators was monitored across the season. Results of this first year of the study will enable us to the refine flower species list and to further select those that promote the most pollinators and natural enemies but are least attractive to pests. Some species including Centaurea cyanus, Achillea millefolium, Phacelia tanacetifolia, Foeniculum vulgare and Coriandrum sativum are promising candidates based on preliminary results on the abundance of visiting pollinators. We further set up additional experiments in UK and Serbia with the goal to assess the impact of crop boundary features and local semi-natural habitats on biological control and pollination services in oilseed rape crops. Throughout the season we simultaneously monitored the abundance of pests, natural enemies and pollinators in crop fields and adjacent grasslands, woods and hedges to determine plant-insect dynamics between these two habitats and the effect on crop yield. Our aim is to better understand role of existing off-crop semi-natural habitats in maintaining ecosystem services of biological control and pollination, and to ultimately enhance them by implementing bespoke flower margins.

The regulations in the European Union for approval of plant-beneficial microorganisms and their corresponding products are currently scattered on several different regulatory frameworks. For instance, while antagonistic microorganisms used in plant pest and disease control are strictly regulated under the plant protection products (PPP) framework, microorganisms known to act through growth stimulation processes (‘plant biostimulants’) are generally covered by various (currently mostly national) regulations in the fertilizers area.

Based on an inventory of current regulatory policies, we formulated options for improved, more harmonized, regulations that can lead to more efficient approval of plant-beneficial microorganisms and increase the availability of microbial products to farmers in the future. These preliminary options were discussed with representatives of major regulatory bodies and interest organizations at an EcoStack workshop. Our informed recommendations for improved regulatory policy were presented in two journal publications.

In brief, a substantial body of evidence shows that the PPP framework has performed poorly for evaluating microorganisms. The assessments need stronger rooting in microbiological sciences, by revisions of the legislation and possibly establishment of units and personnel that are dedicated to microorganisms and microbial PPPs. We also recommend that in the longer run, policy makers and legislators should take steps to develop and implementat new, harmonized regulations that, possibly, cover all plant-beneficial microorganisms under one single framework.

Farmer field days (FFDs) provide the opportunity to relay complex information alongside seeing techniques and technology in practice; this is known to be an effective route for dissemination. 11 FFDs took place in 6 countries (2 in Germany, Finland, France, Italy, and UK; 1 in Spain) in the period 30 November 2020 - 5 July 2021. Due to Covid-19 restrictions, part of the field days were held in the form of online meetings hosted on institutional Zoom and Teams platforms. A considerable number of stakeholders (420) registered and participated to the events, including farmers, bee keepers, agriculture educators, extension service technicians, agronomists, researchers, business representatives, and policy makers. Upload of live power point presentations and video on YouTube and dissemination through social media guaranteed further spread of FFDs contents. Led and supervised by scientists of the consortium, FFDs allowed to disseminate, discuss and have practitioners' feedback on several EcoStack research topics; e.g., intraspecific and interspecific crop plant diversity for pest management and yield, measures to favour pollinators, and plant diversification in cereal crops.

To maintain high productivity in modern agriculture and secure long-term biodiversity in agricultural landscapes it is necessary to know in detail the biology, ecology and ecotoxicological sensitivity to pesticides of non-target organisms, in particular those having key roles in ecosystem service provision − the ESPs. This work package addresses three concerns raised by the current state of the art, namely the lack of knowledge on the ecology of ESPs; the need to understand their ecotoxicological profiles, and the need to evaluate ecotoxicological interactions with any new bio-inspired products or management developed in this project. To meet the objectives, the work addresses these knowledge deficits by developing a database of ecology and biology of the most important ESPs, and evaluating their sensitivity to common pesticides, and potential interactions with new products and management.

Standard operating procedures (SOPs) have been prepared to be used in testing non-target arthropods (NTA) for their sensitivity to pesticides. A large number of ecotoxicological tests have been performed, including Coated Glass Tube (CGT) assays and laboratory tests to construct Species Sensitivity Distribution (SSD) curves and compare different exposure routes/doses. The CGT tests have identified several species populations with reduced sensitivity to -cyhalothrin, while the SSD revealed that some insecticides, when used at recommended application rates, bring unacceptable risk to ESPs. The tests performed so far within WP7 showed, thus, that current regulations do not protect sufficiently the ESPs, and all NTA in general, and should be revised.

Many soil microorganisms living as root symbionts are increasingly used in agriculture since they can promote both plant growth and plant defence barriers, active against pathogens and pests. Several Trichoderma species or strains are indeed effective biocontrol agents of plant pathogens and, more recently, proved to be effective also against insect pests, even though their control activity can be variable. To foster their predictable use under field conditions, we have investigated the impact of the temperature on the performance of insect pests feeding on tomato plants colonized by different Trichoderma strains and maintained at different temperatures. Tomato plants treated with the strain T. atroviride P1 and reared at mild temperatures (19-20°C) are protected from the attack of aphids and caterpillars, while the commercial strain T. harzianum T22 was more efficient at higher temperatures (25°C). These results were consistent with the expression levels of defence genes in experimental plants. Temperature is, therefore, an important parameter conditioning the interaction between a specific fungal strain and the plant, which determines different levels of protection. This finding has remarkable applied implications, shedding light on the pivotal importance of an accurate selection of the fungal strain better performing under the environmental conditions occurring across different geographic areas in Europe.

Entomopathogens are microbials able to infect and kill insects, but they can also colonize endophytically several plant species, promoting their growth and providing protection against pests and pathogens. We have studied these aspects in oilseed and tomato plants endophytically colonized by the entomopathogen Beuaveria bassiana, using both wild and commercial strains. The growth promotion was strain-specific and varied from neutral to negative in oilseed plants and from positive to neutral in tomato plants. In oilseed, endophytic B. bassiana did not affect the pest damage, while in tomato, selected strains were able to reduce the symptoms of grey mold and tomato leaf spot, both in vitro and in planta. Furthermore, aphid survival and fertility were reduced and Spodoptera pupal development was impaired and fertility was reduced although larval development was not affected. In addition, a remarkable reduction of the immune competence of Spodoptera was observed considerably enhancing the killing activity of Bacillus thuringiensis and, potentially, of other natural antagonists. This allows defining more effective pest control strategies based on the exploitation of such synergistic interactions. Although the growth promotion and protection against pests and pathogens vary depending on the crop plant and fungal strain, the use of entomopathogens as endophytes, either alone or in combination with other beneficial microbes, is a promising tool for biocontrol of pests and pathogens.

"In spring 2021, within the framework of the EcoStack project, 2 field days were organised in France on a field experiment involving barley crop variety mixtures undersown with crimson clover. The objective of the field days was to illustrate the innovative undersowing technique implemented by a farmer of a French local actor group network, and to show, thanks to captured specimens, posters, and in-situ observations, what useful insects and spiders can be found in farmers’ fields and what are their role in pest control.

Barley varieties mixtures and undersowing barley with clover can reduce the presence of aphids and eventually increase the presence of natural enemies compared to monocropping of one single barley variety. Aphids are vectors of the very dangerous barley yellow dwarf virus (BYDV). The effects of the two practices on aphid populations, their natural enemies, and virus spread were tested. Preliminary results were discussed in the presence of researchers and the farmer directly involved in the study.

What fascinated mostly the audience was to discover the variety of different beneficial insects and spiders living in agricultural fields. Practitioners were happy to have information on their biology and their positive role in pest regulation. All participants were intrigued. Advisors especially expressed the need to fill their knowledge gaps on functional biodiversity. They need more information on beneficial arthropods and on the practices favouring them, to be able to promote biodiversity in farmer’s fields."

The EcoStack is a multi-actor project based on innovations that can improve European agriculture by exchanging (innovative) farming practices. Innovations, however, can be improved by means of continuous interactions among stakeholders, such as farmers, researchers and advisors from different sectors and member states. To that end, a resource hub and a learning platform are integrated in the EcoStack project. They are dedicated online resource hub and messaging system that enable interested people to actively interact with EcoStack project researchers. The learning platform will also be used to disseminate latest information and results from the EcoStack project.

Two type of access are possible: “open”, where documents for the most general audience are made available for reading and downloading, and “controlled”, requiring registration on the EcoSTack web site (https://www.ecostack-h2020.eu/stakeholder-learning-platform/). Registered stakeholders will be allowed to access two tools: Alfresco (document sharing: http://alfresco.agecon.unina.it) and Mattermost (messaging/discussion/blogging: http://mattermost.agecon.unina.it). A moderator per Country will be selected so that documents and discussions can be done in any country’s own language. Both tools are available online and are managed by the University of Naples Federico II (Italy).

Agricultural strategies to enhance functional biodiversity (EcoStack-strategies) are combinations of beneficial measures, such as structural elements within the agricultural landscape, specific cultivation methods or crop and variety diversity. These measures aim at supporting the positive effects of biodiversity on agricultural production as well as biodiversity itself. They are, however, associated with both benefits and costs for farms as well as for the society. In order to assess and balance these economic impacts, all expected positive and negative effects of the measures must be identified and described in detail. If all these are obvious and preferably quantified, monetary costs and benefits can be estimated.

Positive effects of enhancing agrobiodiversity are, for example, less incidence and infestation of pests and diseases or higher abundance of natural enemies, resulting in more yield stability. Both can result in lower costs for plant protection measures and lower environmental impacts. Measures can lead to higher abundance of pollinators resulting in increased yields and thus higher revenues for farmers. Negative effects include increased labour and machinery input and costs for implementing the measures or lower yields, resulting in lower revenues for farmers.

We aim to provide this crucial information on benefits and costs through factsheets for individual measures as well as combinations and EcoStack-strategies. This will help farmers or other stakeholders to take decisions on available and most effective measures and whether they are applicable and (economically) beneficial for their specific farming system and regional conditions.

ALMaSS (www.almass.dk) is a simulation system for evaluating the impact of management on wildlife, including beneficial organisms at landscape scales. Developed in Denmark, this methodology has been expanded to include Poland, The Netherlands and parts of Portugal. EcoStack added three new countries, namely France, Sweden and Finland. As part of this process the methods for ‘capturing’ landscapes in simulation have been refined. These methods encompass three main processes:

• Landscape data collection – this is the collection and collation of key geographic and farm databases to create the basis for the generation of ALMaSS mapping per country. These data sets, for all but Portugal, allow for a full national coverage of ALMaSS landscapes for simulation. The data needed includes detailed maps, including farm fields (mapped or determined from remote sensing), what crops the farm grows, and details of the animals present on each farm.

• ALMaSS static map generation - done by creating a small program that will combine the information collected to form a static map for input to ALMaSS. Included in this process is the classification of farms into predefined farm types, and specification of the typical crop rotations for each farm type.

• Generation of dynamic elements - includes simulating the regional-specific management of crops, growth responses of crops to management and weather, and inclusion of historical weather records for each country/region modelled.

Once created landscapes, typically 10x10km, can be selected from the database to be simulated in ALMaSS. This provides an important resource for very realistic modelling scenarios of management of agricultural systems and the impact of these on natural systems.

The two crucial questions faced by Ecological Risk Assessment for pesticides is whether current, simplistic bioassays are protective for Ecosystem Service Provider (ESP) species and what effects on ESP communities the currently recommended applications of insecticides may have. The laboratory tests comparing three different routes of exposure to lamba-cyhalothrin: acetone solution-covered Petri dish, Petri dish sprayed with water solution of a pesticide using Potter tower and soil sprayed in the same way, proved, in all four tested species (Coccinella septempunctata, Poecilus cupreus, Bembidion lampros, Calathus fuscipes) the acetone solution on a Petri dish method to be the most sensitive, resulting in median survival times (LT50) significantly lower than in exposure through the sprayed soil. In that sense, the simplest of the three assays gave the most conservative results.

It should be noted, however, that the insecticide caused significantly increased mortality in all four species already at 0.2 Recommended Field Dose (RFD) if the animals were exposed on a Petri dish, and at 1 RFD significant increase in mortality, particularly severe in C. septempunctata and B. lampros, was also recorded on sprayed soil. The Species Sensitivity Distribution (SSD) curves estimated for carabids exposed to cypermethrin showed that the estimated Hazardous Concentration for 5% species (HC5) decreased with exposure time from ca. 0.1 RFD at 5 h and 24 h to 0.05 RFD after 96 h exposure. Besides, the order of species in terms of their sensitivity has changed during the exposure, with Bembidion lampros (one of the ESP species modelled in ALMaSS) being the least sensitive species after 5 h exposure and the most sensitive after 96 h exposure.

Although evolution of resistance to insecticides have been shown in a number of pest species (336 in 2015), the phenomenon seems much less common among pest control agents, with only 38 species with proven resistance to at least one pesticide. The difference may be caused by inferior detoxification mechanisms in pest control agents or, simply, by the less likely detection of resistance generally in ESPs. To test these hypotheses a wide campaign to screen resistance in ESPs is needed.

The CGT test proved to be a fast, effective and inexpensive tool for monitoring resistance of invertebrates to pesticides. The test uses small (ca. 15-20 mL) vials coated with selected pesticide applied as an active ingredient dissolved in acetone and dried afterwards. The pesticide can be applied in several doses, allowing for estimating species sensitivity distribution (SSD) curves for whole communities and identifying resistant populations. The first set of screening tests across the EU, using glass tubes coated with lamba-cyhalothrin, have identified several ESP species populations with reduced sensitivity to the insecticide, namely: Poecilus cupreus in France and Poland, Metallinea properans and Pseudoophonus rufipes in France, Scymnus sp. in Portugal, and Coccinella septemempunctata, Pterostichus melanarius and Dierettiela rapae in the UK.

The existence of resistant populations of ESP species can be considered beneficial but, on the other hand, increased resistance usually connects with some costs of the tolerance, for example reduced fecundity or increased susceptibility to other stressors, including sub-optimal temperatures, drought, starvation, etc., which may be of particular interest in the context of global climate change.

Insect immune barriers are suppressed by parasites and pathogens, which have developed effective virulence strategies and host-regulation mechanisms as a result of co-evolution. Moreover, some predators and parasitoids can produce potent toxins which have very strong and selective insecticide activity. Growing knowledge of the molecular mechanisms underlying these antagonistic interactions enables the development of new bioinspired tools for pest control, mimicking these insect suppression strategies existing in nature. The EcoStack project has identified new bioactive molecules, which are currently being used to develop new bioinsecticides.

The small venom peptide, SFl6, is a potent neurotoxin that modulates the activity of voltage-gated sodium (Nav) channels, disrupting essential neuronal messages. Delivery of this neurotoxin to the site of action will use Bovine Serum Albumin (BSA) as a carrier molecule. BSA is known to enter the plant vascular system via roots and also to cross the insect gut thus delivering the SFl6 neurotoxin to the insect haemocoel and central nervous system. This novel bioinsecticide is highly toxic to two aphid species and will allow development of new pest management strategies.

A dsRNA able to suppress the expression of an insect immune gene, downregulated in parasitized caterpillars, has been designed. Its ingestion by noctuid moth larvae mimics the immunosuppression syndrome induced by a parasitic wasp and remarkably enhances the killing activity of Bacillus thuringiensis as a consequence of increased septicaemia. This paves the way towards the development of new strategies for enhancing Bt efficacy and/or to reduce the dose of its application and hence the related risks of pest resistance.

Fungi and bacteria associated with plants can provide efficient protection against insect pests and diseases, leading to improved plant health and productivity. Beneficial organisms are occurring naturally in soil. Therefore, cultural practices used should be chosen carefully not to destroy the diversity of microorganisms, but to stimulate the beneficial parts of the community. This can be done for example by additions of fresh or composted plant material to the soil. Management of naturally occurring beneficial microorganisms in agricultural cropping systems constitute an interesting possibility for sustainable crop protection, but strategies for how to include such approaches in the cropping practices to utilize their full potential needs to be further developed.

We are studying how organic soil amendments influence the abundance and composition of fungal and bacterial communities in soil and on roots, and how root diseases of olive and tomato can be controlled by such strategies. Both fungal and bacterial communities are greatly influenced by additions of composted as well as fresh plant material. In tomato, we have found that root diseases such as corky root rot, can be suppressed by additions of plant material as it results in increased microbial activity. However, the magnitude and direction of changes in the communities of beneficial microorganisms has been found to depend on the soil. Although the responses of microbial communities vary among soils, the addition of plant material results in disease suppression and improved plant growth.

Stimulating the beneficial microbes by additions of plant material is therefore a promising approach to reduce root diseases and the damage they cause.

We study agricultural practices within the crop to promote ecosystem services. Practices include variety mixtures, mixed crops or companion cropping, undersowing crops and the use of organic mulching materials.

In various combinations of variety mixtures in cereal cultivation, the Agricultural University of Uppsala, Sweden, has demonstrated an influence of one variety on the other mixing partner that has led to a reduced attractiveness of the plants to aphids. The University of Belgrade, Serbia, and the University of Barcelona, Spain, have already been able to confirm lower aphid infestation in field trials with variety mixtures suggested by the Agricultural University of Uppsala.

In oilseed rape cultivation trials by Rothamsted Research, UK, damage caused by the cabbage-stem flea beetle was reduced by undersowing and by straw mulch.

The University of Kassel, Germany, was able to show in potato cultivation that the use of mulch materials made from vetch triticale led to higher yields, less aphid landings and fewer aphids on the plants. In earlier experiments, a reduced transmission of the Potato virus Y by straw mulch was also observed. Higher potato yields with mulch application were also achieved in trials in Bulgaria (Agricultural University Plovdiv) and Bosnia and Herzegovina (University of Banja Luka). At the Agricultural University of Plovdiv, the two-spotted stink bug could be detected as a previously unknown antagonist of the Colorado potato beetle in this region.

The effect of reduced tillage cultivation and the above-mentioned methods on ecosystem services provided by soil organisms is under investigation. We will demonstrate our work during on-farm field days.

We aim to improve ecosystem service provision in-crop by optimizing provision of off-crop resources which provide habitat, food and alternative hosts for crop pollinators and the natural enemies of crop pests: the ecosystem service providers (ESPs).

We sampled pests and ESPs in oilseed rape (OSR) crops and in surrounding semi-natural habitats in two contrasting types of landscapes, complex and simple in Serbia and UK. We used standard entomological methods including pan trapping, pitfall trapping, sweep netting and visual observations. Before and during flowering of OSR we found a greater abundance of pollen beetle pests in OSR crops in simple landscapes while later in the season this trend was reversed; OSR and semi-natural habitats in complex landscapes had noticeably higher pest abundance than in simple landscapes. Parasitoids were more abundant in semi-natural habitats, while ground beetles were most abundant in semi-natural habitats in simple landscapes. Bees were more abundant in semi-natural habitats than in OSR crops, and more abundant in OSR in simple than complex landscapes.

These preliminary results indicate the importance of semi-natural habitats for various groups of insects, especially in simple landscapes. This further strengthens the need to better understand how floral availability influences ESPs.

The spatial and temporal dynamics of arthropods which provide pollination and pest regulation services in crops is affected by both positive (mutualistic) and negative (antagonistic) interactions which are in turn affected by the quality, quantity and location of semi-natural habitats which support them.

We are developing new tools to track ESP dynamics including molecular methods (metabarcoding), and automatic detection and identification of arthropods via image-based techniques. We will use these and traditional entomological methods and ecological network analyses to identify the major pollinators and natural enemies of crop pests that contribute to pollination and pest regulation services in-crop, and we will quantify their effect on crop yield. By linking yield monitor data from GPS-enabled combine harvesters with crop boundary features we will study the relationship between the landscape features found just beyond the field with variations in crop yield within it.

This work will provide new tools and know-how to help farmers manage off-crop agricultural habitats by ‘ecostacking’ habitats which optimise pollination and pest regulation ecosystem service provision, thereby maximising yield and reduce reliance on synthetic agrochemicals.

Farmers in different European countries were asked on their use of environmental-friendly practices, their constraints and their potential. Participants were asked to name practices that they currently carry out to control pests, disease and weeds.

They indicate that currently pesticides are mainly used to control pests, however some biological control is also practiced. Often pests are not the main concern, especially in cereal crops and pasture lands. Sometimes pesticides on are not used on wheat crops because of the low cost-effectiveness of treatments. Resistant crop varieties were cited as an environmental-friendly practice (agroecological practice) that is used to protect against plant diseases. The use of herbicides is still the main method to control weeds. However, mechanical weeding, green manures, false seedbed technique, use of tall wheat varieties and grazing were stated as ways used to control weeds. Crop rotations were also mentioned as an effective practice to control pests, diseases, and weeds. However, the high costs and development of resistant populations and harm to non-target organisms were noted by interviewees as the main disadvantages for using pesticides.

The main disadvantages of environmental friendly practices, as stated by interviewees, were predominately linked to weed management. Issues interviewees mentioned related to the higher investment of time need to control weeds using environmental friendly practices, including finding the right time for field operations.