Final Report Summary - BIOCOMES (Biological control manufacturers in Europe develop novel biological control products to support the implementation of Integrated Pest Management in agriculture and forestry)
Executive Summary:
Objective of BIOCOMES was to provide the essential information for development of plant protection agents (BCA) based on invertebrate biological control agents (parasitoids, entomopathogenic nematodes), baculoviruses for pest control, and bacteria and fungi for pest and fungal disease control. These innovative bio-based plant protection products can be applied by means of seed treatment or pre- and post-harvest applications in open field crops, protected crops and in forestry. The consortium activities aimed at controlling forestry pests like gypsy moth (Lymantria dispar) and pine weevil (Hylobius abietis), aphids in fruit tree crops, lepidopterans in Brassica crops as well as major pests in vegetable production in open field and protected crops like the tomato pinworm (Tuta absoluta), beet armyworm (Spodoptera littoralis, Spodoptera exigua), diamondback moth (Plutella xylostella), Mamestra brassicae and whiteflies (Bemisia tabaci, Trialeuroides vaporarorium). Micro-organisms were used to control fungal diseases caused by Verticilllium longisporum in oilseed rape, powdery mildew (Blumeria graminis) in cereals, Fusarium spp. in corn and cereals, Monilinia spp. (brown rot) in stone fruit and damping-off in tree nurseries.
The BCA developing enterprises of the BIOCOMES consortium selected BCAs and production technologies for development within the project. The following key results were achieved with new BCAs against pests. LdMNPV showed high activity in field trials resulting in increased mortality of Lymantria dispar and reduced leaf damage. Polyhedral inclusion bodies of LdMNPV were produced in vitro in a wide range of different cell culture reactors. Field and glasshouse trials conducted over two years in Italy and Greece have shown promising results for virulent PhopGV isolates against Tuta absoluta in tomato. Isaria fumosorosea reduced white fly populations in tomatoes and peppers in Italy and Greece and Plutella xyllostella on cabbage in Sweden. Released newly selected parasitoids controlled aphids in fruit tree crops allowing reduced insecticide use by growers. The proof of concept confirmed the potential of the parasitoid Telenomus laeviceps as biocontrol agent to control the cabbage moth. Entomopathogenic nematodes caused high parasitisation levels of large pine weevil larvae in large scale field trials. Genetic improvement of virulence and longevity was achieved for Herorhabditis bacteriophora lines which showed high efficacy in field trials against the western corn rootworm in maize. Production technologies for entomopathogenic nematodes were significantly improved allowing upscaling by new separation, washing and storage technologies.
Significant results were also obtained with new BCAs for disease control. Penicillium frequentans Pf909 and Bacillus amyloliquefaciens CPA-8 controlled brown rot of stone fruit similar to chemicals in different European regions. The developed formulation of Trichoderma harzianum TH-1010 is suitable for seed treatment of winter wheat and maize to control Fusarium graminearum. From more than 1200 evaluated fungal isolates, two isolates reduced powdery mildew of wheat in field trials. Effects of bacterial treatments for Verticillium wilt control in oilseed rape were cultivar, strain and disease-pressure specific. Screening for new endophytes of Brassicaceae with antagonistic properties was successful. Paenibacillus polymyxa and Serratia plymuthica have a potential in protection of tree seedlings against damping-off in nurseries.
Newsletters, updates of results and various slideshares, videos and animations illustrating the research activities have been posted via www.biocomes.eu. During the project period there were 154,000 page views and 76,000 visitors on the website.
The BIOCOMES project has shown that it is possible to boost the development of new biological control products in just four years. A large international public-private partnership with industries and scientific institutes made this possible by successfully combining the needed scientific and commercial expertise. Two new biological control products are on the way to registration: one against Tuta absoluta in tomato and one against Fusarium in cereals. Antagonists for brown rot control in stone fruit are also ready for registration. Another biological control agent based on nematodes – for which no registration is needed – has considerable commercial potential to be further developed to control pests in maize and forestry. The entomopathogenic nematodes have significantly been improved by marker assisted breeding and their production technology has significantly been improved to fulfill demands on the growing markets. Parasitoid-based products to control aphids in fruit orchards and a cabbage pest have been developed. Here too, commercial production of this BIOCOMES development is almost certain. Also for most other biocontrol products under development, perspectives are positive and the partners of BIOCOMES are committed to proceed with the development and market introduction of the new biological control products and production technologies.
Project Context and Objectives:
The project ‘Biological control manufacturers in Europe develop novel biological control products to support the implementation of integrated pest management in agriculture and forestry’ was initiated by a consortium of four small- and medium-sized enterprises (SMEs) Andermatt Biocontrol (ABC), e-nema (ENE), OpenNatur (OPEN) and Viridaxis (VIRIDAXIS), and two biocontrol divisions of larger transnational European enterprises CBC (Europe) S.r.l. (BIOGARD; former Intrachem Bio Italia) and Bayer CropScience Biologics (BCSB, former Prophyta). The founding companies first identified biological control agents with high economic potential in various agricultural and horticultural cropping systems and forestry. The consortium then consulted suitable public partners in fundamental and applied S&T and field development to provide complementary scientific and technological input in order to accelerate the introduction into biocontrol markets in agriculture and forestry. General objective of BIOCOMES was to provide the essential information for development of plant protection products based on invertebrate biological control agents (parasitoids, entomopathogenic nematodes; EPN), baculoviruses for pest control, and bacteria and fungi for pest and fungal disease control. These innovative bio-based plant protection products can be applied by means of seed treatment, pre- and post-harvest applications in open field crops, protected crops and in forestry. The BCA developing enterprises of the BIOCOMES consortium selected the following BCAs and production technologies for development within the project.
1. Production technologies for BCAs (WP1 and WP2)
Biomass for microbial BCAs is produced in solid state fermenters or in bioreactors in liquid culture. Considering future markets needs and recent investments of the BCA-producing enterprises, S&T of BIOCOMES focused on 2 production technologies. The objectives were to develop new production technologies, including downstream, storage and formulation technologies, for entomopathogenic viruses (LdMNPV) for control of Lymantria dispar in forestry and nematodes (EPN) and to improve the virulence and storage stability of the EPN Heterorhabitis bacteriophora by breeding. In vitro production of the baculovirus LdMNPV in cell cultures will enable a cost- and time-saving production of baculoviruses independent from costly insect rearings. Improved downstream and storage protocols and genetic improvement of EPN will enable handling of larger nematode quantities, reduce the number of process steps, and minimize losses during processing. It will also enhance shelf life and product quality, will reduce production costs and will enable the development of nematode-based products for large scale open field markets in agriculture and forestry at competitive costs compared with chemical compounds. The specific objectives were:
• To develop a LdMNPV-product based on in vitro production;
• To improve yield and efficiency of LdMNPV in vitro production systems;
• To reduce the LdMNPV production costs to the same level as in vivo production;
• To develop large-scale downstream processing for EPN;
• To improve storage and formulation technologies for EPN;
• To identify the genetic basis and to develop molecular markers to aid cross-breeding for higher desiccation and heat tolerance and virulence;
• To produce a genetically improved strain of H. bacteriophora with prolonged life-span and persistence and increased infectivity.
2. BCAs for agriculture
PhopGV baculoviruses for control of Tuta absoluta in tomato and Phthorimaea operculella and Tecia solanivora in potato (WP4)
Tomato is grown on 272,500 ha. Tuta absoluta is a main pest in the Mediterranean growing areas currently controlled by spraying specific synthetic insecticides. The objective of WP4 was to develop a new BCA based on PhopGV baculovirus suitable for control of Tuta absoluta (Tuab) in tomato as well as Phthorimaea operculella (Phop) and Tecia solanivora (Teso) in potato. ABC envisaged commercial exploitation. The specific objectives were:
• To identify and characterize PhopGV baculoviruses suitable for control of Tuta absoluta as well as Phthorimaea operculella and Tecia solanivora;
• To select the most virulent strain for greenhouse and field testing;
• To optimize the production of the virus by ensuring its genetic and biological stability;
• To optimize the application strategy for the control of insect pests of solanaceous crops;
• To develop a biocontrol agent ready for marketing.
Bacterial seed treatments against Brassica diseases focusing on oilseed rape (WP5)
Oilseed rape is grown on 6,201,000 ha in the EU. Seed treatment with synthetic fungicides against soilborne diseases is common practice. The objective of WP5 was to develop a seed treatment for oilseed rape and Brassica vegetables based on the combined use of already selected strains of Serratia plymuthica HRO-C48 and Paenibacillus polymyxa. ENE envisaged commercial exploitation. The specific objectives were:
• To complement existing knowledge about the BCAs Serratia plymuthica and Paenibacillus polymyxa, and to analyze their interplay;
• To determine crop and cultivar-specific minimum effective doses, impact of soil quality as well as synergistic effects of both BCAs under controlled conditions;
• To develop genome-based optimized protocols for effective fermentation and commercial formulations for seed treatments;
• To evaluate biocontrol effects of both BCAs on oilseed rape (Brassica napus) and Brassica vegetables under commercial conditions.
Trichoderma harzianum DSM25764 seed treatment for control of Fusarium in cereals (WP6)
Maize is grown on 13,590,000 ha in the EU and small-grain cereals on 47,100 thousand ha. Seed treatment with synthetic fungicides is common. Fusarium spp. cause major diseases in all cereals leading to severe losses and mycotoxins in food and feed. It is expected that climate change could result in increased mycotoxin contamination, especially in North West Europe. The objective of WP6 was to develop a seed treatment for maize and other cereals for control of Fusarium spp. based on the already selected strain Trichoderma harzianum DSM25764. BIOGARD envisaged commercial exploitation. The specific objectives were:
• To characterize T. harzianum strain DSM25764 and complement existing knowledge on characteristics of T. harzianum strain DSM25764;
• To determine the minimum effective dose of T. harzianum strain DSM25764 and its effect on Fusarium survival under controlled conditions in soil on roots;
• To develop an effective commercial formulation of T. harzianum strain DSM25764 for seed treatment with optimum shelf-life;
• To evaluate the efficacy and establish the minimum effective dose of T. harzianum strain DSM25764 in open field trials and to develop application strategies in wheat and maize.
Penicillium frequentans 909 for brown rot (Monilinia spp.) control in stone fruit and Bacillus subtilis
CPA-8 for brown rot (Monilinia spp.) control in stone fruit (WP7)
Stone fruit production in the EU comprises 727,700 ha with multiple fungicide treatments per season. The objective of WP7 was to complete the development of the biocontrol agents Penicillium frequentans and Bacillus subtilis CPA-8 to obtain products that provide an effective strategy to control brown rot in stone fruit production. Both BCAs had been previously studied and the research built on existing knowledge to fill gaps on the road to provision of effective products under scale up conditions to develop one sufficient application strategy for growers. OPEN, together with ENE and BCSB, envisaged commercial exploitation. Specific objectives were:
• To characterize the BCAs in order to meet Directive 2001/36 EEC requirements and to develop products suitable to be registered and commercialized;
• To optimize BCA production processes to obtain commercially feasible scaling-up and downstream processing protocols;
• To develop formulation strategies for each BCA to prolong shelf life and improve efficacy against brown rot in stone fruits under practical conditions;
• To develop and validate control strategies based on the BCAs, to determine doses, cost effectiveness and application times adapted to different European stone fruit producing regions.
Fungal BCA for control of powdery mildew in cereals (WP8)
Wheat, barley, rye and triticale are grown on 44,146,000 ha in the EU. Powdery mildew is a key fungal disease in these crops. Powdery mildew is regularly controlled with single or multiple applications of specific mildew or broad-spectrum fungicides across Europe. The objective of WP8 was to develop microbial BCAs for spray applications for control of powdery mildew in wheat and other small grain cereals. Efficacy, mass production and formulation for use by end-users, and safety, sustainability and economics have been considered. BCSB envisaged commercial exploitation. The specific objectives were:
• To build a collection of fungal isolates (~1000) from of powdery mildew pustules as candidate antagonists;
• To select potential candidates using high throughput screening system and data-mining;
• To select antagonists with high efficacy against powdery mildew and suitable characteristics as regards mass production and product formulation;
• To develop a BCA based on newly selected fungal antagonists against powdery mildew in cereals with proven efficacy in multiple field trials and known mode-of action.
Isaria fumosorosea for pest insect control in vegetables (WP9)
The vegetable growing area in the EU amounts to more than 3,000,000 ha. Control of various insect pests by means of synthetic insecticides is common. The overall objective of WP9 was to develop a microbial BCA product based on the entomopathogenic fungus Isaria fumosorosea for control of pest insects of vegetables under different environmental conditions in greenhouse and open field. BCSB envisaged commercial exploitation. The specific objectives were:
• To compare the potential of at least 10 I. fumosorosea isolates of the JKI collection with the already available strain from BCSB;
• To determine the efficacy of 5 selected I. fumosorosea strains under controlled laboratory conditions against 5 key insect pests and 3 beneficial insects;
• To develop a strain-specific marker for identification of 2 I. fumosorosea strains;
• To optimize the efficacy and its reliability under various environmental conditions of 1 optimized formulation of 2 I. fumosorosea strains, applied alone and in combination;
• To evaluate the dose-response of 2 optimized formulations of I. fumosorosea under greenhouse conditions;
• To assess the efficacy of the formulated product containing 1 and/or 2 I. fumosorosea strains under commercial growing conditions in vegetable crops grown in Mediterranean greenhouses and open fields, as well as in Brassica crops grown in the Northern zone.
Parasitoids against aphids in fruit tree crops (WP10)
Apple production in the EU covers 560,000 ha and pear production takes 139,900 ha. Commercial orchards are sprayed with multiple insecticide treatments per season to control major pests, including several aphid species. The objective of WP10 was to develop a parasitoid-based product against aphids in at least one economically important fruit tree crop (apple, pear, cherry, plum). VIRIDAXIS envisaged commercial exploitation. The specific objectives were:
• To identify biologically efficient parasitoids suitable for cost-efficient mass-rearing;
• To integrate the BCA in a total fauna approach within IPM strategies;
• To facilitate the start of implementation of the BCA in pilot projects at growers.
Telenomus laeviceps for control of Mamestra brassicae in Brassica crops (WP11)
Cabbage is grown on 336,000 ha in the EU. Synthetic insecticides are broadly used. Broad-scale implementation of existing products for biological pest control is currently hampered by a lack of efficacy of such products against Mamestra brassicae, which can therefore not be controlled biologically. The overall objective of WP 11 was to develop a novel parasitoid-based product for control of at least one key pest in Brassica vegetable crops with the potential to control additional noctuid pests of major agricultural crops. ABC envisaged commercial exploitation. The specific objectives were:
• To characterize Telenomus sp. population structures in European Brassica vegetable production areas and to determine intraspecific variability of T. laeviceps;
• To optimize parameters relevant for productivity of rearing systems, storage and transport logistics, and field efficacy of T. laeviceps;
• To upscale high quality production of T. laeviceps and to develop efficient delivery systems;
• To integrate the egg parasitoid T. laeviceps in current plant protection strategies in representative production systems of brassica.
3. BCAs for forestry
In the EU, 181.8 Mio ha are used for agricultural production while forestry production covers 178 Mio ha. Pests and diseases cause losses and limit actual production in both production systems. The BCA developing enterprises of BIOCOMES identified 3 products with potential (future) markets for control of two pests and one disease complex. These three products are:
LdMNPV for control of gypsy moth (Lymantria dispar): This pest causes losses in forestry in different climatic zones. It is expected that the pest will spread and increase significantly in Northern Europe and at higher altitudes due to climate change.
Entomopathogenic nematodes (EPN) for control of pine weevil (Hylobius abietis): The potential of EPN for the control of Hylobius abietis has been demonstrated and EPN are used on very limited acreages to control this pest. ENE sees a significant future market potential coming within reach if EPN-BCAs with increased shelf life can be produced.
Microbial BCA for disease control in forestry nurseries: Soilborne pathogens such as Phytophthora plurivora, Fusarium oxysporum and Rhizoctonia solani may potentially cause high losses in seedlings nurseries of various tree species including beech, oak and Scots pine. BIOGARD and ENE selected the antagonists Serratia plymuthica HRO-C48, Paenibacillus polymyxa and Trichoderma harzianum DSM25764 because of their high potential to control soilborne diseases.
The specific objectives were:
• To develop a highly active product containing in vivo produced LdMNPV (later to be replaced by in vitro produced LdMNPV; WP1) with an appropriate formulation for control of gypsy moth on deciduous trees;
• To optimize strain selection and application parameters for EPN against large pine weevil in clear-felled coniferous forests;
• To develop microbial BCAs for suppression of fungal root diseases in forest nurseries.
Project Results:
Substantial progress has been achieved for the development of new biocontrol agents against pest and diseases. The new products based on beneficial strains of viruses, nematodes, bacteria, fungi and parasitoids gave promising results in bioassays and under greenhouse and field conditions. Newsletters, updates of results and various slideshares, videos and animations illustrating the research activities have been posted via www.biocomes.eu. During the project period there were 154,000 page views and 76,000 visitors on the website.
Production technologies for LdMNPV and EPN (WP1)
Production technologies for LdMNPV
Baculovirus products are among the most successful biological insecticides worldwide. Until now, baculoviruses have been produced by infecting larvae of their host insects. Known as in vivo production, this method guarantees consistently high virus quality but is very complex and expensive.
Since the early 1970s, there has been research on ways to multiply baculoviruses in vitro, i.e. in bioreactors on cell cultures obtained from host insects. High production costs and problems relating to virus quality have so far obstructed any conversion into a marketable baculovirus product.
Moreover, only a few insect species have provided productive cell lines. Due to technological advances in cell culture technology, however, this new production method is becoming increasingly attractive.
Although there are still various biological and technical hurdles to overcome, the current trial results within BIOCOMES work package 1 give cause for optimism. They also indicate, that a close cooperation between industry and research is absolutely necessary in the development of new baculovirus products based on in vitro technology.
Infection studies were conducted with a Lymantria dispar cell line. Polyhedral inclusion bodies (PIB) of the Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV) were produced in a wide range of different cell culture reactors. An important focus has been placed on the scalability of the reactor system, which is essential for a cost-effective production strategy. The best overall results were achieved in the wave-mixed SU bioreactor BIOSTAT RM. With cell cultivation systems like iCELLis nano or CELLine, higher PIB concentrations could be reached, but their limited scalability prevents the use as a production system. The LdMNPV PIBs from the wave-mixed SU bioreactor BIOSTAT RM are naturally free of any microbial contamination. Furthermore, the simple downstream processing allows multiple formulations.
The Quality control of the PIBs was done with bioassays on the host insect and TEM microscopy. The bioassays on Lymantria dispar larvae revealed, that the bioactivity of the in vitro produced PIBs was generally low compared to the reference isolate from in vivo production. The activity also varied between different reactor systems and process strategies. TEM microscopy showed, that in vitro produced PIBs were significantly smaller than the in vivo inoculum. In addition, the number of nucleocapsids and virions per OB was decreased and the number of empty OBs was increasing in vitro.
Results from these trials allow the investigation of possible triggers, which influence the PIB activity during the production process. If these triggers can be found and deliberately controlled, the virus quality from in vitro production will be equal to in vivo PIBs. Although many questions remained unanswered, the research progress within BIOCOMES WP1 contributed an important part to a deeper understanding of baculovirus in vitro production for further projects.
Production technologies for EPN
Entomopathogenic nematodes (EPNs) of the genera Heterorhabditis and Steinernema are highly effective against economically important pests. The specific infective juveniles (IJs) can actively seek and infect insects in the soil. Their introduction into pest control markets was a result of the development of in vitro mass production in liquid culture. Today, production is scaled-up to industry size bioreactors of 50 m³. However, production costs are still high due to significant losses of IJs during downstream processing and storage, resulting in application costs approximately 60% higher than chemical control measures. The new products resulting from BIOCOMES are targeted at outdoor markets, like control of the pine weevil (Hylobius abietis) and the corn rootworm (Diabrotica virgifera virgifera). Treatment of such crops needs several hundreds of tons of nematodes per year. Production facilities for these amounts are available. However, the bottlenecks are downstream processing and storage protocols that are able to maintain nematode viability and infectivity. The larger markets have longer transport chains and formulated nematode products therefore also need a longer shelf life. In order to overcome these problems, BIOCOMES aimed at the improvement of harvest technology and a prolongation of the shelf-life of EPN products by technical and genetic approaches
After production of the IJs in liquid culture they need to be separated from the liquid substrate for further storage and delivery to the end-user. During downstream processing the viability and infectivity of the harvested IJs needs to be preserved. During the project, the performance of several separation devices was compared with a disc separator, which had been used so far in the company. An industrial decanter system was able to outperform the standard equipment. Process parameters were improved for the decanter and summarized in a standard operation protocol, now allowing the separation of the IJs from the culture broth without losses at a flow rate of 2.000 l/h, whereas only 400 l/h could be obtained with the separator. For both devices, the survival and infectivity of the harvested IJs revealed no significant differences. Additionally, the decanter turned out to be less labour intensive compared to the separator. Investment costs for both downstream systems are nearly the same.
The IJs are stored at high density in cooled tanks in an isotonic solution. The storage time was so far limited to 6 weeks. During that time the nematodes need to be formulated and sent to the customer. Because of the relative short shelf-life of the nematodes they needed to be produced and delivered on demand. This is a handicap for the market access compared to chemical pesticides, which usually have a shelf-life of several years. An increased survival of the nematodes during storage would give more flexibility for the timing of production and would allow a better capacity utilisation of the bioreactors. Currently the nematodes are stored at densities of up to 400,000/ml. At higher densities the survival in the storage tanks was significantly reduced. When the nematodes are harvested from the bioreactor the density is usually at 200,000/ml and more. This means that at least 50% of the bioreactor volume needs to be provided in cooled storage tanks, which causes high investment and energy costs. This is one factor keeping the product cost for EPNs at a higher level compared to chemicals and can also hamper the scale-up to higher production capacities. Additionally, the IJ mortality during storage can cause further losses and keep the production costs on a high level. Especially detrimental bacterial contaminations can cause severe losses during storage, which, in the worst case, can result in the loss of a whole storage tank with several thousand litres, representing an economical loss of more than 100,000 € per year. The detrimental contaminations are usually accompanied by an ammonia gas and/or hydrogen sulphide smell, indicating anaerobic bacterial metabolism. As soon as microbial contaminations are recognized during storage the nematodes must be separated from the storage solution, washed and re-suspended in fresh storage solution, which is laborious and is causing stress to the nematodes and can reduce the storage time.
The investigations into the liquid storage system have demonstrated that it is possible to store the harvested IJs at a density of 800,000/ml by using higher aerations rates to the nematode storage suspension. This will reduce the investments into additional liquid storage capacities in the future. A new sieving technology was tested and allows further washing steps after separation from the culture broth, which reduces the risk of detrimental microbial contaminations during storage. This sieving machine can also be used to exchange the storage solution during storage or for recycling by a water treatment system. Increasing sulphide concentrations were found to be correlated with the nematode mortality during storage and can be a possible indicator for the contamination with detrimental anaerobic bacteria. However, these benefits are not without tradeoff effects, like higher energy costs for higher aeration rates or investments into new technology.
Before the nematodes can be send to the distributor or end user they need to be formulated in a water dispersible granular carrier, which keeps up a high humidity and guarantees a sufficient gas exchange. The shelf-life of the resulting product is so far limited to 6 weeks only and consequently the product needs to be delivered on demand. This is a handicap for the market access compared to chemical pesticides, which usually have a shelf-life of several years. A longer shelf-life would allow sending bigger product batches to local distributers and thus would reduce logistic efforts and costs. Furthermore, a longer product shelf-life would increase the flexibility for the end user regarding the schedule for the product application. No technical measures could finally be identified during the BIOCOMES project, which could guarantee a longer shelf-life of the formulated product based on the nematode H. bacteriophora. Disinfection of the EPN during liquid storage and directly before formulation can reduce microbial contamination, but a positive influence on the storability of the nematodes could not be recorded.
Two main objectives for the nematode production part of BIOCOMES were the improvement of the storage time of the nematodes in liquid and the shelf-life of the product. These two objectives were not achieved by technical measures. The reasons seem to be biological limitations in the so far commercially used strain of H. bacteriophora. The virulence of the commercial strain EN01 could not be kept up for more than 4 weeks during storage and in the formulated product even though the nematodes could survive for more than 10 weeks. However, it has been demonstrated that strains, which resulted from activities of WP2 within BIOCOMES and were obtained by genetic selection for longevity and virulence, can be kept longer and their higher virulence in maintained during storage in liquid and in the formulated product. The breeding for an improved nematode strain will be continued beyond the BIOCOMES project.
Genetics for trait improvement of entomopathogenic nematodes (WP2)
BIOCOMES WP2 was working on the genetic improvement of beneficial traits of the biological insect control nematode Heterorhabditis bacteriophora. Objective of the project was to produce nematodes with higher virulence and longevity in order to be able to apply less nematodes per area and thus lower the application costs for nematode products. Starting point was the characterisation of the traits survival, virulence, and desiccation-tolerance of nematode Dauer Juveniles (DJs) within a collection of natural strains and isolates. The DJ is a non-feeding developmentally-arrested stage well adapted to long-term survival. This stage is also used in nematode products. It is responsible for insect host finding, invasion into the host, overcoming the insect´s immune defence and finally killing the insect hosts. E-nema (Germany) dedicated the efforts to predict the survival of H. bacteriophora DJs by developing a bioassay based on the resistance to oxidative stress (exposure to hydrogen peroxide H2O2). Oxidative stress is an internal metabolic misbalance that affects aging in all living organisms and its resistance correlated well with nematode survival. The test was complemented by storing the nematodes at different temperatures. In general, DJs stored at 7°C lived longer than DJs stored at 25°C. Under oxidative stress, all DJs lived substantially shorter at both temperatures. By applying this test, the variability on survival along forty H. bacteriophora natural isolates was determined. The results indicated that DJs from certain isolates survived well across all test conditions (best survivors). These individuals were selected as parents for genetic crosses and for genomic analyses.
The project partners at the Volcani Centre (Israel) focused their efforts on characterizing the tolerance to desiccation in a subset of H. bacteriophora natural isolates. Experiments using hygroscopic and evaporative desiccation methods were carried out to survey DJ mortality. The survival of the nematodes was followed over time up to 96 hours after stress induction. Similarly to the observations made under oxidative stress, desiccation-tolerant and –sensitive isolates were determined. With this information in hand, two time-points (24h and 36h) under hygroscopic and evaporative conditions were chosen for molecular analyses of the DJ’s desiccation tolerance in the H. bacteriophora commercial strain.
The H. bacteriophora virulence was evaluated at e-nema. Two host insects were used: i) mealworm (Tenebrio molitor), an easy-to-handle insect, and ii) larvae of the western corn rootworm (WCR, Diabrotica virgifera virgifera), an invasive maize pest. As parameter for virulence, the number of DJs per insect required to kill 50% of an insect population (Lethal dose, LD50) was determined for all isolates. The LD50 against T. molitor did not exactly correlate with the LD50 against WCR. However, a few isolates presented low LD50 (high virulence) for both hosts. Partners at the University of the Azores (Portugal) identified two genetically similar Portuguese H. bacteriophora isolates that had contrasting LD50 against T. molitor.
In a next step, WP2 focused on generating massive genetic sequence information related to desiccation, survival and virulence in H. bacteriophora. This was done by making a full inventory of all expressed genes in DJs under a given condition. Gene expression provides detailed information on genetic constructs coding for proteins which regulate the activity of an organism. The inventory of gene-expression messenger RNA (mRNAs) of DJs exposed to low relative humidity was assessed at the Volcani Centre. DJs under normal conditions were compared against DJs held for 24h and 36h under desiccation stress. The inventory of mRNAs of DJs under oxidative stress was assessed at e-nema in two H. bacteriophora isolates with contrasting longevity. Both isolates were challenged with oxidative stress and 4h hours after stress induction a “snapshot” of their gene expression (transcriptome) was taken. Partners at the University of Azores did the expression inventory along the host infection process. To filter information, experiment setups simulated the host infection process in three ways: i) simulation by a physical stimulus was done by inducing the loosening of an external cuticle, which is only lost in DJs upon host infection, ii) simulation by a chemical stimulus was done by provoking developmental changes in the DJs triggered by a chemical compound, and iii) simulation by a biologic stimulus was done by putting DJs in contact with the haemolymph of the host. The haemolymph in insects is homologous to the human circulatory system. In this way, only haemolymph-exclusive expressed genes were detached from the background-noise caused by the other stimulation conditions.
This large gene expression inventory from virulence, survival and desiccation studies yielded more than ~55.000 distinct expression messages (mRNAs) observed in any of the situations. Approximately 17% of the messages were common for the three approaches, whereas 5% were exclusive to desiccation, 6% for oxidative stress and almost 50% were exclusively seen in the virulence analysis. Each project partner focused on filtering and interpreting this large amount of data to detach the physiologically important messages (genes) from the background information (complete transcriptome) and to try to identify the related genes (gene annotation). Several genes ligated to the catalysis of oxidants were found to be expressed at different magnitude (differentially expressed) after deep analysis of the oxidative stress expression survey. Also, genes involved in protein protection and on stability of cells and tissues appear to have an important role in oxidative stress tolerance. The DJ reaction to desiccation shared some important messenger RNAs with the oxidative stress responses. Genes involved in protein stability, protection of the cells and tissues structure, and genes involved in lipid metabolism were considered relevant for these conditions. The physiological mechanisms behind the virulence process in DJs appear to involve genes of very diverse function. Enzymes (proteases and peptidases) are secreted by the DJs and have the function to degrade proteins of host membranes. They play a major role along the DJ infection process. Additionally, the very comprehensive gene expression surveys were complemented by the search for very small differences in the genomes of H. bacteriophora natural isolates. These small differences are changes in single positions on the DNA. They are referred to as single nucleotide polymorphisms (SNPs) and together they can form “bar-codes” that can differentiate individuals. More than 1.100 SNPs were found in the H. bacteriophora genome. This information was then used to search for links between natural isolates showing particular bar-codes and their virulence and survival. With this approach we identified more than 10 bar-codes (molecular markers) that are related to virulence or survival. In addition, the genome of H. bacteriophora has been sequenced and the results will be used to further characterize genes and markers related to beneficial traits of the biocontrol agent.
In a parallel step to the acquisition of expression information, genetic crosses were done between H. bacteriophora isolates with enhanced survival and virulence. The progeny of the genetic crosses served two main purposes: i) to produce hybrid strains to be tested for their efficacy in lab and field trials and ii) to derive families of highly homogeneous sister lines (recombinant inbred lines), in which the genetic information (genotype) of the parents is differently distributed (segregation). Products from genetic crosses that showed high virulence and survival were evaluated for their efficacy. Comparative laboratory assays evaluated the virulence of these cross products in comparison to DJs of the current nematode commercial line. Subsequently, field-simulation tests were carried out at e-nema to evaluate the efficacy of the DJs under semi-field and finally field conditions. Nematodes were applied in maize field trials in central Europe to evaluate their effectivity against the western corn rootworm and to compare their performance with a commercial strain and chemical control agents (Belem = Cypermethrin). The effect of the DJs application on the pest population and the effect on the maize plants damage was quantified. In the majority of the semi-field and field trials the selected strain performed better than the alterative products. The number of detected adult insects was reduced and less plant damage was determined in plots treated with the new strain, compared to plots treated with the current commercial nematodes. Even more important, plots treated with any of the nematode strains produced better pest reduction and healthier plants than plots treated with the chemical control agent.
Apart from a broad platform of pheno- and genotypic data, BIOCOMES WP2 provided valuable tools (markers) for future genetic improvement of H. bacteriophora for cross-breeding and selection.
BCAs for control of forest pests and diseases (WP3)
Workpackage 3 targeted three biotic threats to forestry, two insects and one disease. The BCAs under development were: a virus for the gypsy moth Lymantria dispar, insect-killing nematodes for the large pine weevil, Hylobius abietis, and various microbial agents for the disease complex responsible for “damping off” in nursery seedlings.
The aim of WP3.1 was to find a highly active Lymantria dispar nuclear polyhedrosis virus (LdMNPV) and build up a large-scale production of LdMNPV with a high level of automatization including the development of an effective and easy to use product formulation. In a first step, an initial L. dispar population was acquired and a small-scale rearing was set up. This provided the basis to test and implement adaptions toward a large-scale production system. This included trials regarding the biology of L. dispar, as well as automatizations on the rearing, virus and downstream side. Furthermore, the quality control for the product was set up. While building up the large-scale production, a virus database has been set up with 15 different strains from all over the world. A new bioassay was developed to compare all the strains and the most effective strains were evaluated. The best two isolates have been used to develop new liquid formulations, which were tested in field trials. Experiments performed by Forest Research Institute (Poland) in WP3.2 aimed to assess the insecticidal activity of gypsy moth Lymantria dispar virus (LdMNPV) formulated by Andermatt Biocontrol. The trials consisted of greenhouse bioassay and field treatments. In greenhouse experiments the caterpillars of gypsy moth were reared on birch (Betula pendula) leaves sprayed with different concentrations of LdMNPV to estimate its biological activity based on the feeding activity and mortality of infected caterpillars. The results of greenhouse experiments showed that the use of LdMNPV reduced feeding intensity and increased mortality of insects up to 87%. The field trials included ground spraying treatments with different concentrations of virus applied to alder (Alnus glutinosa) and birch B. pendula trees colonized by gypsy moth. Due to the limited occurrence of L. dispar populations in Polish forests in 2016-2017, experiments involved placing laboratory-reared caterpillars in mesh sleeves attached to B. pendula and oak (Quercus robur) trees sprayed with different concentrations of LdMNPV. The efficacy of all field treatments was assessed based on insect mortality and on the comparison of damage to leaves collected from treated and untreated trees. Field application of LdMNPV to A. glutinosa trees led to a 4-fold reduction of leaf damage and increased mortality up to 100% of L. dispar caterpillars. The use of LdMNPV to limit populations of L. dispar naturally developing on B. pendula trees resulted in the 4-fold reduction of leaf damage with the highest concentration. Rearing of L. dispar in sleeves attached to birch and oak trees sprayed with two isolates of LdMNPV led to increased mortality of up to 83% of caterpillars and up to 4-fold less leaf damage compared to untreated leaves. The results of greenhouse bioassay and field treatments showed the high activity of LdMNPV which, when applied to deciduous trees resulted in increased mortality of L. dispar and the reduction of damage to leaves caused by gypsy moth caterpillars.
The large pine weevil Hylobius abietis is the most important pest of tree seedlings in replanted coniferous forests in Northern Europe. Adult weevils feed on and can kill newly planted seedling. The weevils develop in stumps of felled trees. In WP 3.3 various strains of entomopathogenic nematodes (EPN) supplied by E-nema were applied to stumps in two regions of Europe with contrasting conditions: Ireland, where soil moisture is usually not a limiting factor but many plantations are on deep peat soils, and Poland, where soil moisture may be a limiting factor for nematode efficacy. Nematodes could parasitise the immature stages and reduce the number of adult weevils emerging. In Poland, Heterorhabditis downesi was the best of four EPN species trialled, with significantly higher parasitism rates than by other species. However, applications did not affect the level of damage caused by H. abietis beetles to Pinus sylvestris seedlings on experimental areas. While nematodes worked surprisingly well (up to 17.5% of weevil larvae were parasitized) despite the drought conditions prevailing during trials and also the great depth at which weevil larvae are typically found in Poland, application was not sufficiently successful to recommend use of nematodes under such conditions. Higher parasitisation levels (up to 70%) were recorded in Ireland where the higher rainfall and shallower distribution of weevils in soil are probably factors favouring greater success of EPN. In small scale trials both Steinernema carpocapsae and H. downesi were equally effective in suppressing pine weevil populations to below the current, informal thresholds of economic damage. EPN were equally efficient in controlling pine weevil in deep peat and in mineral soils typical of conifer plantation sites in northern Europe. Several factors affecting weevil parasitism were examined, including method of application, weevil distribution and stump size. In conclusion, EPN could contribute to reducing weevil populations under a wide range of conditions, as long as the two main conditions of adequate soil moisture and weevils not too deep in soil are met, and their use can be recommended for inclusion in IPM programmes for pine weevil. Results from these trials provide guidance towards choice of EPN species, application method and site characteristics favouring EPN success.
Tree seedlings are produced in forest nurseries, bare-root or container, where they have the best conditions for development. The greatest threat to the planting stock production is a fungal disease, damping-off, caused by a complex of soil-borne pathogens. The pathogens are able to infect a germinating seed or young seedling resulting in a decline. Compared to agriculture, foresters have a limited number of fungicides allowed to use against damping-off and almost no biological control products are available on the market. The aim of WP3.4 was to test three biological control products developed by University of Padova/Biogard and Graz University/E-nema against damping-off. A beneficial fungus Trichoderma harzianum INAT11 was used for soil treatment in order to reduce the amount of the pathogen inoculum in soil as well as to enhance plant resistance. The dose was set at 100g per ha with concentration of fungal spores of 1x1010 CFU/g, applied in aquesous suspension three times during the growing season at 10-day intervals. The two bacterial strains – Paenibacillus polymyxa and Serratia plymuthica were used for seed coating in concentration of 1x107 CFU/ml and 1x109CFU/ml respectively. Additionally, the seedlings were watered twice with bacterial suspension at various concentrations during the growing season. The tests were run in the greenhouse and in two forest nurseries. The criteria for BCA efficacy were as follows: seedling survival rate, biometric parameters of seedlings, fine roots parameters, community structure of mycorrhizal fungi, chlorophyll fluorescence (photosystem II activity) and leaf area index (LAI). The BCA containing T. harzianum successfully suppressed the pathogens in the laboratory tests and reduced damping-off in the greenhouse. In open field trials, it had no consistent impact on the disease nor the other parameters. What is important is that T. harzianum did not have a negative effect on the mycorrhizae, which are necessary for optimal seedling development in forest plantation. Trichoderma harzianum was found to be very sensitive to weather conditions, especially low temperature. Biological control agents containing bacteria successfully reduced damping-off in the greenhouse and in the forest nurseries. There was also an improvement of selected seedling biometric parameters and leaf area index, but at the same time a reduction of mycorrhiza abundance was observed, connected with increased abundance of dead roots. In conclusion, we see a potential of T. harzianum as a biological control agent dedicated for container nurseries, where the seedlings are produced under controlled conditions. In case of P. polymyxa and S. plymuthica there is a need to develop product formulation and its further tests, since there is a potential of these BCAs in protection against damping-off.
PhopGV for control of Tuta absoluta (tomato) and Phthorimaea operculella / Tecia solanivora (potato) (WP4)
Tuta absoluta Merick (Tuab), Phthorimaea operculella Zeller (Phop) and Tecia solanivora Povolny (Teso) and are three lepidopteran species from the Lepidopteran family Gelechiidae causing severe economic damage to important crops, particularly in potatoes and tomatoes. For these three pest species, traditional control methods with chemical insecticides are no longer efficient and baculovirus-based pesticides are a recommended control measure. The aim of WP4 was the development of an efficient baculovirus-based biocontrol agent that is effective against Tuab, Phop, and Teso.
A broad genetic background is essential for the development of a new and effective baculovirus biocontrol agent. Therefore, as a first step, new PhopGV isolates were field-collected in Italy and Greece. A total of 23 different PhopGV isolates were finally available and exchanged among the partners. Small-scale rearings of the pest insects were established and standardized bioassay protocols were developed. Unfortunately, it was not possible to work with the pest Tecia solanivora for quarantine reasons. Adequate screening methods were developed and bioassay series were performed on different pests. All available PhopGV isolates were screened for their activity and a selection of isolates was characterized by molecular means, including PCR and sequencing of selected PhopGV gene fragments, restriction fragment length polymorphism (RFLP) of isolated viral DNA, and next generation sequencing (NGS). Molecular analyses confirmed that PhopGV genomes are highly conserved, but based on NGS whole genome sequencing data, the isolates could be successfully differentiated. Full genome sequences were established for 11 isolates. In general, the isolates only moderately differed in their activity, except for three isolates, which showed a significant higher activity.
The most pathogenic and virulent PhopGV isolates were finally selected for further development of a test product to be used in greenhouse and field experiments. As baculoviruses are produced in vivo, a stable and cost-effective large-scale rearing of Phop was developed, which has been proven to be the most suitable host for the production of the virus. In vivo propagation of PhopGV was continuously optimized for a cost-effective production process. Furthermore, an adequate downstream and formulation process was developed.
Four test products of the most promising PhopGV isolates were developed and chosen for field testing. Small test batches were produced and trial protocols were developed. Several independent field, greenhouse and warehouse trials were conducted over two years in Italy and Greece. Field and glasshouse trials including chemical and non-chemical reference products have shown promising results. Dose-effect relationships as well as significant efficacies were found. The evaluation of an economically feasible and at the same time sufficiently efficient application rate will be crucial for the success of the product. The most active PhopGV isolate allowed a significant damage reduction and a further reduction of the application rates of the test product and the application of a PhopGV product appears to be economically feasible.
Bacterial seed treatments against Brassica diseases with focus on oilseed rape (WP5)
The overall objective of the WP5 project was to develop a sustainable method of protecting oilseed rape and Brassica vegetables against fungal pathogens using seed treatment with beneficial bacteria with the focus on Verticillium wilt. Verticillium wilt is caused Verticillium spp. in a wide range of plants, while V. longisporum is the main cause of Verticillium wilt in oilseed rape (OSR). Serratia plymuthica HRO-C48 and Paenibacillus polymyxa Sb3-1 were previously identified as promising biocontrol agents against Verticillium wilt in oilseed rape. In order to identify the mode of action of the selected biocontrol agents we (1) carried out the genome analysis of the selected BCA; (2) looked into the mechanisms how the selected microorganisms react towards contact with the volatile organic substances of plant pathogenic fungi by using transcriptomics approach and gas/mass spectrometry; (3) studied the colonization patterns of the selected microorganisms in the oilseed rape. Both microorganisms contained a range of genes putatively involved in biocontrol and plant growth promotion, as was shown by genome analysis of S. plymuthica HRO-C48 and P. polymyxa Sb3-1. We found that S. plymuthica HRO-C48 changed its metabolisms towards maintaining energy usage of the cell, reduction of motility and enhancement of the biofilm production upon contact with the volatile organic substances of plant pathogenic fungi V. longisporum, Leptosphaeria maculans and Rhizoctonia solani. The strong in vitro antagonistic effect of S. plymuthica against R. solani correlated with the upregulation of transcripts responsible for antagonistic effects in S. plymuthica HRO-C48. A range of antimicrobial substances was upregulated in P. polymyxa Sb3-1 upon its interaction with Verticillium volatiles, correlating with its strong ability to inhibit the growth of the plant pathogen in vitro. The colonization patterns of biocontrol agents observed by confocal scanning laser microscopy showed that the Serratia cells were either found as clouds surrounding the roots, or they formed large micro-colonies in the root tissue. The Paenibacillus colonies were often detected in the areas surrounding damaged root and leaf tissues or in cavities where they formed large micro-colonies.
We developed strain- and species- specific markers for identification of the applied strains in the soil samples. P. polymyxa Sb3-1 applied to the seeds using pelleting resulted in significant improvement of the lengths of the plants infested with V. longisporum. We found that the effects of the formulation and fermentation with BCA strains was strain- and disease-pressure specific. The selected BCAs were able to reduce disease the symptoms of Plasmodiophora brassica (clubroot). We observed mild to significant reduction of Leptosphaeria maculans infection rate due to the treatments with S. plymuthica HRO-C48 and fungicides in field trials. Verticillium wilt disease reduction was found to be cultivar dependent; i.e. seed treatment with P. polymyxa Sb3-1 reduced Verticillium wilt symptoms in Avatar, but not in Traviata and Sherpa oilseed rape cultivars. This cultivar specificity was confirmed on the seed microbiota level. We found that cultivars Sherpa and Traviata contained higher indigenous diversity within their seeds than Avatar cultivar and were characterized as having a higher colonization resistance against beneficial and pathogenic microorganisms in the field and greenhouse trials.
The unexpected reduction of the germination rate of oilseed rape in one of the field trials due to the treatment with P. polymyxa Sb3-1 and observed phytotoxic effects in vitro lead to the decision to screen endophytes of Brassicaceae for novel V. longisporum antagonists that will replace P. polymyxa Sb3-1 and enhance the effect of the S. plymuthica HRO-C48 by being the co-formulates for the optimized seed treatment against fungal diseases. The selected novel Brassica endophytes had neutral to positive effect on the growth of the oilseed rape seedlings in greenhouse trials. The new endophytic isolate Serratia plymuthica F20 showed strong positive effect against Verticillium wilt in two independent field trials. Further trials are necessary to prove the effectivity of this strain and its combination with S. plymuthica HRO-C48 under adverse field conditions.
In addition, our studies gave new insights into the outbreak of soil-borne diseases. Verticillium spp. is a ubiquitous endophyte in many plants, and probably fulfill important functions, e.g. in the maturity. Only a general loss of microbial diversity or dysbiosis causes an outbreak of the pathogen. We therefore propose that this “microbiome disease” can be suppressed by increasing diversity, which can also be supported by BCAs. They can be applied in complementary seed treatments.
Trichoderma harzianum DSM25764 seed treatment for control of Fusarium in cereals (WP6)
Fusarium head blight of wheat (FHB), Fusarium graminearum, and pink ear rot of maize (PER), F. verticillioides, are two plant diseases causing severe quantitative and qualitative yield losses and mycotoxin contamination in feed and food.
In preliminary studies conducted by University of Padova (Italy), Trichoderma harzianum strain INAT11 (deposit number DSM25764), henceforth strain INAT11, had shown promising activity against FHB and PER. The aim of WP6 was to develop a seed treatment for maize and other cereals for control of Fusarium spp. based on the already selected strain INAT11.
Strain-specific primers for strain INAT11 were obtained by University of Graz (Austria). A strain-specific detection tool for strain INAT11 is now available for use, enabling to specifically identify the strain among other Trichoderma species and strains.
A highly concentrated WP (Wettable Powder) formulation of strain INAT11 was developed. This formulation proved to be appropriate for seed treatment of maize and wheat, and showed acceptable shelf life (no decline in spore viability even after 12 months of storage at 5±2°C).
The knowledge on the characteristics of strain INAT11 was complemented. In particular, the effects of environmental factors (temperature, pH, Water Activity, soil type), as well as effects of the two pathogens (F. verticilloides and F. graminearum) and plant species (maize and wheat) on the growth of strain INAT11 were investigated in laboratory, greenhouse and field studies. The optimal conditions for growth of strain INAT11 under laboratory conditions were warm temperatures (above 10°C), sub-acidic soil pH and high water activity. However, under greenhouse and field conditions, extremely complex interactions among plant species, soil pH, water availability, pathogens and strain INAT11 emerged, and root colonization by strain INAT11 resulted to be influenced by a combination of factors and not just one single factor.
Strain INAT11 showed rhizosphere competence on both target crops, with root colonization levels being in general higher on maize than on wheat in both greenhouse and field studies.
In greenhouse trials, strain INAT11 applied as seed treatment, was able to significantly reduce symptoms of F. graminearum and F. verticillioides infections in respectively wheat and maize on aerial parts. In laboratory tests a variation in the expression of the majority of the markers selected to investigate the induction of systemic resistance was observed, confirming the hypothesis of systemic resistance induced by strain INAT11.
Open-field trials were conducted over two years in Sweden, the Netherlands and Italy (total n. trials: 28). In these trials, the strain INAT11 seed treatment was tested at different application rates in comparison to an untreated control and a chemical reference seed treatment. Strain INAT11 seed treatment was effective in controlling F. graminearum and the foliar disease Septoria sp. in winter wheat, and F. graminearum in maize. Results on the activity against F. graminearum in spring wheat and F. verticilliodes in maize, instead, were not consistent. The minimum effective doses for maize and wheat, which had been proposed based on the results of previous laboratory and greenhouse studies, were effective in controlling F. graminearum on winter wheat and maize also under open-field conditions.
Compatibility studies with conventional plant protection products, commonly applied to seed and at or shortly after sowing, and with stickers, commonly used by stakeholders to make plant protection products adhere to seed, were performed. The studies showed that the combined use of strain INAT11 with fungicides should be avoided due to growth inhibition of the BCA by most of the tested fungicides. Herbicides, insecticides, stickers and bird repellents, instead, may be used in combination with strain INAT11, but compatibility studies should always be performed, if effects are not yet known.
The main foregrounds achieved in this work package are a strain-specific detection tool for strain INAT11, a commercial formulation of strain INAT11 for seed treatment with acceptable shelf life, knowledge on factors (e.g. temperature range for optimum activity, compatibility with conventional plant protection products commonly used on wheat and maize, and many more), which may impair the efficacy of strain INAT11 in the field, and the field trials conducted in compliance with the principles of Good Experimental Practices (GEP) as defined by The European and Mediterranean Plant Protection Organization (EPPO) and with the relevant EPPO guidelines, which can be used for the registration of the formulated product.
Penicillium frequentans 909 and Bacillus subtilis CPA-8 for Monilinia control in stone fruit (WP7)
Brown rot caused by Monilinia spp. is one of the economically most important fungal diseases on stone fruit causing substantial pre-harvest and post-harvest losses worldwide. Alternatively to chemical application, workpackage 7 aimed to complete the development of two biocontrol agents (BCAs) Bacillus amyloliquefaciens CPA-8 and Penicillium frequentans 909 (Pf909), to obtain microorganism-based products that provide an effective strategy to control brown rot disease. Specifically, this work aimed to meet regulation requirements to develop products suitable to be registered and commercialised: molecular maker design, physiological characterisation, mode of action, production and formulation and definition and validation of biocontrol strategies based in both BCAs.
Molecular markers based on RAPD-SCAR methodology or related to ecological adaptations were developed for both BCAs, providing suitable monitoring tools to specifically identify and track them. Moreover, better understanding of the microorganisms’ biology and physiology was achieved focusing on key ecological parameters (temperature, water activity and pH), the susceptibility pattern to different antibiotics or fungicides and toxicity evaluation, providing useful information for registration process. On the one hand, competition for nutrients and space has demonstrated to be the primary mode of action of Pf909 against Monilinia spp. and on the other hand, mode of action of CPA-8 is based on its capability of production of powerful antifungal metabolites such as fengycin-like lipopetides, although volatile organic compounds (VOCs) emitted by this strain have also been described to play an important role. Regarding the production strategies, many criteria were optimized to meet the product quality requirements: culture media based on products and by-products from the food industry and growth conditions (temperature, pH, agitation, aeration, initial inoculum and time duration). Protocols for the mass production of Pf909 (solid state) and CPA-8 (liquid state) have been defined and quality control systems for both processes have been designed. Furthermore, downstream processing protocols were designed to separate the biomass from the spent medium. Once production was solved, formulation process was deeply studied. Oil-diluted forms were successfully developed for Pf909 and different formulation systems have been assessed to formulate CPA-8: liquid, freeze-drying and fluid-bed spray-drying, the latter being the most adequate. Once the best formulation approach was defined, different processing parameters (protectants, adjuvants, and carrier materials) were optimized to obtain products with a stable shelf-life and high performance under commercial conditions. Different storage materials and air packaging conditions were also studied in order to ensure product stability and easy manipulation. Oil-diluted and fluid-bed spray-drying formulations optimized for either Pf909 or CPA-8 in the suitable packaging and stored at cold and room conditions respectively, had at least a shelf life of 12 months and maintained biocontrol efficacy. The spectrum of action of both BCAs has been checked and broadened to all stone fruit species; peaches, nectarines, plums, apricots, cherries, flat peaches and flat nectarines. Furthermore, technical application thresholds for BCA products were determined and a wide tolerance to unfavorable environmental conditions of temperature, humidity and rainfall has been demonstrated for Pf909 and CPA-8 products.
Effective strategies to control brown rot based on the application of both biocontrol products (CPA-8 and Pf909) combined or separately were designed and studied during 2014 and 2015 seasons at field conditions in commercial orchards. Combination of both BCAs did not improve the efficacy of each of them alone and final strategies chosen for validation were based only on one product, the fungal or the bacterial product separately. Doses, calendar of application and effect of adherents and wetting agents were also optimized for the BCA products. Moreover, dispersion and persistence of both BCAs under field and packinghouse conditions were evaluated. A good distribution, persistence and adaptation of CPA-8 to field and postharvest conditions was demonstrated and although Pf909 had less dispersion and persistence than the bacterium, its presence on fruit during season and postharvest was sufficient to control Monilinia spp. The effect of both BCAs on non-target organisms was also studied. All these results are useful and necessary for products registration.
Finally, CPA-8 and Pf909 control strategies have been validated in different stone fruit hosts (peaches, nectarines and cherries) and four different producing countries (Belgium, France, Italy and Spain) over 2 seasons in commercial orchards under EPPO standards. The results obtained indicated that the degree of biocontrol products activity was dependent on the level of inoculum pressure present in the orchard. Three different scenarios have been defined: Scenario 1 (No disease), in which there was no presence of inoculum in the field and consequently no treatments were needed. Scenario 2 (Uncontrollable disease), in which high pressure of inoculum in the field and extremely adverse meteorological conditions occurred, and treatments in general (biologicals or chemicals) are not worthwhile. And finally, Scenario 3 (Controllable disease), in which it is worthwhile and possible to control the disease. This was the case in 62.5% of trials conducted in this study, demonstrating that under low-medium or medium-high inoculum pressure, BCAs products strategies were able to control brown rot even similar to chemicals.
These encouraging results allow to conclude that formulated products of CPA-8 and Pf909 developed in BIOCOMES project are ready to be registered and commercially available to control brown rot in a near future, providing a valuable tool for farmers to produce stone fruit more competitive and cover consumers demand for safer and more environmental friendly products.
The main foregrounds achieved in this workpackage are the formulated products based on CPA-8 and Pf909 ready to be registered and commercialized, the completely defined optimized production and formulation processes for both BCAs, the quality control protocols that are easily implemented to other BCAs and the data set of all field trials in different producing countries that can be used for the registration documentation.
Fungal BCA for control of powdery mildew in cereals (WP8)
The objective of WP8 was to develop microbial biological control agents for spray applications for control of powdery mildew in wheat and other small grain cereals. In a first step, a collection of more than 1200 fungal isolates from powdery mildew pustules has been built up. Leaf samples of cereals, grasses and other plant species with powdery mildew pustules were collected from March to July 2014. From 504 leaf samples, 1237 fungal isolates were obtained from powdery mildew pustules. 889 isolates with regular colony development and abundant spore production were tested in a pre-screening for the following criteria: no growth at 36°C (safety), germination and growth at 5°C (cold tolerance), germination and growth at -7 MPa and at -13 MPa (drought tolerance) and growth after exposure to UVb (UVb tolerance). 748 Isolates fulfilled the pre-screening criteria. From these isolates, 732 could be identified. They belong to 86 different fungal taxonomical groups. However, more than 60% of the isolates belonged to various species within the genus Cladosporium. In the next step, another set of 27 isolates was excluded because they belonged to species which were considered as not safe for humans, animals or plants. The major group of these isolates belongs to various Fusarium spp. which are plant pathogenic and known mycotoxin producers. With the remaining most suitable 185 candidate antagonists a series of bioassays under controlled conditions has been conducted to quantify the effect of those candidate antagonists on powdery mildew. Eight bioassays were conducted with winter wheat plants cv. Julius in which 11 to 26 candidates were tested per assay. In total, 143 different isolates belonging to 42 taxonomical groups were assessed. The remaining 42 isolates, originally selected during the pre-screening program, were excluded from further testing in bioassays because they did not produce sufficient amounts of spores and thus are not suitable for mass production.
From the 143 isolates tested in bioassays on winter wheat, some isolates showed antagonism against Blumeria graminis f.sp. tritici. Based on the overall results, 10 isolates were selected for first small-scale field experiments to compare their potential for powdery mildew control under open field conditions. With these isolates a series of 10 small-scale experiments with potted spring wheat was conducted in the period between 2 May and 21 June 2016 to assess the potential of the applied antagonists under different climatic conditions and different conditions favouring powdery mildew development. Overall results of experiments with potted spring wheat showed that isolates of Tilletiopsis pallescens and Cladosporium spp. have a potential to reduce powdery mildew on wheat. This was shown occasionally during 7 experiments where disease levels were low, but more consistent in 3 other experiments where disease levels allowed a better estimation of treatment effects. Also in 2016 a preliminary small-scale field experiment in a spring wheat crop has been performed. Applications of T. pallescens isolates BC0441 and BC0850 significantly reduced the number of powdery mildew pustules on flag leaves, the leaf coverage with powdery mildew and the growth rate of the mildew epidemic.
In 2017 the 2 most promising strains BC0850 and BC0902 of Tilletiopsis pallescens and strain BC0707 of Cladosporium delicatulum have been tested in 8 field trials this year in Sweden and the Netherlands. Field trials showed significant effects for lower powdery mildew severity for BC0902 in 2 trials in the Netherlands and in 1 trial in Sweden; for BC0850 in 1 trial in Sweden and for Cladosporium strain BC0707 in 1 trial in the Netherlands. Unfortunately, viability tests and bioassays performed with inocula of T. pallescens used in the field trials showed that their quality was poor. Consequently, results of the field trials cannot be used to evaluate the biocontrol potential of these antagonists.
First studies on the mode of action of the T. pallescens isolates indicate that hyperparasitism and production of enzymes degrading fungal cell walls are involved. No signs for induced resistance were found in treated wheat.
Isaria fumosorosea for pest insect control in vegetables (WP9)
The objective of the scientific work in this workpage was to develop a microbial biocontrol agent product, based on the entomopathogenic fungus Isaria fumosorosea for the control of pest insects under different Field and Greenhouse conditions.
It was examined whether the already existing potential of a prototype sample based on an Isaria fumosorosea isolate can be improved. First of all, the strengths and weaknesses of the existing isolate were investigated. Objective was to add a, in its strength and weaknesses complementary isolate of I. fumosorosea to a new formulation in order to compensate the weaknesses and to increase the activity and lastly the efficacy. For this purpose, the growth behavior at six different temperatures within the range of 5 – 37 °C of multiple strains of an existing isolate collection of Isaria fumosorosea belonging to a project partner were evaluated. Also the sporulation behavior of the strains in solid-state fermentation was compared. Randomly Amplified Polymorphic DNA (RAPD) was developed as an identification method of the evaluated strains.
The isolates proven to be of value for the further development were then tested under defined conditions in laboratory climatic chambers for their control effect on whitefly (Bemesia tabaci, Trialeuroides vaporarorium), amyworm (Spodoptera littoralis, Spodoptera exigua) and diamondback moth (Plutella xylostella).
As a result after 1.5 years of investigations, a second isolate was selected for the further development under field and greenhouse conditions. Together with the initial strain from the prototype product this strain was tested in two different formulations (four variants) in the first field test year. The concentration of the microbial pest control agent (MPCA) in these formulations was initially chosen to be very high in order to better visualize the control effect on Bemesia tabaci and Plutella xylostellea.
Storage stability studies of the two formulations showed significant differences in shelf life regardless of the strain used and thus also served as a decision criterion for selection of the formulation in the further field tests.
The field and greenhouse tests were conducted at three different sites (Italy, Greece and Sweden) according to GEP standards and EPPO guidelines PP1 / 152. 135 and 181 performed over 2.5 years. In each case, the white fly trials in Greece and Italy demonstrated a significant reduction in the insect population with a lower initial incidence in tomatoes and peppers. In Sweden, the natural potential of infestation with Plutella xyllostella was used to demonstrate the effect of I. fumosorosea product patterns on different varieties of cabbage. In one experimental year, the necessary disease pressure at favorable weather conditions for the disease and antagonistic MPCA could lead to a reduction of Plutella by 40-50%.
The joint project work and in particular the comprehensive comparison of data available from field and greenhouse results under field conditions are of high value for any further decision on the further development work. At this point of time, the achieved results are providing a guideline and direction for the further product development.
Parasitoids against aphids in fruit tree crops (WP10)
Parasitic wasps are efficient biocontrol agents used against aphids in many different horticultural crops. Biological control of aphids with parasitoids (Hymenoptera, Braconidae and Aphelinidae) is a common practice in covered vegetable and ornamental that has been used successfully over several decades now. However, these beneficial insects have been yet little used in open field crops. In pome and stone fruits, integrated pest management (IPM) is now well developed and there is a strong need for an efficient biological aphid control tool to further develop the IPM strategies. The aim of the WP 10 of the Biocomes project is to develop a parasitoid-based product for the control of aphid infestations in commercial fruit tree orchards. Therefore a first objective of the work-package was to identify the best species of parasitic wasps to be used as Biocontrol agent (BCA) for the control of aphids in fruit tree crops and to mass-rear them for use in preventive releases of parasitoids. Then, a second objective was to develop a complete and optimized release strategy to integrate the product in a total fauna approach within IPM strategies in commercial fruit orchards.
In the two first years of the Biocomes project, it has been shown that aphids of fruit tree crops are naturally attacked by many species of parasitic wasps. From the different species of parasitoids observed in the field, a list of the 10 most promising species as potential new biocontrol agents against aphids in fruit tree crops was identified: Aphelinus mali, Binodoxys angelicae, Diaeretiella rapae, Ephedrus dysaphidis, Ephedrus persicae, Lipolexis gracilis, Lysiphlebus fabarum, Monoctonus cerasi, Monoctonus mali, E. plagiator and Praon abjectum. Species already available at Viridaxis such as Aphidius colemani, A. matricariae, Ephedrus cerasicola and Aphelinus abdominalis also proved to be potentially useful in the control of aphids in fruit tree crops.
Meantime, data on fauna (beneficials and other pests) and current crop protection practices (treatments, chemical compatibilities ...) in fruit tree crops were collected and analyzed. Regarding biological control possibilities of the most common pests associated with fruit tree crops and the potential integration of preventive parasitoid releases to control aphids in these crops, it was decided to focus only on stone fruit trees. Indeed, production tests showed that Viridaxis technology could not be easily adapted to the mass-rearing of key parasitoid species of the apple-tree associated aphid complex, such as Aphelinus mali, the only parasitoid able to control the woolly apple aphid, Eriosoma lanigerum. Furthermore, pear trees are attacked by another pest species than aphids (Cacopsylla pyri) for which no reliable biological control method is yet available.
In order to select the most appropriate parasitoid species to use for the control of aphid infestations in stone fruit orchards, an intensive screening of efficiency was carried out to assess the potential of all parasitoid species of interest against the aphid spectrum attacking these crops. This screening phase was undertaken within a gradual 3-steps approach (laboratory tests ; cage trials ; field trials). Efficiency tests were first performed under strictly controlled conditions, with laboratory tests leading to cage trials if proved successful. These tests successfully showed that aphid spectrum of stone fruits could be controlled efficiently by a mix of parasitoid species already produced or which could be mass-reared at Viridaxis.
Concomitantly, mass-rearing possibilities were assessed for the most promising parasitoid species in order to develop the mixes of parasitoid species to be used during field trials in cherry and peach orchards. Two species of parasitic wasps (Ephedrus plagiator and Diaeretiella rapae) were selected to enter in a scaling-up process. For the two species, the best production parameters were determined and both of them can now be produced at a big scale. However, production and application costs remain high, which limits the commercial use of these two species for now. Additionally to these species, a third one, Praon abjectum showed promising results at a small-scale production and could also be mass-reared if needed in near future.
The final step of the WP10 of the Biocomes project was then to develop, validate and optimize a strategy based on successive preventive releases of parasitoids during field trials carried out in commercial fruit tree orchards. Consequently, three years of consecutive field trials were performed, first in cherry tree orchards in Belgium (2015) and then in peach tree orchards in Spain (2016-2017). Based on the results obtained from these field trials, an integrated protocol was elaborated and successfully optimized for the use of the new BCA product within existing IPM strategies. The use of parasitoids either strongly reduced the aphid population in organic orchards where primary inoculum was high at the beginning of the season or kept the damages under the tolerance threshold for conventionally managed orchards. Overall, the release strategy also led to a reduction in the number of insecticide treatments used in the plots, both in conventional and organic management practices. Indeed, in mean, 2 treatments were avoided in the plots concerned by parasitoid releases in comparison with control orchards. However, despite an overall reduction of about a factor 2 along the 3 years of trials, the cost of the strategy remains too high for conventional peach productions where cheap chemical solutions exist and can offer a total control of aphids. More interest can however be given into higher added-value organic fruit productions, where existing biopesticide strategies (neem oil, natural pyrethrins, ...) do not always offer a satisfying control of aphids and can impair the efficiency of naturally occurring beneficials such as Syrphidae, Coccinelidae or Chrysopidae.
Telenomus laeviceps for control of Mamestra brassicae in brassica crops (WP11)
To characterize the Telenomus laeviceps population structures in European Brassica vegetable production areas and to determine the intraspecific variability of T. laeviceps, i) a species-specific qPCR marker was developed, ii) a reference species collection was established and confirmed by an external taxonomist, and iii) two field sampling campaigns were conducted in 2015 and 2017. The newly developed TaqMan qPCR marker for T. laeviceps was proven to be highly specific to T. laeviceps and was used to determine the species of the field sampled egg parasitoids. During the sampling campaign 2015 we failed to find T. laeviceps in our partner’s countries (Spain, Italy and Sweden) in intensively managed agricultural sites. In 2017, the sampling was extended to low input farming areas, set-asides and natural habitats. In Sweden parasitoids T. laeviceps was identified for the first time and confirmed by PCR analysis (validation based on morphological traits ongoing) providing the prerequisite for release permits and thus expanding the area for commercial use.
In order to successfully rear T. laeviceps at commercial scale and use it in M. brassicae control, detailed knowledge on its biology is required. We conducted experiments to study aspects of the T. laeviceps biology useful for building up an efficient rearing and for a further use of this parasitoid as a biocontrol agent. We investigated the influence of female density, temperature and egg deprivation time on the parasitation rate and sex ratio of the progeny. Results of these trials made it possible to develop a stable and efficient lab-scale rearing. From there on we built up a stable large scale production with a high level of automatization. Parallel to the production up-scaling, a field delivery system for T. laeviceps was developed.
Pilot field trials with the novel formulation of T. laeviceps were conducted in 2015 in a commercial organic cabbage field in Switzerland. T. laeviceps was released in the field in two densities and the parasitation rate monitored through exposition of cabbage moth eggs. The trap eggs were exposed in the field for two days, recollected and the number of parasitized eggs counted. We measured an increase in the mean parasitation rate over the whole season up to 36 %, against the 5% measured in previous years without any T. laeviceps releases (parasitation rate due to wild T. laeviceps). The maximal parasitation rate measured in 2015 reached 70 %. The number of parasitoids released during this first trial was very high to provide the proof of concept. In subsequent trials, reduce densities of parasitoids were used to improve economic viability of BCA release. Costs for current insecticide applications provided the base for calculation. This low density formulation was used in the field trials of 2016 and 2017. During two consecutive growing seasons we conducted field trials to: i) evaluate the efficacy of T. laeviceps in the control of the cabbage moth compared to standard insecticide and ii) integrate T. laeviceps in an existing plant protection strategy for cabbage crops.
Efficacy was tested in field trials conducted in 2016 and 2017 in organic cabbage fields in Switzerland. In each field we defined a plot with T. laeviceps releases, an insecticide treated plot and an untreated control plot. Compared to 2015, the amount of parasitoids per release were reduced to an economically feasible density. Parasitoids were released the first time two weeks after cabbage was planted in the field, depending on the cabbage variety between week 22 and 28. The second release was conducted five weeks after the first release. To quantify the economic impact of T. laeviceps on the product compared to the other two treatments, 12 cabbage heads per plot were harvested. The following variables were recorded: weight of the whole cabbage head (biomass without roots), weight of the cabbage without side leaves, number of side leaves, final weight and number of damaged head leaves.
In both years, the measured parasitation rate did not meet our expectations, with mean values ranging from 0 % to 0.75 %. No difference was found in the weight of the cabbage heads between the three treatments, indicating that neither insecticide nor parasitoid release had a severe impact on yield.
T. laeviceps potentially benefits from nectar providing plants planted or sown near the cabbage field. To quantify the putative impact, T. laeviceps was combined in integration trials in 2016 and 2017 to habitat management programs already established for the brassica production in Switzerland. In the trials conducted in 2016, parasitoids were released in fields with flower strip and companion plants, fields with only flower strip and in control fields without addition of flowers. The flower strip sown is commercially available and its composition specifically designed to promote beneficial insects against cabbage pests, such as the cabbage moth or the small white (Pieris rapae). T. laeviceps was released in the same density as in the above described efficacy trials. The setup of the field trials 2017 were the same as the one of 2016, with the exception that the treatment with only the flowering strip was not tested based on the results of the previous year. In 2016 the total parasitation rate (T. laeviceps and Trichogramma spp.) was significantly higher in the treatment with the flower strip and companion plants compared to the control, respectively 11.71 ± 2.77 % and 3.67 ± 1.19 %. No difference was found between the treatment with only flower strip (7.11 ± 1.93 %) and the other two treatments. In 2017, no difference was found in the total parasitation rate (T. laeviceps and Trichogramma spp.) between the two treatments, 12 ± 2 % for the control and 10 ± 2 % in the treatment with flower provision. Also the parasitation rate of T. laeviceps was not significantly different between the control and the flower treatment, respectively 6 ± 1 % and 4 ± 1 %.
In summary, a mass rearing of Telenomus laeviceps and its host Mamestra brassicae were successfully established and a functional field delivery system for the BCA developed. A survey conducted over two years in Sweden, Italy and Spain demonstrated that T. laeviceps is rarer than anticipated and its occurrence in intensively managed areas very scarce. Therefore, different sampling strategies and methodology were developed and successfully applied in Sweden, where T. laeviceps was collected in 2017. Further efforts are still needed for Italy and Spain. The occurrence of T. laeviceps is a prerequisite for approval of mass releases. Therefore, further researches are needed to expand the potential market of the BCA. We demonstrated that Telenomus laeviceps is an effective BCA under favorable conditions. A rearing system for Telenomus laeviceps, as well as a suitable field delivery system was successfully developed and evaluated in the field. The season 2015 was successful in proving the proof of concept, with a significant increase of the parasitation rate due to mass release of T. laeviceps. However, trials with reduction of the amount of parasitoids were not successful, due to unexpected quality problems of mass reared parasitoids but potentially also due to yet unknown factors. In consequence, the efficiency of T. laeviceps was strongly reduced. The parasitation rate measured was also dependent on the plant protection measures applied in the field. These results indicate that there is still some work to be done to find a suitable density to release the parasitoid and efficiently integrate them in current plant protection systems.
Implementation and ensuring exploitation and impact of production technologies and 11 BCAs developed (WP12)
Risk assessment
Published information, internal data from project partners and data generated in BIOCOMES were collected and assessed for their relevance for risk assessment and regulatory requirements. Recommendations were given to the project partners which experimental data could be generated within BIOCOMES. Such data were generated and provided by the partners. Literature information was also used to further select potential candidates as biocontrol agents, and to exclude potentially critical strains such as producers of potentially toxic metabolites or pathogens of plants or other non-target organisms.
The data that were compiled during the project are appropriate to address many of the requirements for the approval of the microorganisms as active ingredients and the registration of the products. Further data need to be generated in all cases, such as the physical-chemical and technical properties of the products, toxicity, pathogenicity and infectivity of the microorganisms and effects on non-target organisms. Such studies need to be conducted according to internationally agreed guidelines and under GLP. These studies were not within the scope of BIOCOMES. Further recommendations were given to how to generate missing data. Finally, based on existing data, a strategy for the preparation of the dossier for active ingredient approval was proposed for each strain. These strategies need to be further discussed with authorities in the rapporteur member states that can be chosen in the EU.
No major concern with regard to the risk assessment or regulatory requirements was identified for the strains that were selected. The 9 strains are appropriate as active ingredients in plant protection products according to Regulation (EC) 1107/2009 and an approval as a “low risk substance” seems realistic. The regulatory process is still very consuming. Dossier preparation for the first strains can start in 2018 and first products could be on the market in 2023.
The approach chosen in BIOCOMES turned out to be very effective. Regulatory requirements and the risk assessment were considered early in the selection phase and the development, which avoided development of microorganisms that might have potentially negative effects on humans, non-target organisms, or the environment. This approach lead to a more focused selection of strains and consequently reduced time and costs for the development.
The data generated for the risk assessment are the key information for the dossier for the approval of the active ingredients and the registration of the products, which are pre-requisites for the access to the market. The evaluation has demonstrated that no unacceptable risk is anticipated from the use of the products. This should allow a relatively rapid access to the market, where the products developed in BIOCOMES should be able to replace existing products which potentially have higher effects on human health, other non target organisms and the environment. Consequently, the products will allow food production in a more sustainable way with lower risks for humans and the environment.
Ecological sustainability
Life Cycle Assessment (LCA) has become an integral part of planning processes in industry as well as for energy and infrastructure systems. An essential part of any LCA which evaluates the pressure on the environment executed by the life cycle of a product or service is the Life Cycle Impact Assessment (LCIA). The Sustainable Process Index (SPI) is a LCIA method evaluating environmental impacts generated by individual processes steps in a life cycle. This method is a member of the ecological footprint family. Furthermore, it is compatible with the procedure of the life cycle analyses described in the EN ISO 140xx series. The SPI calculates the ecological footprint as the cumulative area to embed the whole life cycle of an industrial production process as well as the use phase of a product sustainably into the biosphere. It describes relevant ecological pressures of a process including all emerging emissions, the pre-chain and product usage. Material and energy flows of a product, which are extracted from and/or dissipated to the ecosphere, are compared to natural flows.
The SPI method has been implemented in the open access tool SPIonWeb, freely available on http://spionweb.tugraz.at/. With this tool it is possible to create whole life cycles by building up process chains, which can be updated and improved easily. Only a web browser is needed to sign up and use SPIonWeb, it is independent from any operating system. As results, the user gets the SPI Footprint, CO2-lifecycle-emissions and the global warming potential (GWP) of the whole life cycle.
Ecological evaluation had a double role within this project: on the one hand ecological evaluation during process development allowed the partners to identify “environmental hotspots” in the process life cycle. This allows integrating ecological considerations in the technological development, avoiding or improving process steps exerting high ecological pressures and thus increasing the ecological performance of the final product.
On the other hand high ecological performance is a crucial selling point for BCA, this information helps to generate competitive products. Ecological evaluation provides a useful instrument to support market access by highlighting these particular advantages of BCAs. For the this second role as market access support an evaluation of a broad array of different conventional pest control agents has been realised to provide a solid base line against which the performance of the BCAs developed in this project was compared.
Sekem Energy GmbH provided the consortium the methodological tool as well as the evaluation capacity to utilise ecological evaluation within the project to fulfil both roles. In the case of the biological control agents (BCAs) within BIOCOMES, evaluation takes place during the process development phase. Validity and precision of the evaluation results is of course critically dependent on the available data. Evaluation therefore could only be realised in close cooperation with partners. These results allowed a preliminary estimation of the ecological impact of the BCAs in question, compared to conventional competitors as well as the identification of ecological hotspots along the process chain. In order to obtain appropriate data, a detailed data requirement sheet has been sent and filled in together with the partners. On the basis of these data 11 BCA production processes have been ecological evaluated: Lymantria dispar nucleopolyhedrovirus (LdMNPV), Entomopathogenic nematodes (EPN), Serratia plymuthica HRO-C48, Trichoderma harzianum DSM25764, Penicillium frequentans 909, Bacillus amyloliquefaciens CPA-8, Cladosporium delicatulum, Tilletiopsis pallescens, Isaria fumusorosea, parasitic wasp Aphidius colemani, and Telenomus laeviceps.
Economic sustainability
The economic sustainability of 11 BCAs in BIOCOMES has been evaluated by means of marketing evaluation of each BCA in its specific market. Based on product and market information supplied by the industrial partners in BIOCOMES an internal and external analysis has been compiled and a SWOT matrix has been produced for each BCA. The internal analysis comprises an evaluation and semi-quantitative rating of specific product characteristics (strengths and weaknesses) of the BCA and the external analysis comprises an evaluation and semi-quantitative rating of specific market characteristics (market opportunities and threats) for each BCA. The influence of the external market conditions on each of the product characteristics has been thoroughly evaluated and rated in an initial confrontation matrix, taking into account the relative importance of each of the factors in the SWOT matrix. Key marketing issues are then derived from the confrontation matrix through sorting the totals of the ratings. Derived key market opportunities which synergize with product strengths can be profitably utilized in marketing while the main derived market threats and product weaknesses can be given special attention during further development of the BCA after completion of BIOCOMES to reduce possible negative economic impacts. Strategic options and recommendations are discussed for each of the BCAs to minimize possible weaknesses and to avoid market threats.
Potential Impact:
BIOCOMES contributed significantly towards the expected overall impact of WP2013: (i) Reinforcing the EU’s sciences base in the bio-economy by Building a European Knowledge Based Bio-Economy based on ‘Europe 2020 – A strategy for smart, sustainable and inclusive growth’ and the Innovation Union Flagship initiative with the overall priority of bio-resource efficiency, and (ii) Providing tools to maximize the impact of research and innovation on European societies and economies consistent with the common strategic priorities of Horizon 2020: focus on societal challenges, competitiveness and excellent research. BIOCOMES also contributed significantly towards the expected specific impacts listed in the Work Programme 2013 (WP2013) in relation to the topic of KBBE.2013.1.2-5: (i) Generation of knowledge and innovative solutions for pest and disease management - targeted to new biological control products that are effective and can readily and sustainably be introduced into the market taking into account the requirements of registration under EU and national legislation including risk assessment, and (ii) Support of policy (i.e. Directive 2009/128/EC) laying down that all farmers will have to apply the general principles of integrated pest management by January, 2014.
The project contributed to the development of 11 completely new biological control agents for application in major crops and forestry and transformational improvement of 2 industrial production technologies. The project had been initiated by 6 leading European manufacturers of biocontrol products who selected the targeted new innovative solutions of BIOCOMES. Major criteria for selection were: (i) Economic sustainability on the expected future markets, (ii) Ecological sustainability with low negative impact on the environment, and (iii) High bio-resource efficiency. Major markets for plant protection products in European agriculture, horticulture and forestry were targeted. The market-driven work programme of BIOCOMES, the competence and heritage of 10 European SMEs, 3 larger enterprises, all qualified as front-runners of the industry, and the strong co-operation with 14 experienced S&T-partners guaranteed that BIOCOMES will have a strong and significant impact on European agriculture, horticulture and forestry at the economic, environmental and social level.
Economic impact of BIOCOMES
Short-term, the SMEs and INDs in the consortium had a direct economic benefit because BIOCOMES significantly increased their capacity for research and development of BCAs. In total, 64 additional researches had been recruited by project partners during the course of the project. Mid-term, the increase in production, marketing and sales of new biological control agents will strengthen the economic basis of the SMEs involved and gives them room for expansion of their commercial operations. Important aspect is that each BCA within BIOCOMES is selected by the partner (SME or IND) after thorough evaluation of commercial parameters: market potential, economic production characteristics, field efficacy, and the assessment of environmental risks. Long-term, all stakeholders, farmers, forestry industry, European biocontrol industry, citizens and governments will benefit from the market-driven approach of BIOCOMES generating a major increase in new sustainable alternatives for pest and pathogen control for the main agricultural sectors coming available under Regulation 1107/2009.
The BIOCOMES consortium consisted of a group of dedicated SMEs and larger industries with long-term experience in the development of BCAs, all active and based in the European biocontrol industry. The intensive co-operation between leading SMEs, RTD providers and recognized registration experts giving guidance with respect to regulatory issues during product development created synergies on effective product development within and beyond BIOCOMES.
Environmental impact of BIOCOMES
The innovative solutions for pest and pathogen management of BIOCOMES will (after registration) lead to a strong reduction of the use of chemical agents in the European Union. Firstly, the sustainable alternatives of BIOCOMES will decrease the amount of residues of chemical agents on agricultural and fresh produce which is beneficial to human health. A second direct effect is that the exposure of soil, (ground) water and air to residues will also be significantly reduced. In addition to these direct effects, the reduction of the use of chemical agents will induce beneficial effects on the population density of natural enemies and pollinators in support of sustainable production of agricultural products in the European Union.
Social impact of BIOCOMES
The increase in the use of BCAs and the subsequent reduction in use of pesticides projected from BIOCOMES will reduce the exposure of workers in agriculture and forestry as well as consumers to toxic chemical agents. Moreover, the generation and dissemination of broad knowledge on new sustainable solutions for pest and pathogen management based on BCAs will improve the acceptance of these products in society. This was specifically supported by the communication of BIOCOMES via animations and videos.
The average costs of the development of a BCA are estimated at $3-5 million (compared to $250 million for a new synthetic pesticide). Market entry takes approximately 3 years for a BCA in the USA (compared to 10 years for a synthetic pesticide) and, due to different registration requirements, a few years longer in the EU. BIOCOMES contributed significantly to the development and implementation of 11 new BCAs and 2 new industrial production technologies with a project duration of 4 years and total budget of 12 Mio€. S&T strategies had been chosen that led to product development close to implementation for all innovations., e.g. for two microbial BCAs registration has already been initiated by industries, two macrobial BCAs are ready for marketing and one improved production technology is already implemented by industry. Involved industries are all highly experienced in product development and launching of BCAs into markets. Depending on the progress in R&T and economic assessments, industries already invested and will further invest in product development. Follow-up research in national and pan-European IPM programs and networks on the integration of the new BCAs in control strategies of pests and diseases in major agricultural and horticultural crops and different forest systems will ensure the implementation of the results of BIOCOMES.
Support of policy to apply the general principles of integrated pest management (Directive 2009/128/EC)
The project contributed to the development of 11 new BCAs to the stage close to implementation. BCAs and targeted diseases and pests had been selected by the industrial participants (SME or IND) after thorough evaluation of commercial parameters including market potential. Main targeted production areas were arable crops, field-grown vegetable crops, fruit production and forestry in all European climatic zones. The new BCAs will partly substitute the use of major groups of insecticides and fungicides. In particular, EPN will substitute the use of soil insecticides (WPs 1, 2 and 3). The microbial insecticides (WPs 4 and 9) and macrobials (WPs 10 and 11) in BIOCOMES will replace canopy treatments with insecticides. This will have a major impact especially on the use of neonicotinoid insecticides which are suspected to contribute to the colony collapse syndrome in bees and are on the list of compounds to be phased out. Furthermore, the use of pyrethroids, which have major negative impacts on soil life (which is why they are only authorized for use every three years) will partly be substituted. Biological fungicides will replace synthetic fungicides used for seed and seedling treatments (WPs 3, 5 and 6). Foliage treatments with microbial BCAs will replace synthetic fungicides in major fruit (WP7) and arable crops (WP8). This will have a major impact on the currently strong dependency on and broad use of fungicides, including endocrine-disrupting azole fungicides which are also listed to be phased out.
At the end of BIOCOMES, new BCAs reached the stage for implementation in IPM programs which already exist or are under development in various national and international IPM initiatives.
BIOCOMES pro-actively linked project activities with IPM networks. Two members of the External Advisory Board (EAB) are international senior experts in implementation of IPM solutions in agriculture and forestry. They continuously evaluated project progress and advised BIOCOMES participants to ensure that strong links with IPM networks were established during the project period. The ambition is to provide IPM networks with prototype formulations of the BIOCOMES BCAs soon after the project. In this way, many stakeholders will be reached early in the pre-implementation phase. Stakeholders as ‘problem owners’ such as farmers’ unions, NGOs, governmental bodies, traders, retailers or water boards were all important members of IPM knowledge dissemination networks.
Building a European Knowledge Based Bio-Economy
The European biological control manufacturers are operating on a market with particular growth potential. The BCA market at present represents 4.0% of the worldwide overall pesticide market. Sales of BCAs in Europe represent 14% of the BCA world market. Sales within the EU in 2008 are estimated at 79 Mio€ for beneficial insects, 12 Mio€ for beneficial nematodes and 52 Mio€ for microbial biocontrol agents including beneficial viruses, bacteria and fungi. The global biopesticides market has grown exponentially in recent years. Key drivers for this growth are the increasing pressure on the use of synthetic pesticides because of their toxicity, maximum residue levels (MRLs) and negative impact on the environment in combination with the emergence of an increasing number of efficient biocontrol solutions. This in particular applies for greenhouse crops with Spanish vegetable production as a strong example. Global annual growth rates for BCA markets are estimated at 16% compared to a growth rate of 5% for the global pesticides market. BIOCOMES especially targeted the markets in open crops, including arable crops and forestry where use of BCAs is far behind compared to use in protected crops. 71% of the acreage of protected crops in Europe (148 thousand ha) is treated with BCAs whereas only 3% of field vegetables (3,060 thousand ha), 10 % of grapes (3,280 thousand ha), 5% of fruits (2,880 thousand ha), less than 1% of arable crops (108,975 thousand ha) and less than 0.1% of forestry (1,020,000 thousand ha) are treated with BCAs (Blum et al, 2011). Field crops including oilseed crops are expected to be the emerging crop segments in biopesticide markets. Considering the huge acreages grown with arable and open field vegetable crops and forests, enormous markets are still not reached by BCAs offering excellent opportunities for increasing sales of BCAs. This market-driven choice of BCAs together with the estimated growth rates for use of BCAs clearly demonstrates the significant impact that BIOCOMES had and will have on the market positions of the involved biocontrol SMEs and larger biocontrol industries as part of the European Knowledge Based Bio-Economy.
The involved bio-economy enterprises expect a significant increase in production and sales of BCAs, also resulting in an increase in employment of qualified employees. The BIOCOMES project contributed in 2 ways. Industries will jointly develop new BCAs using complementary expertise of the network of industrial and S&T participants. Examples were discovery of new BCA strains, risk assessments, and field trials. The substantial EU contribution enabled industries to invest both into major developments of new technologies and into new BCAs. This opened opportunities for transformational rather than incremental steps in business development of the involved bio-economy industries. Implementation of the new technologies also will allow a transformational rather than incremental step in the use of BCAs in European agriculture, horticulture and forestry as economically and ecologically viable alternatives to synthetic pesticides.
The expected technological breakthroughs, in combination with the general advantages of BCAs over synthetic pesticides, will foster the implementation of BCAs. Examples of such advantages are: lower costs of R&D, incremental growth of markets for IPM and organic production, residue-free production, high labor and harvest flexibility (because of short re-entry times), low environmental impact and sustainable production independent on chemistry or fossil resources. Biocontrol enterprises expect annual revenues of BCAs developed in BIOCOMES of >1 Mio€ in the years after market implementation with increasing market penetration during the next decade leading to an estimated annual turnover of 2.5 to 25 Mio€ per BCA.
The European Knowledge Based Bio-Economy represented in BIOCOMES has been an excellent network consisting of 6 enterprises active as BCA manufacturers, 7 enterprises and 14 scientific research institutions, all supporting the development of BCAs with their specific expertise. Besides the BCA manufacturers, all other bio-economy participants had benefit from their participation in BIOCOMES. GAB is the leading European consultancy partner for registration issues and risk assessment studies of BCAs. GAB had profit from the project collaboration through a large increase in knowledge on selection of microbial strains, the development of fermentation technologies and appropriate formulations. Furthermore, the contact with field research institutions gave GAB the opportunity to gain insight into the development strategies for the use of BCAs under field conditions. GAB had profit from this knowledge also by transferring it to evaluating authorities to facilitate the registration of microbial plant protection products. SEK encompassed life cycle assessment data for cradle-to-grave ecological optimization. The participation in BIOCOMES strengthened the position of SEK as consultancy partner for ecological evaluations in the broader bio-economy. Participants AGPL, ARA, COILLTE, HEFA, and PCFRUIT evaluated new crop protection products under field conditions and integrated these together with producers and growers as end users in their cropping systems. Participation in BIOCOMES strengthened their expertise in testing BCAs, and their collaboration with the biocontrol industry and the biocontrol science. The internationally well-recognized research experts in various aspects of biological control present in BIOCOMES had profit from the collaboration by deepening their expertise in their particular research area, by educating young scientists, and by the synergisms arising at many levels when different participants collaborate within WPs and across WPs. The structured Platforms (P1 to P5) across WPs enabled and stimulated all participants to achieve this goal. The focus of BIOCOMES on the development of economic sustainable industrial solutions trained scientists to develop rational scientific approaches to meet the goals of the industry. BIOCOMES contributed also in this way to the building of a European Knowledge Based Bio-Economy, thus strengthening the European research area.
Providing tools to maximize the impact of research and innovation on European societies and economies and bridging towards Horizon 2020.
BIOCOMES contributed to the three integrated elements of Horizon 2020. The project contributed to Smart growth by developing new opportunities for the biocontrol industry which directly contributed to the European bio-economy (see above). Furthermore, young scientists have been educated and trained in a dedicated program to empower the future development of a bio-economy based on knowledge and innovation. In this program young scientists have directly been involved in project activities with a total of 22 PhD positions. Additionally, 16 young scientists have been trained in a workshop in the development of economic and ecologically sustainable BCAs. This workshop had been organized by a group of industry and research participants and also included visits to two production plants of participants. BIOCOMES will make a significant contribution to Sustainable growth by a more efficient, greener and more competitive agriculture and forestry industry. BCAs as ‘green’ products have been developed with strong emphasis on economic sustainability, guaranteed by the strong guidance of the project by the BCA industry. Continuous risk assessment, life cycle assessment and assessment of bio-resource efficiency for each technology developed by highly qualified specialists (GAB, SEK) demonstrated the strong emphasis of BIOCOMES on ecological sustainability of the BCAs. BIOCOMES contributed to Inclusive growth by creating ample opportunities for new employment in growing bio-economy SMEs. Furthermore, BIOCOMES generated opportunities for the production of low or no-residue agricultural and horticultural products in all EU zones, which contributes strongly to the competitiveness of the European agricultural and forestry industry on a global scale.
The specific potential impact of the different BCAs and production technologies developed by BIOCOMES is presented below:
Production technologies for LdMNPV and EPN (WP1)
Production technologies for LdMNPV
In vitro production of baculoviruses contributes to an improved employee safety in the production companies. Whereas in insect rearings complex safety precautions have to be taken to protect employees from wing scales and stinging hairs, these issues are completely absent in cell culture production systems. Baculoviruses can be produced location-independently, because cell cultures can be established in any suitable laboratory. Extensive quarantine measures are not needed any more when working with non-native insect species. Furthermore, it would enable us to produce baculoviruses from hard-to-breed pests. The in vitro production technology of baculoviruses leads to a significantly reduced microbial contamination level in the final product due to higher hygiene standards and the absence of naturally occurring larval gut microbiota. Multiple formulations are possible, which allows easier storage and application of the product for the customers. If the productions costs can be reduced to a lower level than in vivo production, this could also open up a new field of application in terms of markets, crops and customers.
Production technologies for EPN
The market for EPN is increasing continuously, which has led to investment into larger bioreactor volumes. The establishment of the decanter technology for EPN downstream processing and the ability to store the nematodes at higher densities will enable an upscaling of the production process at much lower investment cost. This and the reduction of labour costs for the downstreaming process and reduced losses by microbial contaminations will reduce the costs for EPN products in the future. This will make EPN more competitive compared to chemical insecticides and will allow to enter the outdoor markets, like control of the corn rootworm (Diabrotica virgifera virgifera).
Genetic s for trait improvement of entomopathogenic nematodes (WP2)
The generation of genetically improved nematode strain with increase resistance to stress, prolonged longevity, field persistence and product shelf life and enhanced virulence will increase product stability, quality during transport to the user and allow the used to use less nematodes per area. This will significantly reduce the application costs and enable the use of the nematodes also in large scale out-door cropping systems. These innovative nematode products will provide farmers with alternative, environmentally friendly management tools. The results of the project will fill gaps caused by phasing out synthetic chemical pesticides, will reduce exposure of applicants to dangerous compounds and contribute to reduction of pesticide residues in food and feed. The project has enabled the SME e-nema GmbH to introduce molecular techniques to professionalise the future breeding programme and support biotechnological production also of other biocontrol agents.
BCAs for control of forest pests and diseases (WP3)
Plantation forestry provides a number of important goods and services including wood products, climate change mitigation through carbon sequestration, biodiversity benefits and recreation. Pests and diseases are a major threat to the sector. Directive 2009/128/EC limits the use of chemical pesticides to reduce their impact on human health and the environment. In addition, EU forests are subject to national laws and international commitments to ensure their sustainability. Sustainable forest management (SFM) is monitored and confirmed by certification processes, and this imposes additional requirements on forest managers to reduce the use of chemical pesticides. This project has taken major steps towards providing biological solutions to three major biotic threats to Europe’s forestry. Cypermethrin and alpha-cypermethrin are being phased out for control of pine weevil Hylobius abietis under SFM, and the proposed replacement chemicals are in the neonicotinoid class. EPN can suppress weevil populations, and EPN species produced by e-nema can be recommended for incorporation into integrated pest management programs for this pest, thus replacing the use of chemical pesticides on sites suitable for their application. Gypsy moth Lymantria dispar occurs in many types of forest and urban landscape on mainland Europe. Outbreaks cause damage in up to thousands of hectares. Stands of oak, gypsy moth’s preferred species, are often located in Natura 2000 areas, where the use of chemical insecticides is prohibited. Gypsy moth also attacks roadside trees, whose chemical protection is banned under EU law. The development of a biological formulation based on virus characterized by high pathogenicity for the host, will provide effective protection of forests threatened by the gypsy moth. Damping-off is the most important cause of losses during transplant production. The disease may cause disruption in the continuity of nursery production and losses must be supplemented by purchasing suitable material from an external source. As much manual work is involved in the production of seedlings, the economic importance is high. Many fungicides are registered for other crops but not for forest plants. This means that there is a strong need for biological control agents as alternatives for damping-off control in forest nurseries.
PhopGV for control of Tuta absoluta (tomato) and Phthorimaea operculella / Tecia solanivora (potato) (WP4)
Advanced market analyses revealed a substantial potential for a PhopGV product against the pest Tuab. For Phop, the market potential is more limited. However, the results from the field trials 2017 showed that one single isolate seems to be one of the most pathogenic and virulent one for both Phop and Tuab larvae.
More field trials will be conducted to further increase the efficacy by optimization of application rate and time, as well as the application strategy. The production process will be further optimized to reduce the production costs.
All data necessary for the registration dossier will be generated and the registration process started. For a successful implementation of the product in the market, it is important to develop an adequate marketing strategy in close collaboration with distributors. The registration dossier for the new product for Tuab control will be compiled and it is expected to be submitted as soon as possible. The new agent will extend the control options for T. absoluta by an efficient, sustainable and environmentally friendly alternative. Tomato growers will have an urgently needed tool for resistance management, as resistance to chemicals occurs more and more frequently.
Bacterial seed treatments against Brassica diseases with focus on oilseed rape (WP5)
We found that the way how plants react to the bacterial seed treatments is disease-pressure and cultivar-specific. While we found positive antifungal effect of the bacterial seed treatments on one oilseed rape cultivar, the same treatment resulted in negative emergence rate and corresponding loss of yield in another cultivar. The study of the seed microbiome of the three commercially used cultivars of oilseed rape showed strong differences in the seed microbiome structure between the cultivars. Therefore, we conclude that the structure of the seed microbiome is an important factor in the development of colonization resistance against pathogens. It also has a strong influence on the response of seedlings to biological seed treatments. These novel insights into seed microbiome structure will enable the development of next generation strategies combining both biocontrol and breeding approaches to address world agricultural challenges. On the other hand, cultivar-specific seed treatments will create new market opportunities and strengthen competitiveness and growth of SMEs.
Trichoderma harzianum DSM25764 seed treatment for control of Fusarium in cereals (WP6)
The new seed treatment product based on strain INAT11 capable of controlling F. graminearum in wheat and maize and most likely also other fungal diseases (for example, Septoria sp. in wheat) can constitute a sustainable plant protection product, in compliance with Dir. 2009/128/EC. Fusarium control in maize is currently based on Good Agricultural Practices (GAP), but with partial control only; on wheat, instead, fungicide treatments (DeMethylation Inhibitors and Strobilurin analogues) are used. Once placed on the market, strain INAT11 could become an additional environmentally friendly disease control option for wheat growers and a completely novel control tool for maize growers, which could help to improve food and feed security due to reduction in mycotoxin contamination. However, notwithstanding the promising results obtained against F. gramineaum on wheat and maize and Septoria sp., further field testing with strain INAT11 must be performed against Fusarium sp., and also against other pathogens in cereals, in order to collect more information on the market potential of strain INAT11. A detailed Data Gap analysis for the preparation of the dossier for the Annex I inclusion of strain INAT11 and the registration of the wettable powder formulation has been carried out, and the results obtained and the knowledge gained within BIOCOMES can be exploited for dossier preparation. However, these data must be completed with additional information to fulfil all data requirements. Once all the information will be available, an advanced market analyses will be performed to evaluate whether the introduction on the market of strain INAT11 could actually be cost effective.
Penicillium frequentans 909 and Bacillus subtilis CPA-8 for Monilinia control in stone fruit (WP7)
WP7 has provided two new biocontrol formulated products based on the bacterium Bacillus amyloliquefaciens CPA-8 and the filamentous fungus Penicillium frequentans Pf909 ready to be registered and commercialized. These new tools are effective to control brown rot on stone fruit and can be easily implemented in a standard commercial strategy. They will allow growers to produce high quality stone fruits covering consumers demand for safer and environment friendly products. The impact of these foregrounds will positively affect all the end-users related with stone fruits: growers, retailers and consumers.
Otherwise, the completely defined optimized production and formulation processes for both BCAs, the quality control protocols that can be easily implemented to other BCAs and the data set of all field trials in different producing countries that can be used for the registration documentation, are other foregrounds achieved in this WP.
Opennatur is looking for partners to share registration of CPA-8, Bayer Crop Science has declined to be involved in the Pf909 registration process, and other companies are in negotiation in order to go ahead with the registration process for developed products. Only final toxicological studies and last economic evaluations are needed to compile documentation necessary in both microorganisms.
Fungal BCA for control of powdery mildew in cereals (WP8)
Wheat, barley, rye and triticale are grown on 44,146,000 ha in the EU. Powdery mildew is a key fungal disease in these crops. Powdery mildew is regularly controlled with single or multiple applications of specific mildew or broad-spectrum fungicides across Europe. In WP8 potential BCAs for spray applications for control of powdery mildew in wheat have been selected. Selected isolates of Tilletiopsis pallescens have shown significant effects for lower powdery mildew severity on wheat in bioassays and in (small scale) field trials. Once mass production, formulation and shelf life of these isolates meet the industrial standards for production and storage, these isolates have a good potential to become a BCA of powdery mildew in cereals.
Isaria fumosorosea for pest insect control in vegetables (WP9)
The fungal entomopathogen Isaria fumosorosea (formally Paecilomocyces fumosoroseus) was first described by Wize in 1904 and subject of many scientific studies. The objective of this project was to modify the existing prototype product based on I. fumosorosea from one project partner. Specifically an extension of the applicability and performance under different and broader environmental and climatic conditions was targeted.
The characterization of host range aspects on fungal growth at different environmental conditions, the sporulation under fermentation conditions, the biological efficacy against several pest insects and beneficial insects were compared for ten different Isaria fumosorosea strain candidates at the beginning of the project. After an intensive screening cascade one strain candidate was selected for further development and formulated in the same way as the existing prototype product. Both candidates were evaluated under laboratory conditions prior to initiating field trials. Strength and weaknesses of the formulations were evaluated in field- and greenhouse experiments in different vegetables over a period of 2 ½ years. Three project partners responsible, performed in total 24 field trials under GEP and according to EPPO guidelines in only 2 ½-project years. The excellent cooperation of all six project partners and the expansion of the already existing experiences by dealing with the pests insect and fungal microorganisms Isaria were essential for the success of this project.
As outcome following can be stated: both Isaria fumosorosea isolates applied as formulated products on whitefly (Bemesia tabaci, Trialeurodes vaporarorium) and diamondback moth (Plutella xylostell) showed partially statistically significant medium level control effects in cash crops (tomato and sweet pepper), which is a good result for a biocontrol agent. The reliability in appropriate climatic conditions is acceptable, whereas climatic influences on the efficacy are currently still a problem. The efficacy data ascertained in this project is a comprehensive base for the further development of a microbial biocontrol product for the market.
Parasitoids against aphids in fruit tree crops (WP10)
A new strategy for the biocontrol of aphids in fruit tree orchards was developed by the WP10 within the Biocomes project. This strategy is based on several successive releases of aphid parasitoids carried out in a preventive manner early in the spring. Field trials showed that the strategy is efficient for aphid control and that aphid infestations can be significantly reduced with the use of parasitoids. This biocontrol strategy allows an overall reduction in the number of insecticides applied in orchards concerned by the parasitoid releases. Releases can be performed easily and safely by the end-users and represent therefore a true alternative to the use of pesticides for the control of aphids. The reduction in the number of insecticides used in the orchards also induces an increase in aphid predator populations which work in a synergic manner with released parasitoids. Negative impacts of chemical treatments on the natural ecosystems and water resources can therefore be limited and fruits are also less susceptible to be contaminated with harmful pesticide residues.
Telenomus laeviceps for control of Mamestra brassicae in brassica crops (WP11)
During the project, we could demonstrate the potential benefits by using T. laeviceps in the control of the cabbage moth. The density of released parasitoids needs to be adjusted, in order to reach the desired effects. This parasitoid is also well suited to be integrated in conservation biocontrol programs, with low input plant protection regimes. Field trials showed that this parasitoid could benefit from nectar resources provided through habitat management. Releases of T. laeviceps in more intensively cultivated brassica fields need to be further explored since harmful effects of the standard insecticides may be limiting for usage of the parasitoid.
Implementation and ensuring exploitation and impact of production technologies and 11 BCAs developed (WP12)
Risk assessment
Data that are required for the regulatory approval of the microorganisms and their products were generated and collected during the project. These data represent the basis of the dossier that needs to be submitted to regulatory authorities. In additions, recommendations were given how to address data requirements that are not covered yet. This will allow to compile the dossier in a relatively short time and to accelerate the access to the market. It was demonstrated that no unacceptable risk is anticipated for the use of these products in crop protection. The products are expected to replace products which potentially have a higher impact on human health, the environment or non-target organisms.
Ecological sustainability
Detailed ecological pressures along the whole production life cycle for 11 BCAs have been elaborated using SPIonWeb (http://spionweb.tugraz.at/) and measures to lower the ecological impact within the production phase have been proposed. It is shown that the used fossil based energy is the main driver for the overall footprint in most of the production processes. In some exceptional cases, also materials like polystyrene and substances for disinfectant can cause relevant ecological pressures. Furthermore, the positive impact of using renewable energy within the production of some producers is presented.
As final step, application scenarios for specific agricultural products and specific diseases/pests were created and comparisons of BCA to conventional alternatives based on applications to these plants were drawn. It is shown that in most of these comparisons the BCAs perform ecological better than their conventional competitors do. Additionally it is presented that in some scenarios the application to the fields (in particular the machine use) is a considerable contributor to the ecological impact of pest control.
Economic sustainability
Analysis of the economic sustainability has been performed in a standardized way by means of a strategic SWOT analysis for each of the BCAs developed in BIOCOMES. Main market threats and bottlenecks for development and marketing are described for each BCA. Generally speaking, analysis of the BCAs under development shows a reasonable to good economic sustainability, the strengths of the BCAs in BIOCOMES fit well with the market requirements and opportunities. Several BCAs are already in an advanced stage of development and could go already into application for active ingredient approval mid 2018 or in 2019. Other BCAs for specific niche markets are also particularly suited for purpose and meet the needs of market and society. Common conditions to promote the adoption of the BCAs in Integrated Pest Management (IPM) in this evaluation of the economic sustainability are: (1) selectivity of / compatibility with chemical active substances, (2) production costs of the BCA and (3) regulatory hurdles of which the costs of preparation of the registration dossier and uncertainties in the timeline for evaluation are the main issues. If the target disease/pest of the BCA is the key disease/pest in a crop, crop management can be adapted for the BCA which will promote its efficacy and commercial development. If market threats are either avoidable or can be influenced, counteractive actions are described to reduce the impact of threats on the development of the BCA.
Main dissemination activities and exploitation of results
The new biological opportunities and project results of BIOCOMES were communicated with IPM networks and stakeholders developing new IPM methods and technologies through various communication channels. Awareness of the different new biocontrol solutions, which were under development in BIOCOMES, and of further steps needed for each product and their potential for use in future IPM has been raised. It is expected that various BIOCOMES BCAs will directly be included in upcoming IPM projects on the development of new IPM strategies.
End users and influences of end users have been informed about the contribution of the BIOCOMES products and the important role of biological pest control in IPM via our website, the newsletters and the slideshare presentations. The BIOCOMES website fulfilled an important and successful role in this which is underpinned by the number of visits that has been increased ever since the launch of the website. During the project period, the BIOCOMES website had 154.233 page views by 75.878 visitors. The website will be active until end of 2020.
In our 13 newsletters we were able to focus on specific research updates and other relevant items concerning biological control developments and obstacles which enabled the dissemination of the project results. This was very much appreciated by the subscribers, resulting in a substantial and regular grow of the number of subscribers to 477 subscribers for the final newsletter.
The 3 animated videos explaining what is biological control and how the BIOCOMES BCAs are tackling several diseases and pests and have contributed 1) to explain why the use of biological control products is important and how it works and 2) how specific BIOCOMES products are working and thus controlling pests and diseases.
The 14 flyers with the final results of the developed BCAs and technologies show their advantages in integrated pest management and in economic value and environmental effects in agriculture and forestry. These flyers are interesting for the scientific public including IPM networks, but also for the biological control industry and regulatory and certification authorities organizations and policy makers.
A video on the public-private partnership for new biocontrol products and 16 videos on specific BIOCOMES results are important dissemination tools of BIOCOMES. The videos are in a relatively easy to understand way informing end users and their influencers and the general public about the final results. The videos can be accessed via the BIOCOMES website and the YouTube channel of BIOCOMES.
Individual BIOCOMES partners were very active in dissemination of project objectives, progress and results. In total, there were 384 dissemination activities during the course of the project . Additionally, 24 peer reviewed publications, 52 papers in proceedings of conferences, 2 articles in edited books, and 26 thesis had been finalized at the end of the project and a substantial number of further publications are expected during the year after project termination.
Links to BIOCOMES dissemination materials
Website www.biocomes.eu (all other dissemination materials can be reached via this website)
Newsletters (13): Newsletter 1 (February 2015), Newsletter 2, (April 2015), Newsletter 3 (July 2015), Newsletter 4 (October 2015), Newsletter 5 (January 2016), Newsletter 6 (April 2016), Newsletter 7 (August 2016), Newsletter 8 (November 2016), Newsletter 9 (February 2017), Newsletter 10 (April 2017), Newsletter 11 (July 2017), Newsletter 12 (October 2017), Newsletter 13 (December 2017)
Slideshares (9): 11 new biological control products; Gypsy moth: field experiences; Fusarium; Control of aphids with parasitoids; Cabbage moth; Powdery mildew; Gypsy moth; Seed treatment development for the control of Verticillium wilt; Brown rot on stone fruits.
Animations (3): What is biological control?; Biological control of diseases; Biological control of pests
Flyers (14): Monilinia control in stone fruit; Fungal root diseases; Seed treatment for control of Fusarium in cereals; New antagonists for powdery mildew control in cereals; Bacterial seed treatments against Brassica diseases; Parasitoids against aphids in fruit tree crops; Control of Mamestra brassicae in brassica crops; Lymantria dispar for control of forest pests and diseases; EPN for pine weevil control in reforestation; Control of tomato leaf miner and potato moths; Isaria fumosorosea for pest insect control in vegetables; Production technologies for LdMNPV; Genetics for trait improvement of EPN.
Videos (16): YouTube channel of BIOCOMES with:
BIOCOMES: A public-private partnership for new biocontrol products;
Animations: What is biological control?; Biological control of diseases; Biological control of pests
Results on: Economic stability, Ecological sustainability and Risk assessment of the BCAs
Results on: BIOCOMES research on biological control of tomato leaf miner and potato moths; BIOCOMES research on biological control of Verticillium wilt; BIOCOMES research on biological control of large pine weevil in forests; BIOCOMES research on biological control of aphids in fruit orchards; BIOCOMES research on biological control of whitefly in greenhouses; BIOCOMES research on biological control of brown rot in stone fruit; BIOCOMES research on biological control of fusarium in cereals; BIOCOMES research on biological control of fungal root diseases; BIOCOMES research on biological control of gypsy moth; BIOCOMES research on biological control of cabbage moth; BIOCOMES research on entomopathogenic nematodes; BIOCOMES research on entomopathogenic viruses; BIOCOMES research on biological control of powdery mildew in cereals.
Exploitation of results
The BIOCOMES project has shown that it is possible to boost the development of new biological control products in just four years. A large international public-private partnership with industries and scientific institutes made this possible by successfully combining the needed scientific and commercial expertise.
Two new biological control products are on the way to registration. One against Tuta absoluta in tomato and one against Fusarium in cereals. Another biological control agent based on nematodes – for which no registration is needed – has considerable commercial potential to be further developed to control pests in maize and forestry. The entomopathogenic nematodes have significantly been improved by marker assisted breeding and their production technology has significantly been improved to fulfill demands on the growing markets. Parasitoid-based products to control aphids in fruit orchards and a cabbage pest have been developed. Here too, commercial production of this BIOCOMES development is almost certain. Also for most other biocontrol products under development, perspectives are positive and the partners of BIOCOMES are committed to proceed with the development and market introduction of the new biological control products and production technologies.
Another very important outcome of BIOCOMES might be termed ‘process knowledge’. This resulted in bringing all fourteen research institutes and thirteen companies to the table in a constructive way and being able to avoid potential conflicts regarding intellectual property rights. The BIOCOMES project has been able to close the gap between the needs for research (research and technology development for good scientific publications) and the needs for industry (high probability for a substantial improvement or a new product).
List of Websites:
Public website: www.biocomes.eu
Contact details:
Dr. Jürgen Köhl
Wageningen University & Research
(former Dienst Landbouwkundig Onderzoek - DLO)
Droevendaalsesteeg 1
P.O. Box 16
6700 AA Wageningen
The Netherlands
jurgen.kohl@wur.nl
Objective of BIOCOMES was to provide the essential information for development of plant protection agents (BCA) based on invertebrate biological control agents (parasitoids, entomopathogenic nematodes), baculoviruses for pest control, and bacteria and fungi for pest and fungal disease control. These innovative bio-based plant protection products can be applied by means of seed treatment or pre- and post-harvest applications in open field crops, protected crops and in forestry. The consortium activities aimed at controlling forestry pests like gypsy moth (Lymantria dispar) and pine weevil (Hylobius abietis), aphids in fruit tree crops, lepidopterans in Brassica crops as well as major pests in vegetable production in open field and protected crops like the tomato pinworm (Tuta absoluta), beet armyworm (Spodoptera littoralis, Spodoptera exigua), diamondback moth (Plutella xylostella), Mamestra brassicae and whiteflies (Bemisia tabaci, Trialeuroides vaporarorium). Micro-organisms were used to control fungal diseases caused by Verticilllium longisporum in oilseed rape, powdery mildew (Blumeria graminis) in cereals, Fusarium spp. in corn and cereals, Monilinia spp. (brown rot) in stone fruit and damping-off in tree nurseries.
The BCA developing enterprises of the BIOCOMES consortium selected BCAs and production technologies for development within the project. The following key results were achieved with new BCAs against pests. LdMNPV showed high activity in field trials resulting in increased mortality of Lymantria dispar and reduced leaf damage. Polyhedral inclusion bodies of LdMNPV were produced in vitro in a wide range of different cell culture reactors. Field and glasshouse trials conducted over two years in Italy and Greece have shown promising results for virulent PhopGV isolates against Tuta absoluta in tomato. Isaria fumosorosea reduced white fly populations in tomatoes and peppers in Italy and Greece and Plutella xyllostella on cabbage in Sweden. Released newly selected parasitoids controlled aphids in fruit tree crops allowing reduced insecticide use by growers. The proof of concept confirmed the potential of the parasitoid Telenomus laeviceps as biocontrol agent to control the cabbage moth. Entomopathogenic nematodes caused high parasitisation levels of large pine weevil larvae in large scale field trials. Genetic improvement of virulence and longevity was achieved for Herorhabditis bacteriophora lines which showed high efficacy in field trials against the western corn rootworm in maize. Production technologies for entomopathogenic nematodes were significantly improved allowing upscaling by new separation, washing and storage technologies.
Significant results were also obtained with new BCAs for disease control. Penicillium frequentans Pf909 and Bacillus amyloliquefaciens CPA-8 controlled brown rot of stone fruit similar to chemicals in different European regions. The developed formulation of Trichoderma harzianum TH-1010 is suitable for seed treatment of winter wheat and maize to control Fusarium graminearum. From more than 1200 evaluated fungal isolates, two isolates reduced powdery mildew of wheat in field trials. Effects of bacterial treatments for Verticillium wilt control in oilseed rape were cultivar, strain and disease-pressure specific. Screening for new endophytes of Brassicaceae with antagonistic properties was successful. Paenibacillus polymyxa and Serratia plymuthica have a potential in protection of tree seedlings against damping-off in nurseries.
Newsletters, updates of results and various slideshares, videos and animations illustrating the research activities have been posted via www.biocomes.eu. During the project period there were 154,000 page views and 76,000 visitors on the website.
The BIOCOMES project has shown that it is possible to boost the development of new biological control products in just four years. A large international public-private partnership with industries and scientific institutes made this possible by successfully combining the needed scientific and commercial expertise. Two new biological control products are on the way to registration: one against Tuta absoluta in tomato and one against Fusarium in cereals. Antagonists for brown rot control in stone fruit are also ready for registration. Another biological control agent based on nematodes – for which no registration is needed – has considerable commercial potential to be further developed to control pests in maize and forestry. The entomopathogenic nematodes have significantly been improved by marker assisted breeding and their production technology has significantly been improved to fulfill demands on the growing markets. Parasitoid-based products to control aphids in fruit orchards and a cabbage pest have been developed. Here too, commercial production of this BIOCOMES development is almost certain. Also for most other biocontrol products under development, perspectives are positive and the partners of BIOCOMES are committed to proceed with the development and market introduction of the new biological control products and production technologies.
Project Context and Objectives:
The project ‘Biological control manufacturers in Europe develop novel biological control products to support the implementation of integrated pest management in agriculture and forestry’ was initiated by a consortium of four small- and medium-sized enterprises (SMEs) Andermatt Biocontrol (ABC), e-nema (ENE), OpenNatur (OPEN) and Viridaxis (VIRIDAXIS), and two biocontrol divisions of larger transnational European enterprises CBC (Europe) S.r.l. (BIOGARD; former Intrachem Bio Italia) and Bayer CropScience Biologics (BCSB, former Prophyta). The founding companies first identified biological control agents with high economic potential in various agricultural and horticultural cropping systems and forestry. The consortium then consulted suitable public partners in fundamental and applied S&T and field development to provide complementary scientific and technological input in order to accelerate the introduction into biocontrol markets in agriculture and forestry. General objective of BIOCOMES was to provide the essential information for development of plant protection products based on invertebrate biological control agents (parasitoids, entomopathogenic nematodes; EPN), baculoviruses for pest control, and bacteria and fungi for pest and fungal disease control. These innovative bio-based plant protection products can be applied by means of seed treatment, pre- and post-harvest applications in open field crops, protected crops and in forestry. The BCA developing enterprises of the BIOCOMES consortium selected the following BCAs and production technologies for development within the project.
1. Production technologies for BCAs (WP1 and WP2)
Biomass for microbial BCAs is produced in solid state fermenters or in bioreactors in liquid culture. Considering future markets needs and recent investments of the BCA-producing enterprises, S&T of BIOCOMES focused on 2 production technologies. The objectives were to develop new production technologies, including downstream, storage and formulation technologies, for entomopathogenic viruses (LdMNPV) for control of Lymantria dispar in forestry and nematodes (EPN) and to improve the virulence and storage stability of the EPN Heterorhabitis bacteriophora by breeding. In vitro production of the baculovirus LdMNPV in cell cultures will enable a cost- and time-saving production of baculoviruses independent from costly insect rearings. Improved downstream and storage protocols and genetic improvement of EPN will enable handling of larger nematode quantities, reduce the number of process steps, and minimize losses during processing. It will also enhance shelf life and product quality, will reduce production costs and will enable the development of nematode-based products for large scale open field markets in agriculture and forestry at competitive costs compared with chemical compounds. The specific objectives were:
• To develop a LdMNPV-product based on in vitro production;
• To improve yield and efficiency of LdMNPV in vitro production systems;
• To reduce the LdMNPV production costs to the same level as in vivo production;
• To develop large-scale downstream processing for EPN;
• To improve storage and formulation technologies for EPN;
• To identify the genetic basis and to develop molecular markers to aid cross-breeding for higher desiccation and heat tolerance and virulence;
• To produce a genetically improved strain of H. bacteriophora with prolonged life-span and persistence and increased infectivity.
2. BCAs for agriculture
PhopGV baculoviruses for control of Tuta absoluta in tomato and Phthorimaea operculella and Tecia solanivora in potato (WP4)
Tomato is grown on 272,500 ha. Tuta absoluta is a main pest in the Mediterranean growing areas currently controlled by spraying specific synthetic insecticides. The objective of WP4 was to develop a new BCA based on PhopGV baculovirus suitable for control of Tuta absoluta (Tuab) in tomato as well as Phthorimaea operculella (Phop) and Tecia solanivora (Teso) in potato. ABC envisaged commercial exploitation. The specific objectives were:
• To identify and characterize PhopGV baculoviruses suitable for control of Tuta absoluta as well as Phthorimaea operculella and Tecia solanivora;
• To select the most virulent strain for greenhouse and field testing;
• To optimize the production of the virus by ensuring its genetic and biological stability;
• To optimize the application strategy for the control of insect pests of solanaceous crops;
• To develop a biocontrol agent ready for marketing.
Bacterial seed treatments against Brassica diseases focusing on oilseed rape (WP5)
Oilseed rape is grown on 6,201,000 ha in the EU. Seed treatment with synthetic fungicides against soilborne diseases is common practice. The objective of WP5 was to develop a seed treatment for oilseed rape and Brassica vegetables based on the combined use of already selected strains of Serratia plymuthica HRO-C48 and Paenibacillus polymyxa. ENE envisaged commercial exploitation. The specific objectives were:
• To complement existing knowledge about the BCAs Serratia plymuthica and Paenibacillus polymyxa, and to analyze their interplay;
• To determine crop and cultivar-specific minimum effective doses, impact of soil quality as well as synergistic effects of both BCAs under controlled conditions;
• To develop genome-based optimized protocols for effective fermentation and commercial formulations for seed treatments;
• To evaluate biocontrol effects of both BCAs on oilseed rape (Brassica napus) and Brassica vegetables under commercial conditions.
Trichoderma harzianum DSM25764 seed treatment for control of Fusarium in cereals (WP6)
Maize is grown on 13,590,000 ha in the EU and small-grain cereals on 47,100 thousand ha. Seed treatment with synthetic fungicides is common. Fusarium spp. cause major diseases in all cereals leading to severe losses and mycotoxins in food and feed. It is expected that climate change could result in increased mycotoxin contamination, especially in North West Europe. The objective of WP6 was to develop a seed treatment for maize and other cereals for control of Fusarium spp. based on the already selected strain Trichoderma harzianum DSM25764. BIOGARD envisaged commercial exploitation. The specific objectives were:
• To characterize T. harzianum strain DSM25764 and complement existing knowledge on characteristics of T. harzianum strain DSM25764;
• To determine the minimum effective dose of T. harzianum strain DSM25764 and its effect on Fusarium survival under controlled conditions in soil on roots;
• To develop an effective commercial formulation of T. harzianum strain DSM25764 for seed treatment with optimum shelf-life;
• To evaluate the efficacy and establish the minimum effective dose of T. harzianum strain DSM25764 in open field trials and to develop application strategies in wheat and maize.
Penicillium frequentans 909 for brown rot (Monilinia spp.) control in stone fruit and Bacillus subtilis
CPA-8 for brown rot (Monilinia spp.) control in stone fruit (WP7)
Stone fruit production in the EU comprises 727,700 ha with multiple fungicide treatments per season. The objective of WP7 was to complete the development of the biocontrol agents Penicillium frequentans and Bacillus subtilis CPA-8 to obtain products that provide an effective strategy to control brown rot in stone fruit production. Both BCAs had been previously studied and the research built on existing knowledge to fill gaps on the road to provision of effective products under scale up conditions to develop one sufficient application strategy for growers. OPEN, together with ENE and BCSB, envisaged commercial exploitation. Specific objectives were:
• To characterize the BCAs in order to meet Directive 2001/36 EEC requirements and to develop products suitable to be registered and commercialized;
• To optimize BCA production processes to obtain commercially feasible scaling-up and downstream processing protocols;
• To develop formulation strategies for each BCA to prolong shelf life and improve efficacy against brown rot in stone fruits under practical conditions;
• To develop and validate control strategies based on the BCAs, to determine doses, cost effectiveness and application times adapted to different European stone fruit producing regions.
Fungal BCA for control of powdery mildew in cereals (WP8)
Wheat, barley, rye and triticale are grown on 44,146,000 ha in the EU. Powdery mildew is a key fungal disease in these crops. Powdery mildew is regularly controlled with single or multiple applications of specific mildew or broad-spectrum fungicides across Europe. The objective of WP8 was to develop microbial BCAs for spray applications for control of powdery mildew in wheat and other small grain cereals. Efficacy, mass production and formulation for use by end-users, and safety, sustainability and economics have been considered. BCSB envisaged commercial exploitation. The specific objectives were:
• To build a collection of fungal isolates (~1000) from of powdery mildew pustules as candidate antagonists;
• To select potential candidates using high throughput screening system and data-mining;
• To select antagonists with high efficacy against powdery mildew and suitable characteristics as regards mass production and product formulation;
• To develop a BCA based on newly selected fungal antagonists against powdery mildew in cereals with proven efficacy in multiple field trials and known mode-of action.
Isaria fumosorosea for pest insect control in vegetables (WP9)
The vegetable growing area in the EU amounts to more than 3,000,000 ha. Control of various insect pests by means of synthetic insecticides is common. The overall objective of WP9 was to develop a microbial BCA product based on the entomopathogenic fungus Isaria fumosorosea for control of pest insects of vegetables under different environmental conditions in greenhouse and open field. BCSB envisaged commercial exploitation. The specific objectives were:
• To compare the potential of at least 10 I. fumosorosea isolates of the JKI collection with the already available strain from BCSB;
• To determine the efficacy of 5 selected I. fumosorosea strains under controlled laboratory conditions against 5 key insect pests and 3 beneficial insects;
• To develop a strain-specific marker for identification of 2 I. fumosorosea strains;
• To optimize the efficacy and its reliability under various environmental conditions of 1 optimized formulation of 2 I. fumosorosea strains, applied alone and in combination;
• To evaluate the dose-response of 2 optimized formulations of I. fumosorosea under greenhouse conditions;
• To assess the efficacy of the formulated product containing 1 and/or 2 I. fumosorosea strains under commercial growing conditions in vegetable crops grown in Mediterranean greenhouses and open fields, as well as in Brassica crops grown in the Northern zone.
Parasitoids against aphids in fruit tree crops (WP10)
Apple production in the EU covers 560,000 ha and pear production takes 139,900 ha. Commercial orchards are sprayed with multiple insecticide treatments per season to control major pests, including several aphid species. The objective of WP10 was to develop a parasitoid-based product against aphids in at least one economically important fruit tree crop (apple, pear, cherry, plum). VIRIDAXIS envisaged commercial exploitation. The specific objectives were:
• To identify biologically efficient parasitoids suitable for cost-efficient mass-rearing;
• To integrate the BCA in a total fauna approach within IPM strategies;
• To facilitate the start of implementation of the BCA in pilot projects at growers.
Telenomus laeviceps for control of Mamestra brassicae in Brassica crops (WP11)
Cabbage is grown on 336,000 ha in the EU. Synthetic insecticides are broadly used. Broad-scale implementation of existing products for biological pest control is currently hampered by a lack of efficacy of such products against Mamestra brassicae, which can therefore not be controlled biologically. The overall objective of WP 11 was to develop a novel parasitoid-based product for control of at least one key pest in Brassica vegetable crops with the potential to control additional noctuid pests of major agricultural crops. ABC envisaged commercial exploitation. The specific objectives were:
• To characterize Telenomus sp. population structures in European Brassica vegetable production areas and to determine intraspecific variability of T. laeviceps;
• To optimize parameters relevant for productivity of rearing systems, storage and transport logistics, and field efficacy of T. laeviceps;
• To upscale high quality production of T. laeviceps and to develop efficient delivery systems;
• To integrate the egg parasitoid T. laeviceps in current plant protection strategies in representative production systems of brassica.
3. BCAs for forestry
In the EU, 181.8 Mio ha are used for agricultural production while forestry production covers 178 Mio ha. Pests and diseases cause losses and limit actual production in both production systems. The BCA developing enterprises of BIOCOMES identified 3 products with potential (future) markets for control of two pests and one disease complex. These three products are:
LdMNPV for control of gypsy moth (Lymantria dispar): This pest causes losses in forestry in different climatic zones. It is expected that the pest will spread and increase significantly in Northern Europe and at higher altitudes due to climate change.
Entomopathogenic nematodes (EPN) for control of pine weevil (Hylobius abietis): The potential of EPN for the control of Hylobius abietis has been demonstrated and EPN are used on very limited acreages to control this pest. ENE sees a significant future market potential coming within reach if EPN-BCAs with increased shelf life can be produced.
Microbial BCA for disease control in forestry nurseries: Soilborne pathogens such as Phytophthora plurivora, Fusarium oxysporum and Rhizoctonia solani may potentially cause high losses in seedlings nurseries of various tree species including beech, oak and Scots pine. BIOGARD and ENE selected the antagonists Serratia plymuthica HRO-C48, Paenibacillus polymyxa and Trichoderma harzianum DSM25764 because of their high potential to control soilborne diseases.
The specific objectives were:
• To develop a highly active product containing in vivo produced LdMNPV (later to be replaced by in vitro produced LdMNPV; WP1) with an appropriate formulation for control of gypsy moth on deciduous trees;
• To optimize strain selection and application parameters for EPN against large pine weevil in clear-felled coniferous forests;
• To develop microbial BCAs for suppression of fungal root diseases in forest nurseries.
Project Results:
Substantial progress has been achieved for the development of new biocontrol agents against pest and diseases. The new products based on beneficial strains of viruses, nematodes, bacteria, fungi and parasitoids gave promising results in bioassays and under greenhouse and field conditions. Newsletters, updates of results and various slideshares, videos and animations illustrating the research activities have been posted via www.biocomes.eu. During the project period there were 154,000 page views and 76,000 visitors on the website.
Production technologies for LdMNPV and EPN (WP1)
Production technologies for LdMNPV
Baculovirus products are among the most successful biological insecticides worldwide. Until now, baculoviruses have been produced by infecting larvae of their host insects. Known as in vivo production, this method guarantees consistently high virus quality but is very complex and expensive.
Since the early 1970s, there has been research on ways to multiply baculoviruses in vitro, i.e. in bioreactors on cell cultures obtained from host insects. High production costs and problems relating to virus quality have so far obstructed any conversion into a marketable baculovirus product.
Moreover, only a few insect species have provided productive cell lines. Due to technological advances in cell culture technology, however, this new production method is becoming increasingly attractive.
Although there are still various biological and technical hurdles to overcome, the current trial results within BIOCOMES work package 1 give cause for optimism. They also indicate, that a close cooperation between industry and research is absolutely necessary in the development of new baculovirus products based on in vitro technology.
Infection studies were conducted with a Lymantria dispar cell line. Polyhedral inclusion bodies (PIB) of the Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV) were produced in a wide range of different cell culture reactors. An important focus has been placed on the scalability of the reactor system, which is essential for a cost-effective production strategy. The best overall results were achieved in the wave-mixed SU bioreactor BIOSTAT RM. With cell cultivation systems like iCELLis nano or CELLine, higher PIB concentrations could be reached, but their limited scalability prevents the use as a production system. The LdMNPV PIBs from the wave-mixed SU bioreactor BIOSTAT RM are naturally free of any microbial contamination. Furthermore, the simple downstream processing allows multiple formulations.
The Quality control of the PIBs was done with bioassays on the host insect and TEM microscopy. The bioassays on Lymantria dispar larvae revealed, that the bioactivity of the in vitro produced PIBs was generally low compared to the reference isolate from in vivo production. The activity also varied between different reactor systems and process strategies. TEM microscopy showed, that in vitro produced PIBs were significantly smaller than the in vivo inoculum. In addition, the number of nucleocapsids and virions per OB was decreased and the number of empty OBs was increasing in vitro.
Results from these trials allow the investigation of possible triggers, which influence the PIB activity during the production process. If these triggers can be found and deliberately controlled, the virus quality from in vitro production will be equal to in vivo PIBs. Although many questions remained unanswered, the research progress within BIOCOMES WP1 contributed an important part to a deeper understanding of baculovirus in vitro production for further projects.
Production technologies for EPN
Entomopathogenic nematodes (EPNs) of the genera Heterorhabditis and Steinernema are highly effective against economically important pests. The specific infective juveniles (IJs) can actively seek and infect insects in the soil. Their introduction into pest control markets was a result of the development of in vitro mass production in liquid culture. Today, production is scaled-up to industry size bioreactors of 50 m³. However, production costs are still high due to significant losses of IJs during downstream processing and storage, resulting in application costs approximately 60% higher than chemical control measures. The new products resulting from BIOCOMES are targeted at outdoor markets, like control of the pine weevil (Hylobius abietis) and the corn rootworm (Diabrotica virgifera virgifera). Treatment of such crops needs several hundreds of tons of nematodes per year. Production facilities for these amounts are available. However, the bottlenecks are downstream processing and storage protocols that are able to maintain nematode viability and infectivity. The larger markets have longer transport chains and formulated nematode products therefore also need a longer shelf life. In order to overcome these problems, BIOCOMES aimed at the improvement of harvest technology and a prolongation of the shelf-life of EPN products by technical and genetic approaches
After production of the IJs in liquid culture they need to be separated from the liquid substrate for further storage and delivery to the end-user. During downstream processing the viability and infectivity of the harvested IJs needs to be preserved. During the project, the performance of several separation devices was compared with a disc separator, which had been used so far in the company. An industrial decanter system was able to outperform the standard equipment. Process parameters were improved for the decanter and summarized in a standard operation protocol, now allowing the separation of the IJs from the culture broth without losses at a flow rate of 2.000 l/h, whereas only 400 l/h could be obtained with the separator. For both devices, the survival and infectivity of the harvested IJs revealed no significant differences. Additionally, the decanter turned out to be less labour intensive compared to the separator. Investment costs for both downstream systems are nearly the same.
The IJs are stored at high density in cooled tanks in an isotonic solution. The storage time was so far limited to 6 weeks. During that time the nematodes need to be formulated and sent to the customer. Because of the relative short shelf-life of the nematodes they needed to be produced and delivered on demand. This is a handicap for the market access compared to chemical pesticides, which usually have a shelf-life of several years. An increased survival of the nematodes during storage would give more flexibility for the timing of production and would allow a better capacity utilisation of the bioreactors. Currently the nematodes are stored at densities of up to 400,000/ml. At higher densities the survival in the storage tanks was significantly reduced. When the nematodes are harvested from the bioreactor the density is usually at 200,000/ml and more. This means that at least 50% of the bioreactor volume needs to be provided in cooled storage tanks, which causes high investment and energy costs. This is one factor keeping the product cost for EPNs at a higher level compared to chemicals and can also hamper the scale-up to higher production capacities. Additionally, the IJ mortality during storage can cause further losses and keep the production costs on a high level. Especially detrimental bacterial contaminations can cause severe losses during storage, which, in the worst case, can result in the loss of a whole storage tank with several thousand litres, representing an economical loss of more than 100,000 € per year. The detrimental contaminations are usually accompanied by an ammonia gas and/or hydrogen sulphide smell, indicating anaerobic bacterial metabolism. As soon as microbial contaminations are recognized during storage the nematodes must be separated from the storage solution, washed and re-suspended in fresh storage solution, which is laborious and is causing stress to the nematodes and can reduce the storage time.
The investigations into the liquid storage system have demonstrated that it is possible to store the harvested IJs at a density of 800,000/ml by using higher aerations rates to the nematode storage suspension. This will reduce the investments into additional liquid storage capacities in the future. A new sieving technology was tested and allows further washing steps after separation from the culture broth, which reduces the risk of detrimental microbial contaminations during storage. This sieving machine can also be used to exchange the storage solution during storage or for recycling by a water treatment system. Increasing sulphide concentrations were found to be correlated with the nematode mortality during storage and can be a possible indicator for the contamination with detrimental anaerobic bacteria. However, these benefits are not without tradeoff effects, like higher energy costs for higher aeration rates or investments into new technology.
Before the nematodes can be send to the distributor or end user they need to be formulated in a water dispersible granular carrier, which keeps up a high humidity and guarantees a sufficient gas exchange. The shelf-life of the resulting product is so far limited to 6 weeks only and consequently the product needs to be delivered on demand. This is a handicap for the market access compared to chemical pesticides, which usually have a shelf-life of several years. A longer shelf-life would allow sending bigger product batches to local distributers and thus would reduce logistic efforts and costs. Furthermore, a longer product shelf-life would increase the flexibility for the end user regarding the schedule for the product application. No technical measures could finally be identified during the BIOCOMES project, which could guarantee a longer shelf-life of the formulated product based on the nematode H. bacteriophora. Disinfection of the EPN during liquid storage and directly before formulation can reduce microbial contamination, but a positive influence on the storability of the nematodes could not be recorded.
Two main objectives for the nematode production part of BIOCOMES were the improvement of the storage time of the nematodes in liquid and the shelf-life of the product. These two objectives were not achieved by technical measures. The reasons seem to be biological limitations in the so far commercially used strain of H. bacteriophora. The virulence of the commercial strain EN01 could not be kept up for more than 4 weeks during storage and in the formulated product even though the nematodes could survive for more than 10 weeks. However, it has been demonstrated that strains, which resulted from activities of WP2 within BIOCOMES and were obtained by genetic selection for longevity and virulence, can be kept longer and their higher virulence in maintained during storage in liquid and in the formulated product. The breeding for an improved nematode strain will be continued beyond the BIOCOMES project.
Genetics for trait improvement of entomopathogenic nematodes (WP2)
BIOCOMES WP2 was working on the genetic improvement of beneficial traits of the biological insect control nematode Heterorhabditis bacteriophora. Objective of the project was to produce nematodes with higher virulence and longevity in order to be able to apply less nematodes per area and thus lower the application costs for nematode products. Starting point was the characterisation of the traits survival, virulence, and desiccation-tolerance of nematode Dauer Juveniles (DJs) within a collection of natural strains and isolates. The DJ is a non-feeding developmentally-arrested stage well adapted to long-term survival. This stage is also used in nematode products. It is responsible for insect host finding, invasion into the host, overcoming the insect´s immune defence and finally killing the insect hosts. E-nema (Germany) dedicated the efforts to predict the survival of H. bacteriophora DJs by developing a bioassay based on the resistance to oxidative stress (exposure to hydrogen peroxide H2O2). Oxidative stress is an internal metabolic misbalance that affects aging in all living organisms and its resistance correlated well with nematode survival. The test was complemented by storing the nematodes at different temperatures. In general, DJs stored at 7°C lived longer than DJs stored at 25°C. Under oxidative stress, all DJs lived substantially shorter at both temperatures. By applying this test, the variability on survival along forty H. bacteriophora natural isolates was determined. The results indicated that DJs from certain isolates survived well across all test conditions (best survivors). These individuals were selected as parents for genetic crosses and for genomic analyses.
The project partners at the Volcani Centre (Israel) focused their efforts on characterizing the tolerance to desiccation in a subset of H. bacteriophora natural isolates. Experiments using hygroscopic and evaporative desiccation methods were carried out to survey DJ mortality. The survival of the nematodes was followed over time up to 96 hours after stress induction. Similarly to the observations made under oxidative stress, desiccation-tolerant and –sensitive isolates were determined. With this information in hand, two time-points (24h and 36h) under hygroscopic and evaporative conditions were chosen for molecular analyses of the DJ’s desiccation tolerance in the H. bacteriophora commercial strain.
The H. bacteriophora virulence was evaluated at e-nema. Two host insects were used: i) mealworm (Tenebrio molitor), an easy-to-handle insect, and ii) larvae of the western corn rootworm (WCR, Diabrotica virgifera virgifera), an invasive maize pest. As parameter for virulence, the number of DJs per insect required to kill 50% of an insect population (Lethal dose, LD50) was determined for all isolates. The LD50 against T. molitor did not exactly correlate with the LD50 against WCR. However, a few isolates presented low LD50 (high virulence) for both hosts. Partners at the University of the Azores (Portugal) identified two genetically similar Portuguese H. bacteriophora isolates that had contrasting LD50 against T. molitor.
In a next step, WP2 focused on generating massive genetic sequence information related to desiccation, survival and virulence in H. bacteriophora. This was done by making a full inventory of all expressed genes in DJs under a given condition. Gene expression provides detailed information on genetic constructs coding for proteins which regulate the activity of an organism. The inventory of gene-expression messenger RNA (mRNAs) of DJs exposed to low relative humidity was assessed at the Volcani Centre. DJs under normal conditions were compared against DJs held for 24h and 36h under desiccation stress. The inventory of mRNAs of DJs under oxidative stress was assessed at e-nema in two H. bacteriophora isolates with contrasting longevity. Both isolates were challenged with oxidative stress and 4h hours after stress induction a “snapshot” of their gene expression (transcriptome) was taken. Partners at the University of Azores did the expression inventory along the host infection process. To filter information, experiment setups simulated the host infection process in three ways: i) simulation by a physical stimulus was done by inducing the loosening of an external cuticle, which is only lost in DJs upon host infection, ii) simulation by a chemical stimulus was done by provoking developmental changes in the DJs triggered by a chemical compound, and iii) simulation by a biologic stimulus was done by putting DJs in contact with the haemolymph of the host. The haemolymph in insects is homologous to the human circulatory system. In this way, only haemolymph-exclusive expressed genes were detached from the background-noise caused by the other stimulation conditions.
This large gene expression inventory from virulence, survival and desiccation studies yielded more than ~55.000 distinct expression messages (mRNAs) observed in any of the situations. Approximately 17% of the messages were common for the three approaches, whereas 5% were exclusive to desiccation, 6% for oxidative stress and almost 50% were exclusively seen in the virulence analysis. Each project partner focused on filtering and interpreting this large amount of data to detach the physiologically important messages (genes) from the background information (complete transcriptome) and to try to identify the related genes (gene annotation). Several genes ligated to the catalysis of oxidants were found to be expressed at different magnitude (differentially expressed) after deep analysis of the oxidative stress expression survey. Also, genes involved in protein protection and on stability of cells and tissues appear to have an important role in oxidative stress tolerance. The DJ reaction to desiccation shared some important messenger RNAs with the oxidative stress responses. Genes involved in protein stability, protection of the cells and tissues structure, and genes involved in lipid metabolism were considered relevant for these conditions. The physiological mechanisms behind the virulence process in DJs appear to involve genes of very diverse function. Enzymes (proteases and peptidases) are secreted by the DJs and have the function to degrade proteins of host membranes. They play a major role along the DJ infection process. Additionally, the very comprehensive gene expression surveys were complemented by the search for very small differences in the genomes of H. bacteriophora natural isolates. These small differences are changes in single positions on the DNA. They are referred to as single nucleotide polymorphisms (SNPs) and together they can form “bar-codes” that can differentiate individuals. More than 1.100 SNPs were found in the H. bacteriophora genome. This information was then used to search for links between natural isolates showing particular bar-codes and their virulence and survival. With this approach we identified more than 10 bar-codes (molecular markers) that are related to virulence or survival. In addition, the genome of H. bacteriophora has been sequenced and the results will be used to further characterize genes and markers related to beneficial traits of the biocontrol agent.
In a parallel step to the acquisition of expression information, genetic crosses were done between H. bacteriophora isolates with enhanced survival and virulence. The progeny of the genetic crosses served two main purposes: i) to produce hybrid strains to be tested for their efficacy in lab and field trials and ii) to derive families of highly homogeneous sister lines (recombinant inbred lines), in which the genetic information (genotype) of the parents is differently distributed (segregation). Products from genetic crosses that showed high virulence and survival were evaluated for their efficacy. Comparative laboratory assays evaluated the virulence of these cross products in comparison to DJs of the current nematode commercial line. Subsequently, field-simulation tests were carried out at e-nema to evaluate the efficacy of the DJs under semi-field and finally field conditions. Nematodes were applied in maize field trials in central Europe to evaluate their effectivity against the western corn rootworm and to compare their performance with a commercial strain and chemical control agents (Belem = Cypermethrin). The effect of the DJs application on the pest population and the effect on the maize plants damage was quantified. In the majority of the semi-field and field trials the selected strain performed better than the alterative products. The number of detected adult insects was reduced and less plant damage was determined in plots treated with the new strain, compared to plots treated with the current commercial nematodes. Even more important, plots treated with any of the nematode strains produced better pest reduction and healthier plants than plots treated with the chemical control agent.
Apart from a broad platform of pheno- and genotypic data, BIOCOMES WP2 provided valuable tools (markers) for future genetic improvement of H. bacteriophora for cross-breeding and selection.
BCAs for control of forest pests and diseases (WP3)
Workpackage 3 targeted three biotic threats to forestry, two insects and one disease. The BCAs under development were: a virus for the gypsy moth Lymantria dispar, insect-killing nematodes for the large pine weevil, Hylobius abietis, and various microbial agents for the disease complex responsible for “damping off” in nursery seedlings.
The aim of WP3.1 was to find a highly active Lymantria dispar nuclear polyhedrosis virus (LdMNPV) and build up a large-scale production of LdMNPV with a high level of automatization including the development of an effective and easy to use product formulation. In a first step, an initial L. dispar population was acquired and a small-scale rearing was set up. This provided the basis to test and implement adaptions toward a large-scale production system. This included trials regarding the biology of L. dispar, as well as automatizations on the rearing, virus and downstream side. Furthermore, the quality control for the product was set up. While building up the large-scale production, a virus database has been set up with 15 different strains from all over the world. A new bioassay was developed to compare all the strains and the most effective strains were evaluated. The best two isolates have been used to develop new liquid formulations, which were tested in field trials. Experiments performed by Forest Research Institute (Poland) in WP3.2 aimed to assess the insecticidal activity of gypsy moth Lymantria dispar virus (LdMNPV) formulated by Andermatt Biocontrol. The trials consisted of greenhouse bioassay and field treatments. In greenhouse experiments the caterpillars of gypsy moth were reared on birch (Betula pendula) leaves sprayed with different concentrations of LdMNPV to estimate its biological activity based on the feeding activity and mortality of infected caterpillars. The results of greenhouse experiments showed that the use of LdMNPV reduced feeding intensity and increased mortality of insects up to 87%. The field trials included ground spraying treatments with different concentrations of virus applied to alder (Alnus glutinosa) and birch B. pendula trees colonized by gypsy moth. Due to the limited occurrence of L. dispar populations in Polish forests in 2016-2017, experiments involved placing laboratory-reared caterpillars in mesh sleeves attached to B. pendula and oak (Quercus robur) trees sprayed with different concentrations of LdMNPV. The efficacy of all field treatments was assessed based on insect mortality and on the comparison of damage to leaves collected from treated and untreated trees. Field application of LdMNPV to A. glutinosa trees led to a 4-fold reduction of leaf damage and increased mortality up to 100% of L. dispar caterpillars. The use of LdMNPV to limit populations of L. dispar naturally developing on B. pendula trees resulted in the 4-fold reduction of leaf damage with the highest concentration. Rearing of L. dispar in sleeves attached to birch and oak trees sprayed with two isolates of LdMNPV led to increased mortality of up to 83% of caterpillars and up to 4-fold less leaf damage compared to untreated leaves. The results of greenhouse bioassay and field treatments showed the high activity of LdMNPV which, when applied to deciduous trees resulted in increased mortality of L. dispar and the reduction of damage to leaves caused by gypsy moth caterpillars.
The large pine weevil Hylobius abietis is the most important pest of tree seedlings in replanted coniferous forests in Northern Europe. Adult weevils feed on and can kill newly planted seedling. The weevils develop in stumps of felled trees. In WP 3.3 various strains of entomopathogenic nematodes (EPN) supplied by E-nema were applied to stumps in two regions of Europe with contrasting conditions: Ireland, where soil moisture is usually not a limiting factor but many plantations are on deep peat soils, and Poland, where soil moisture may be a limiting factor for nematode efficacy. Nematodes could parasitise the immature stages and reduce the number of adult weevils emerging. In Poland, Heterorhabditis downesi was the best of four EPN species trialled, with significantly higher parasitism rates than by other species. However, applications did not affect the level of damage caused by H. abietis beetles to Pinus sylvestris seedlings on experimental areas. While nematodes worked surprisingly well (up to 17.5% of weevil larvae were parasitized) despite the drought conditions prevailing during trials and also the great depth at which weevil larvae are typically found in Poland, application was not sufficiently successful to recommend use of nematodes under such conditions. Higher parasitisation levels (up to 70%) were recorded in Ireland where the higher rainfall and shallower distribution of weevils in soil are probably factors favouring greater success of EPN. In small scale trials both Steinernema carpocapsae and H. downesi were equally effective in suppressing pine weevil populations to below the current, informal thresholds of economic damage. EPN were equally efficient in controlling pine weevil in deep peat and in mineral soils typical of conifer plantation sites in northern Europe. Several factors affecting weevil parasitism were examined, including method of application, weevil distribution and stump size. In conclusion, EPN could contribute to reducing weevil populations under a wide range of conditions, as long as the two main conditions of adequate soil moisture and weevils not too deep in soil are met, and their use can be recommended for inclusion in IPM programmes for pine weevil. Results from these trials provide guidance towards choice of EPN species, application method and site characteristics favouring EPN success.
Tree seedlings are produced in forest nurseries, bare-root or container, where they have the best conditions for development. The greatest threat to the planting stock production is a fungal disease, damping-off, caused by a complex of soil-borne pathogens. The pathogens are able to infect a germinating seed or young seedling resulting in a decline. Compared to agriculture, foresters have a limited number of fungicides allowed to use against damping-off and almost no biological control products are available on the market. The aim of WP3.4 was to test three biological control products developed by University of Padova/Biogard and Graz University/E-nema against damping-off. A beneficial fungus Trichoderma harzianum INAT11 was used for soil treatment in order to reduce the amount of the pathogen inoculum in soil as well as to enhance plant resistance. The dose was set at 100g per ha with concentration of fungal spores of 1x1010 CFU/g, applied in aquesous suspension three times during the growing season at 10-day intervals. The two bacterial strains – Paenibacillus polymyxa and Serratia plymuthica were used for seed coating in concentration of 1x107 CFU/ml and 1x109CFU/ml respectively. Additionally, the seedlings were watered twice with bacterial suspension at various concentrations during the growing season. The tests were run in the greenhouse and in two forest nurseries. The criteria for BCA efficacy were as follows: seedling survival rate, biometric parameters of seedlings, fine roots parameters, community structure of mycorrhizal fungi, chlorophyll fluorescence (photosystem II activity) and leaf area index (LAI). The BCA containing T. harzianum successfully suppressed the pathogens in the laboratory tests and reduced damping-off in the greenhouse. In open field trials, it had no consistent impact on the disease nor the other parameters. What is important is that T. harzianum did not have a negative effect on the mycorrhizae, which are necessary for optimal seedling development in forest plantation. Trichoderma harzianum was found to be very sensitive to weather conditions, especially low temperature. Biological control agents containing bacteria successfully reduced damping-off in the greenhouse and in the forest nurseries. There was also an improvement of selected seedling biometric parameters and leaf area index, but at the same time a reduction of mycorrhiza abundance was observed, connected with increased abundance of dead roots. In conclusion, we see a potential of T. harzianum as a biological control agent dedicated for container nurseries, where the seedlings are produced under controlled conditions. In case of P. polymyxa and S. plymuthica there is a need to develop product formulation and its further tests, since there is a potential of these BCAs in protection against damping-off.
PhopGV for control of Tuta absoluta (tomato) and Phthorimaea operculella / Tecia solanivora (potato) (WP4)
Tuta absoluta Merick (Tuab), Phthorimaea operculella Zeller (Phop) and Tecia solanivora Povolny (Teso) and are three lepidopteran species from the Lepidopteran family Gelechiidae causing severe economic damage to important crops, particularly in potatoes and tomatoes. For these three pest species, traditional control methods with chemical insecticides are no longer efficient and baculovirus-based pesticides are a recommended control measure. The aim of WP4 was the development of an efficient baculovirus-based biocontrol agent that is effective against Tuab, Phop, and Teso.
A broad genetic background is essential for the development of a new and effective baculovirus biocontrol agent. Therefore, as a first step, new PhopGV isolates were field-collected in Italy and Greece. A total of 23 different PhopGV isolates were finally available and exchanged among the partners. Small-scale rearings of the pest insects were established and standardized bioassay protocols were developed. Unfortunately, it was not possible to work with the pest Tecia solanivora for quarantine reasons. Adequate screening methods were developed and bioassay series were performed on different pests. All available PhopGV isolates were screened for their activity and a selection of isolates was characterized by molecular means, including PCR and sequencing of selected PhopGV gene fragments, restriction fragment length polymorphism (RFLP) of isolated viral DNA, and next generation sequencing (NGS). Molecular analyses confirmed that PhopGV genomes are highly conserved, but based on NGS whole genome sequencing data, the isolates could be successfully differentiated. Full genome sequences were established for 11 isolates. In general, the isolates only moderately differed in their activity, except for three isolates, which showed a significant higher activity.
The most pathogenic and virulent PhopGV isolates were finally selected for further development of a test product to be used in greenhouse and field experiments. As baculoviruses are produced in vivo, a stable and cost-effective large-scale rearing of Phop was developed, which has been proven to be the most suitable host for the production of the virus. In vivo propagation of PhopGV was continuously optimized for a cost-effective production process. Furthermore, an adequate downstream and formulation process was developed.
Four test products of the most promising PhopGV isolates were developed and chosen for field testing. Small test batches were produced and trial protocols were developed. Several independent field, greenhouse and warehouse trials were conducted over two years in Italy and Greece. Field and glasshouse trials including chemical and non-chemical reference products have shown promising results. Dose-effect relationships as well as significant efficacies were found. The evaluation of an economically feasible and at the same time sufficiently efficient application rate will be crucial for the success of the product. The most active PhopGV isolate allowed a significant damage reduction and a further reduction of the application rates of the test product and the application of a PhopGV product appears to be economically feasible.
Bacterial seed treatments against Brassica diseases with focus on oilseed rape (WP5)
The overall objective of the WP5 project was to develop a sustainable method of protecting oilseed rape and Brassica vegetables against fungal pathogens using seed treatment with beneficial bacteria with the focus on Verticillium wilt. Verticillium wilt is caused Verticillium spp. in a wide range of plants, while V. longisporum is the main cause of Verticillium wilt in oilseed rape (OSR). Serratia plymuthica HRO-C48 and Paenibacillus polymyxa Sb3-1 were previously identified as promising biocontrol agents against Verticillium wilt in oilseed rape. In order to identify the mode of action of the selected biocontrol agents we (1) carried out the genome analysis of the selected BCA; (2) looked into the mechanisms how the selected microorganisms react towards contact with the volatile organic substances of plant pathogenic fungi by using transcriptomics approach and gas/mass spectrometry; (3) studied the colonization patterns of the selected microorganisms in the oilseed rape. Both microorganisms contained a range of genes putatively involved in biocontrol and plant growth promotion, as was shown by genome analysis of S. plymuthica HRO-C48 and P. polymyxa Sb3-1. We found that S. plymuthica HRO-C48 changed its metabolisms towards maintaining energy usage of the cell, reduction of motility and enhancement of the biofilm production upon contact with the volatile organic substances of plant pathogenic fungi V. longisporum, Leptosphaeria maculans and Rhizoctonia solani. The strong in vitro antagonistic effect of S. plymuthica against R. solani correlated with the upregulation of transcripts responsible for antagonistic effects in S. plymuthica HRO-C48. A range of antimicrobial substances was upregulated in P. polymyxa Sb3-1 upon its interaction with Verticillium volatiles, correlating with its strong ability to inhibit the growth of the plant pathogen in vitro. The colonization patterns of biocontrol agents observed by confocal scanning laser microscopy showed that the Serratia cells were either found as clouds surrounding the roots, or they formed large micro-colonies in the root tissue. The Paenibacillus colonies were often detected in the areas surrounding damaged root and leaf tissues or in cavities where they formed large micro-colonies.
We developed strain- and species- specific markers for identification of the applied strains in the soil samples. P. polymyxa Sb3-1 applied to the seeds using pelleting resulted in significant improvement of the lengths of the plants infested with V. longisporum. We found that the effects of the formulation and fermentation with BCA strains was strain- and disease-pressure specific. The selected BCAs were able to reduce disease the symptoms of Plasmodiophora brassica (clubroot). We observed mild to significant reduction of Leptosphaeria maculans infection rate due to the treatments with S. plymuthica HRO-C48 and fungicides in field trials. Verticillium wilt disease reduction was found to be cultivar dependent; i.e. seed treatment with P. polymyxa Sb3-1 reduced Verticillium wilt symptoms in Avatar, but not in Traviata and Sherpa oilseed rape cultivars. This cultivar specificity was confirmed on the seed microbiota level. We found that cultivars Sherpa and Traviata contained higher indigenous diversity within their seeds than Avatar cultivar and were characterized as having a higher colonization resistance against beneficial and pathogenic microorganisms in the field and greenhouse trials.
The unexpected reduction of the germination rate of oilseed rape in one of the field trials due to the treatment with P. polymyxa Sb3-1 and observed phytotoxic effects in vitro lead to the decision to screen endophytes of Brassicaceae for novel V. longisporum antagonists that will replace P. polymyxa Sb3-1 and enhance the effect of the S. plymuthica HRO-C48 by being the co-formulates for the optimized seed treatment against fungal diseases. The selected novel Brassica endophytes had neutral to positive effect on the growth of the oilseed rape seedlings in greenhouse trials. The new endophytic isolate Serratia plymuthica F20 showed strong positive effect against Verticillium wilt in two independent field trials. Further trials are necessary to prove the effectivity of this strain and its combination with S. plymuthica HRO-C48 under adverse field conditions.
In addition, our studies gave new insights into the outbreak of soil-borne diseases. Verticillium spp. is a ubiquitous endophyte in many plants, and probably fulfill important functions, e.g. in the maturity. Only a general loss of microbial diversity or dysbiosis causes an outbreak of the pathogen. We therefore propose that this “microbiome disease” can be suppressed by increasing diversity, which can also be supported by BCAs. They can be applied in complementary seed treatments.
Trichoderma harzianum DSM25764 seed treatment for control of Fusarium in cereals (WP6)
Fusarium head blight of wheat (FHB), Fusarium graminearum, and pink ear rot of maize (PER), F. verticillioides, are two plant diseases causing severe quantitative and qualitative yield losses and mycotoxin contamination in feed and food.
In preliminary studies conducted by University of Padova (Italy), Trichoderma harzianum strain INAT11 (deposit number DSM25764), henceforth strain INAT11, had shown promising activity against FHB and PER. The aim of WP6 was to develop a seed treatment for maize and other cereals for control of Fusarium spp. based on the already selected strain INAT11.
Strain-specific primers for strain INAT11 were obtained by University of Graz (Austria). A strain-specific detection tool for strain INAT11 is now available for use, enabling to specifically identify the strain among other Trichoderma species and strains.
A highly concentrated WP (Wettable Powder) formulation of strain INAT11 was developed. This formulation proved to be appropriate for seed treatment of maize and wheat, and showed acceptable shelf life (no decline in spore viability even after 12 months of storage at 5±2°C).
The knowledge on the characteristics of strain INAT11 was complemented. In particular, the effects of environmental factors (temperature, pH, Water Activity, soil type), as well as effects of the two pathogens (F. verticilloides and F. graminearum) and plant species (maize and wheat) on the growth of strain INAT11 were investigated in laboratory, greenhouse and field studies. The optimal conditions for growth of strain INAT11 under laboratory conditions were warm temperatures (above 10°C), sub-acidic soil pH and high water activity. However, under greenhouse and field conditions, extremely complex interactions among plant species, soil pH, water availability, pathogens and strain INAT11 emerged, and root colonization by strain INAT11 resulted to be influenced by a combination of factors and not just one single factor.
Strain INAT11 showed rhizosphere competence on both target crops, with root colonization levels being in general higher on maize than on wheat in both greenhouse and field studies.
In greenhouse trials, strain INAT11 applied as seed treatment, was able to significantly reduce symptoms of F. graminearum and F. verticillioides infections in respectively wheat and maize on aerial parts. In laboratory tests a variation in the expression of the majority of the markers selected to investigate the induction of systemic resistance was observed, confirming the hypothesis of systemic resistance induced by strain INAT11.
Open-field trials were conducted over two years in Sweden, the Netherlands and Italy (total n. trials: 28). In these trials, the strain INAT11 seed treatment was tested at different application rates in comparison to an untreated control and a chemical reference seed treatment. Strain INAT11 seed treatment was effective in controlling F. graminearum and the foliar disease Septoria sp. in winter wheat, and F. graminearum in maize. Results on the activity against F. graminearum in spring wheat and F. verticilliodes in maize, instead, were not consistent. The minimum effective doses for maize and wheat, which had been proposed based on the results of previous laboratory and greenhouse studies, were effective in controlling F. graminearum on winter wheat and maize also under open-field conditions.
Compatibility studies with conventional plant protection products, commonly applied to seed and at or shortly after sowing, and with stickers, commonly used by stakeholders to make plant protection products adhere to seed, were performed. The studies showed that the combined use of strain INAT11 with fungicides should be avoided due to growth inhibition of the BCA by most of the tested fungicides. Herbicides, insecticides, stickers and bird repellents, instead, may be used in combination with strain INAT11, but compatibility studies should always be performed, if effects are not yet known.
The main foregrounds achieved in this work package are a strain-specific detection tool for strain INAT11, a commercial formulation of strain INAT11 for seed treatment with acceptable shelf life, knowledge on factors (e.g. temperature range for optimum activity, compatibility with conventional plant protection products commonly used on wheat and maize, and many more), which may impair the efficacy of strain INAT11 in the field, and the field trials conducted in compliance with the principles of Good Experimental Practices (GEP) as defined by The European and Mediterranean Plant Protection Organization (EPPO) and with the relevant EPPO guidelines, which can be used for the registration of the formulated product.
Penicillium frequentans 909 and Bacillus subtilis CPA-8 for Monilinia control in stone fruit (WP7)
Brown rot caused by Monilinia spp. is one of the economically most important fungal diseases on stone fruit causing substantial pre-harvest and post-harvest losses worldwide. Alternatively to chemical application, workpackage 7 aimed to complete the development of two biocontrol agents (BCAs) Bacillus amyloliquefaciens CPA-8 and Penicillium frequentans 909 (Pf909), to obtain microorganism-based products that provide an effective strategy to control brown rot disease. Specifically, this work aimed to meet regulation requirements to develop products suitable to be registered and commercialised: molecular maker design, physiological characterisation, mode of action, production and formulation and definition and validation of biocontrol strategies based in both BCAs.
Molecular markers based on RAPD-SCAR methodology or related to ecological adaptations were developed for both BCAs, providing suitable monitoring tools to specifically identify and track them. Moreover, better understanding of the microorganisms’ biology and physiology was achieved focusing on key ecological parameters (temperature, water activity and pH), the susceptibility pattern to different antibiotics or fungicides and toxicity evaluation, providing useful information for registration process. On the one hand, competition for nutrients and space has demonstrated to be the primary mode of action of Pf909 against Monilinia spp. and on the other hand, mode of action of CPA-8 is based on its capability of production of powerful antifungal metabolites such as fengycin-like lipopetides, although volatile organic compounds (VOCs) emitted by this strain have also been described to play an important role. Regarding the production strategies, many criteria were optimized to meet the product quality requirements: culture media based on products and by-products from the food industry and growth conditions (temperature, pH, agitation, aeration, initial inoculum and time duration). Protocols for the mass production of Pf909 (solid state) and CPA-8 (liquid state) have been defined and quality control systems for both processes have been designed. Furthermore, downstream processing protocols were designed to separate the biomass from the spent medium. Once production was solved, formulation process was deeply studied. Oil-diluted forms were successfully developed for Pf909 and different formulation systems have been assessed to formulate CPA-8: liquid, freeze-drying and fluid-bed spray-drying, the latter being the most adequate. Once the best formulation approach was defined, different processing parameters (protectants, adjuvants, and carrier materials) were optimized to obtain products with a stable shelf-life and high performance under commercial conditions. Different storage materials and air packaging conditions were also studied in order to ensure product stability and easy manipulation. Oil-diluted and fluid-bed spray-drying formulations optimized for either Pf909 or CPA-8 in the suitable packaging and stored at cold and room conditions respectively, had at least a shelf life of 12 months and maintained biocontrol efficacy. The spectrum of action of both BCAs has been checked and broadened to all stone fruit species; peaches, nectarines, plums, apricots, cherries, flat peaches and flat nectarines. Furthermore, technical application thresholds for BCA products were determined and a wide tolerance to unfavorable environmental conditions of temperature, humidity and rainfall has been demonstrated for Pf909 and CPA-8 products.
Effective strategies to control brown rot based on the application of both biocontrol products (CPA-8 and Pf909) combined or separately were designed and studied during 2014 and 2015 seasons at field conditions in commercial orchards. Combination of both BCAs did not improve the efficacy of each of them alone and final strategies chosen for validation were based only on one product, the fungal or the bacterial product separately. Doses, calendar of application and effect of adherents and wetting agents were also optimized for the BCA products. Moreover, dispersion and persistence of both BCAs under field and packinghouse conditions were evaluated. A good distribution, persistence and adaptation of CPA-8 to field and postharvest conditions was demonstrated and although Pf909 had less dispersion and persistence than the bacterium, its presence on fruit during season and postharvest was sufficient to control Monilinia spp. The effect of both BCAs on non-target organisms was also studied. All these results are useful and necessary for products registration.
Finally, CPA-8 and Pf909 control strategies have been validated in different stone fruit hosts (peaches, nectarines and cherries) and four different producing countries (Belgium, France, Italy and Spain) over 2 seasons in commercial orchards under EPPO standards. The results obtained indicated that the degree of biocontrol products activity was dependent on the level of inoculum pressure present in the orchard. Three different scenarios have been defined: Scenario 1 (No disease), in which there was no presence of inoculum in the field and consequently no treatments were needed. Scenario 2 (Uncontrollable disease), in which high pressure of inoculum in the field and extremely adverse meteorological conditions occurred, and treatments in general (biologicals or chemicals) are not worthwhile. And finally, Scenario 3 (Controllable disease), in which it is worthwhile and possible to control the disease. This was the case in 62.5% of trials conducted in this study, demonstrating that under low-medium or medium-high inoculum pressure, BCAs products strategies were able to control brown rot even similar to chemicals.
These encouraging results allow to conclude that formulated products of CPA-8 and Pf909 developed in BIOCOMES project are ready to be registered and commercially available to control brown rot in a near future, providing a valuable tool for farmers to produce stone fruit more competitive and cover consumers demand for safer and more environmental friendly products.
The main foregrounds achieved in this workpackage are the formulated products based on CPA-8 and Pf909 ready to be registered and commercialized, the completely defined optimized production and formulation processes for both BCAs, the quality control protocols that are easily implemented to other BCAs and the data set of all field trials in different producing countries that can be used for the registration documentation.
Fungal BCA for control of powdery mildew in cereals (WP8)
The objective of WP8 was to develop microbial biological control agents for spray applications for control of powdery mildew in wheat and other small grain cereals. In a first step, a collection of more than 1200 fungal isolates from powdery mildew pustules has been built up. Leaf samples of cereals, grasses and other plant species with powdery mildew pustules were collected from March to July 2014. From 504 leaf samples, 1237 fungal isolates were obtained from powdery mildew pustules. 889 isolates with regular colony development and abundant spore production were tested in a pre-screening for the following criteria: no growth at 36°C (safety), germination and growth at 5°C (cold tolerance), germination and growth at -7 MPa and at -13 MPa (drought tolerance) and growth after exposure to UVb (UVb tolerance). 748 Isolates fulfilled the pre-screening criteria. From these isolates, 732 could be identified. They belong to 86 different fungal taxonomical groups. However, more than 60% of the isolates belonged to various species within the genus Cladosporium. In the next step, another set of 27 isolates was excluded because they belonged to species which were considered as not safe for humans, animals or plants. The major group of these isolates belongs to various Fusarium spp. which are plant pathogenic and known mycotoxin producers. With the remaining most suitable 185 candidate antagonists a series of bioassays under controlled conditions has been conducted to quantify the effect of those candidate antagonists on powdery mildew. Eight bioassays were conducted with winter wheat plants cv. Julius in which 11 to 26 candidates were tested per assay. In total, 143 different isolates belonging to 42 taxonomical groups were assessed. The remaining 42 isolates, originally selected during the pre-screening program, were excluded from further testing in bioassays because they did not produce sufficient amounts of spores and thus are not suitable for mass production.
From the 143 isolates tested in bioassays on winter wheat, some isolates showed antagonism against Blumeria graminis f.sp. tritici. Based on the overall results, 10 isolates were selected for first small-scale field experiments to compare their potential for powdery mildew control under open field conditions. With these isolates a series of 10 small-scale experiments with potted spring wheat was conducted in the period between 2 May and 21 June 2016 to assess the potential of the applied antagonists under different climatic conditions and different conditions favouring powdery mildew development. Overall results of experiments with potted spring wheat showed that isolates of Tilletiopsis pallescens and Cladosporium spp. have a potential to reduce powdery mildew on wheat. This was shown occasionally during 7 experiments where disease levels were low, but more consistent in 3 other experiments where disease levels allowed a better estimation of treatment effects. Also in 2016 a preliminary small-scale field experiment in a spring wheat crop has been performed. Applications of T. pallescens isolates BC0441 and BC0850 significantly reduced the number of powdery mildew pustules on flag leaves, the leaf coverage with powdery mildew and the growth rate of the mildew epidemic.
In 2017 the 2 most promising strains BC0850 and BC0902 of Tilletiopsis pallescens and strain BC0707 of Cladosporium delicatulum have been tested in 8 field trials this year in Sweden and the Netherlands. Field trials showed significant effects for lower powdery mildew severity for BC0902 in 2 trials in the Netherlands and in 1 trial in Sweden; for BC0850 in 1 trial in Sweden and for Cladosporium strain BC0707 in 1 trial in the Netherlands. Unfortunately, viability tests and bioassays performed with inocula of T. pallescens used in the field trials showed that their quality was poor. Consequently, results of the field trials cannot be used to evaluate the biocontrol potential of these antagonists.
First studies on the mode of action of the T. pallescens isolates indicate that hyperparasitism and production of enzymes degrading fungal cell walls are involved. No signs for induced resistance were found in treated wheat.
Isaria fumosorosea for pest insect control in vegetables (WP9)
The objective of the scientific work in this workpage was to develop a microbial biocontrol agent product, based on the entomopathogenic fungus Isaria fumosorosea for the control of pest insects under different Field and Greenhouse conditions.
It was examined whether the already existing potential of a prototype sample based on an Isaria fumosorosea isolate can be improved. First of all, the strengths and weaknesses of the existing isolate were investigated. Objective was to add a, in its strength and weaknesses complementary isolate of I. fumosorosea to a new formulation in order to compensate the weaknesses and to increase the activity and lastly the efficacy. For this purpose, the growth behavior at six different temperatures within the range of 5 – 37 °C of multiple strains of an existing isolate collection of Isaria fumosorosea belonging to a project partner were evaluated. Also the sporulation behavior of the strains in solid-state fermentation was compared. Randomly Amplified Polymorphic DNA (RAPD) was developed as an identification method of the evaluated strains.
The isolates proven to be of value for the further development were then tested under defined conditions in laboratory climatic chambers for their control effect on whitefly (Bemesia tabaci, Trialeuroides vaporarorium), amyworm (Spodoptera littoralis, Spodoptera exigua) and diamondback moth (Plutella xylostella).
As a result after 1.5 years of investigations, a second isolate was selected for the further development under field and greenhouse conditions. Together with the initial strain from the prototype product this strain was tested in two different formulations (four variants) in the first field test year. The concentration of the microbial pest control agent (MPCA) in these formulations was initially chosen to be very high in order to better visualize the control effect on Bemesia tabaci and Plutella xylostellea.
Storage stability studies of the two formulations showed significant differences in shelf life regardless of the strain used and thus also served as a decision criterion for selection of the formulation in the further field tests.
The field and greenhouse tests were conducted at three different sites (Italy, Greece and Sweden) according to GEP standards and EPPO guidelines PP1 / 152. 135 and 181 performed over 2.5 years. In each case, the white fly trials in Greece and Italy demonstrated a significant reduction in the insect population with a lower initial incidence in tomatoes and peppers. In Sweden, the natural potential of infestation with Plutella xyllostella was used to demonstrate the effect of I. fumosorosea product patterns on different varieties of cabbage. In one experimental year, the necessary disease pressure at favorable weather conditions for the disease and antagonistic MPCA could lead to a reduction of Plutella by 40-50%.
The joint project work and in particular the comprehensive comparison of data available from field and greenhouse results under field conditions are of high value for any further decision on the further development work. At this point of time, the achieved results are providing a guideline and direction for the further product development.
Parasitoids against aphids in fruit tree crops (WP10)
Parasitic wasps are efficient biocontrol agents used against aphids in many different horticultural crops. Biological control of aphids with parasitoids (Hymenoptera, Braconidae and Aphelinidae) is a common practice in covered vegetable and ornamental that has been used successfully over several decades now. However, these beneficial insects have been yet little used in open field crops. In pome and stone fruits, integrated pest management (IPM) is now well developed and there is a strong need for an efficient biological aphid control tool to further develop the IPM strategies. The aim of the WP 10 of the Biocomes project is to develop a parasitoid-based product for the control of aphid infestations in commercial fruit tree orchards. Therefore a first objective of the work-package was to identify the best species of parasitic wasps to be used as Biocontrol agent (BCA) for the control of aphids in fruit tree crops and to mass-rear them for use in preventive releases of parasitoids. Then, a second objective was to develop a complete and optimized release strategy to integrate the product in a total fauna approach within IPM strategies in commercial fruit orchards.
In the two first years of the Biocomes project, it has been shown that aphids of fruit tree crops are naturally attacked by many species of parasitic wasps. From the different species of parasitoids observed in the field, a list of the 10 most promising species as potential new biocontrol agents against aphids in fruit tree crops was identified: Aphelinus mali, Binodoxys angelicae, Diaeretiella rapae, Ephedrus dysaphidis, Ephedrus persicae, Lipolexis gracilis, Lysiphlebus fabarum, Monoctonus cerasi, Monoctonus mali, E. plagiator and Praon abjectum. Species already available at Viridaxis such as Aphidius colemani, A. matricariae, Ephedrus cerasicola and Aphelinus abdominalis also proved to be potentially useful in the control of aphids in fruit tree crops.
Meantime, data on fauna (beneficials and other pests) and current crop protection practices (treatments, chemical compatibilities ...) in fruit tree crops were collected and analyzed. Regarding biological control possibilities of the most common pests associated with fruit tree crops and the potential integration of preventive parasitoid releases to control aphids in these crops, it was decided to focus only on stone fruit trees. Indeed, production tests showed that Viridaxis technology could not be easily adapted to the mass-rearing of key parasitoid species of the apple-tree associated aphid complex, such as Aphelinus mali, the only parasitoid able to control the woolly apple aphid, Eriosoma lanigerum. Furthermore, pear trees are attacked by another pest species than aphids (Cacopsylla pyri) for which no reliable biological control method is yet available.
In order to select the most appropriate parasitoid species to use for the control of aphid infestations in stone fruit orchards, an intensive screening of efficiency was carried out to assess the potential of all parasitoid species of interest against the aphid spectrum attacking these crops. This screening phase was undertaken within a gradual 3-steps approach (laboratory tests ; cage trials ; field trials). Efficiency tests were first performed under strictly controlled conditions, with laboratory tests leading to cage trials if proved successful. These tests successfully showed that aphid spectrum of stone fruits could be controlled efficiently by a mix of parasitoid species already produced or which could be mass-reared at Viridaxis.
Concomitantly, mass-rearing possibilities were assessed for the most promising parasitoid species in order to develop the mixes of parasitoid species to be used during field trials in cherry and peach orchards. Two species of parasitic wasps (Ephedrus plagiator and Diaeretiella rapae) were selected to enter in a scaling-up process. For the two species, the best production parameters were determined and both of them can now be produced at a big scale. However, production and application costs remain high, which limits the commercial use of these two species for now. Additionally to these species, a third one, Praon abjectum showed promising results at a small-scale production and could also be mass-reared if needed in near future.
The final step of the WP10 of the Biocomes project was then to develop, validate and optimize a strategy based on successive preventive releases of parasitoids during field trials carried out in commercial fruit tree orchards. Consequently, three years of consecutive field trials were performed, first in cherry tree orchards in Belgium (2015) and then in peach tree orchards in Spain (2016-2017). Based on the results obtained from these field trials, an integrated protocol was elaborated and successfully optimized for the use of the new BCA product within existing IPM strategies. The use of parasitoids either strongly reduced the aphid population in organic orchards where primary inoculum was high at the beginning of the season or kept the damages under the tolerance threshold for conventionally managed orchards. Overall, the release strategy also led to a reduction in the number of insecticide treatments used in the plots, both in conventional and organic management practices. Indeed, in mean, 2 treatments were avoided in the plots concerned by parasitoid releases in comparison with control orchards. However, despite an overall reduction of about a factor 2 along the 3 years of trials, the cost of the strategy remains too high for conventional peach productions where cheap chemical solutions exist and can offer a total control of aphids. More interest can however be given into higher added-value organic fruit productions, where existing biopesticide strategies (neem oil, natural pyrethrins, ...) do not always offer a satisfying control of aphids and can impair the efficiency of naturally occurring beneficials such as Syrphidae, Coccinelidae or Chrysopidae.
Telenomus laeviceps for control of Mamestra brassicae in brassica crops (WP11)
To characterize the Telenomus laeviceps population structures in European Brassica vegetable production areas and to determine the intraspecific variability of T. laeviceps, i) a species-specific qPCR marker was developed, ii) a reference species collection was established and confirmed by an external taxonomist, and iii) two field sampling campaigns were conducted in 2015 and 2017. The newly developed TaqMan qPCR marker for T. laeviceps was proven to be highly specific to T. laeviceps and was used to determine the species of the field sampled egg parasitoids. During the sampling campaign 2015 we failed to find T. laeviceps in our partner’s countries (Spain, Italy and Sweden) in intensively managed agricultural sites. In 2017, the sampling was extended to low input farming areas, set-asides and natural habitats. In Sweden parasitoids T. laeviceps was identified for the first time and confirmed by PCR analysis (validation based on morphological traits ongoing) providing the prerequisite for release permits and thus expanding the area for commercial use.
In order to successfully rear T. laeviceps at commercial scale and use it in M. brassicae control, detailed knowledge on its biology is required. We conducted experiments to study aspects of the T. laeviceps biology useful for building up an efficient rearing and for a further use of this parasitoid as a biocontrol agent. We investigated the influence of female density, temperature and egg deprivation time on the parasitation rate and sex ratio of the progeny. Results of these trials made it possible to develop a stable and efficient lab-scale rearing. From there on we built up a stable large scale production with a high level of automatization. Parallel to the production up-scaling, a field delivery system for T. laeviceps was developed.
Pilot field trials with the novel formulation of T. laeviceps were conducted in 2015 in a commercial organic cabbage field in Switzerland. T. laeviceps was released in the field in two densities and the parasitation rate monitored through exposition of cabbage moth eggs. The trap eggs were exposed in the field for two days, recollected and the number of parasitized eggs counted. We measured an increase in the mean parasitation rate over the whole season up to 36 %, against the 5% measured in previous years without any T. laeviceps releases (parasitation rate due to wild T. laeviceps). The maximal parasitation rate measured in 2015 reached 70 %. The number of parasitoids released during this first trial was very high to provide the proof of concept. In subsequent trials, reduce densities of parasitoids were used to improve economic viability of BCA release. Costs for current insecticide applications provided the base for calculation. This low density formulation was used in the field trials of 2016 and 2017. During two consecutive growing seasons we conducted field trials to: i) evaluate the efficacy of T. laeviceps in the control of the cabbage moth compared to standard insecticide and ii) integrate T. laeviceps in an existing plant protection strategy for cabbage crops.
Efficacy was tested in field trials conducted in 2016 and 2017 in organic cabbage fields in Switzerland. In each field we defined a plot with T. laeviceps releases, an insecticide treated plot and an untreated control plot. Compared to 2015, the amount of parasitoids per release were reduced to an economically feasible density. Parasitoids were released the first time two weeks after cabbage was planted in the field, depending on the cabbage variety between week 22 and 28. The second release was conducted five weeks after the first release. To quantify the economic impact of T. laeviceps on the product compared to the other two treatments, 12 cabbage heads per plot were harvested. The following variables were recorded: weight of the whole cabbage head (biomass without roots), weight of the cabbage without side leaves, number of side leaves, final weight and number of damaged head leaves.
In both years, the measured parasitation rate did not meet our expectations, with mean values ranging from 0 % to 0.75 %. No difference was found in the weight of the cabbage heads between the three treatments, indicating that neither insecticide nor parasitoid release had a severe impact on yield.
T. laeviceps potentially benefits from nectar providing plants planted or sown near the cabbage field. To quantify the putative impact, T. laeviceps was combined in integration trials in 2016 and 2017 to habitat management programs already established for the brassica production in Switzerland. In the trials conducted in 2016, parasitoids were released in fields with flower strip and companion plants, fields with only flower strip and in control fields without addition of flowers. The flower strip sown is commercially available and its composition specifically designed to promote beneficial insects against cabbage pests, such as the cabbage moth or the small white (Pieris rapae). T. laeviceps was released in the same density as in the above described efficacy trials. The setup of the field trials 2017 were the same as the one of 2016, with the exception that the treatment with only the flowering strip was not tested based on the results of the previous year. In 2016 the total parasitation rate (T. laeviceps and Trichogramma spp.) was significantly higher in the treatment with the flower strip and companion plants compared to the control, respectively 11.71 ± 2.77 % and 3.67 ± 1.19 %. No difference was found between the treatment with only flower strip (7.11 ± 1.93 %) and the other two treatments. In 2017, no difference was found in the total parasitation rate (T. laeviceps and Trichogramma spp.) between the two treatments, 12 ± 2 % for the control and 10 ± 2 % in the treatment with flower provision. Also the parasitation rate of T. laeviceps was not significantly different between the control and the flower treatment, respectively 6 ± 1 % and 4 ± 1 %.
In summary, a mass rearing of Telenomus laeviceps and its host Mamestra brassicae were successfully established and a functional field delivery system for the BCA developed. A survey conducted over two years in Sweden, Italy and Spain demonstrated that T. laeviceps is rarer than anticipated and its occurrence in intensively managed areas very scarce. Therefore, different sampling strategies and methodology were developed and successfully applied in Sweden, where T. laeviceps was collected in 2017. Further efforts are still needed for Italy and Spain. The occurrence of T. laeviceps is a prerequisite for approval of mass releases. Therefore, further researches are needed to expand the potential market of the BCA. We demonstrated that Telenomus laeviceps is an effective BCA under favorable conditions. A rearing system for Telenomus laeviceps, as well as a suitable field delivery system was successfully developed and evaluated in the field. The season 2015 was successful in proving the proof of concept, with a significant increase of the parasitation rate due to mass release of T. laeviceps. However, trials with reduction of the amount of parasitoids were not successful, due to unexpected quality problems of mass reared parasitoids but potentially also due to yet unknown factors. In consequence, the efficiency of T. laeviceps was strongly reduced. The parasitation rate measured was also dependent on the plant protection measures applied in the field. These results indicate that there is still some work to be done to find a suitable density to release the parasitoid and efficiently integrate them in current plant protection systems.
Implementation and ensuring exploitation and impact of production technologies and 11 BCAs developed (WP12)
Risk assessment
Published information, internal data from project partners and data generated in BIOCOMES were collected and assessed for their relevance for risk assessment and regulatory requirements. Recommendations were given to the project partners which experimental data could be generated within BIOCOMES. Such data were generated and provided by the partners. Literature information was also used to further select potential candidates as biocontrol agents, and to exclude potentially critical strains such as producers of potentially toxic metabolites or pathogens of plants or other non-target organisms.
The data that were compiled during the project are appropriate to address many of the requirements for the approval of the microorganisms as active ingredients and the registration of the products. Further data need to be generated in all cases, such as the physical-chemical and technical properties of the products, toxicity, pathogenicity and infectivity of the microorganisms and effects on non-target organisms. Such studies need to be conducted according to internationally agreed guidelines and under GLP. These studies were not within the scope of BIOCOMES. Further recommendations were given to how to generate missing data. Finally, based on existing data, a strategy for the preparation of the dossier for active ingredient approval was proposed for each strain. These strategies need to be further discussed with authorities in the rapporteur member states that can be chosen in the EU.
No major concern with regard to the risk assessment or regulatory requirements was identified for the strains that were selected. The 9 strains are appropriate as active ingredients in plant protection products according to Regulation (EC) 1107/2009 and an approval as a “low risk substance” seems realistic. The regulatory process is still very consuming. Dossier preparation for the first strains can start in 2018 and first products could be on the market in 2023.
The approach chosen in BIOCOMES turned out to be very effective. Regulatory requirements and the risk assessment were considered early in the selection phase and the development, which avoided development of microorganisms that might have potentially negative effects on humans, non-target organisms, or the environment. This approach lead to a more focused selection of strains and consequently reduced time and costs for the development.
The data generated for the risk assessment are the key information for the dossier for the approval of the active ingredients and the registration of the products, which are pre-requisites for the access to the market. The evaluation has demonstrated that no unacceptable risk is anticipated from the use of the products. This should allow a relatively rapid access to the market, where the products developed in BIOCOMES should be able to replace existing products which potentially have higher effects on human health, other non target organisms and the environment. Consequently, the products will allow food production in a more sustainable way with lower risks for humans and the environment.
Ecological sustainability
Life Cycle Assessment (LCA) has become an integral part of planning processes in industry as well as for energy and infrastructure systems. An essential part of any LCA which evaluates the pressure on the environment executed by the life cycle of a product or service is the Life Cycle Impact Assessment (LCIA). The Sustainable Process Index (SPI) is a LCIA method evaluating environmental impacts generated by individual processes steps in a life cycle. This method is a member of the ecological footprint family. Furthermore, it is compatible with the procedure of the life cycle analyses described in the EN ISO 140xx series. The SPI calculates the ecological footprint as the cumulative area to embed the whole life cycle of an industrial production process as well as the use phase of a product sustainably into the biosphere. It describes relevant ecological pressures of a process including all emerging emissions, the pre-chain and product usage. Material and energy flows of a product, which are extracted from and/or dissipated to the ecosphere, are compared to natural flows.
The SPI method has been implemented in the open access tool SPIonWeb, freely available on http://spionweb.tugraz.at/. With this tool it is possible to create whole life cycles by building up process chains, which can be updated and improved easily. Only a web browser is needed to sign up and use SPIonWeb, it is independent from any operating system. As results, the user gets the SPI Footprint, CO2-lifecycle-emissions and the global warming potential (GWP) of the whole life cycle.
Ecological evaluation had a double role within this project: on the one hand ecological evaluation during process development allowed the partners to identify “environmental hotspots” in the process life cycle. This allows integrating ecological considerations in the technological development, avoiding or improving process steps exerting high ecological pressures and thus increasing the ecological performance of the final product.
On the other hand high ecological performance is a crucial selling point for BCA, this information helps to generate competitive products. Ecological evaluation provides a useful instrument to support market access by highlighting these particular advantages of BCAs. For the this second role as market access support an evaluation of a broad array of different conventional pest control agents has been realised to provide a solid base line against which the performance of the BCAs developed in this project was compared.
Sekem Energy GmbH provided the consortium the methodological tool as well as the evaluation capacity to utilise ecological evaluation within the project to fulfil both roles. In the case of the biological control agents (BCAs) within BIOCOMES, evaluation takes place during the process development phase. Validity and precision of the evaluation results is of course critically dependent on the available data. Evaluation therefore could only be realised in close cooperation with partners. These results allowed a preliminary estimation of the ecological impact of the BCAs in question, compared to conventional competitors as well as the identification of ecological hotspots along the process chain. In order to obtain appropriate data, a detailed data requirement sheet has been sent and filled in together with the partners. On the basis of these data 11 BCA production processes have been ecological evaluated: Lymantria dispar nucleopolyhedrovirus (LdMNPV), Entomopathogenic nematodes (EPN), Serratia plymuthica HRO-C48, Trichoderma harzianum DSM25764, Penicillium frequentans 909, Bacillus amyloliquefaciens CPA-8, Cladosporium delicatulum, Tilletiopsis pallescens, Isaria fumusorosea, parasitic wasp Aphidius colemani, and Telenomus laeviceps.
Economic sustainability
The economic sustainability of 11 BCAs in BIOCOMES has been evaluated by means of marketing evaluation of each BCA in its specific market. Based on product and market information supplied by the industrial partners in BIOCOMES an internal and external analysis has been compiled and a SWOT matrix has been produced for each BCA. The internal analysis comprises an evaluation and semi-quantitative rating of specific product characteristics (strengths and weaknesses) of the BCA and the external analysis comprises an evaluation and semi-quantitative rating of specific market characteristics (market opportunities and threats) for each BCA. The influence of the external market conditions on each of the product characteristics has been thoroughly evaluated and rated in an initial confrontation matrix, taking into account the relative importance of each of the factors in the SWOT matrix. Key marketing issues are then derived from the confrontation matrix through sorting the totals of the ratings. Derived key market opportunities which synergize with product strengths can be profitably utilized in marketing while the main derived market threats and product weaknesses can be given special attention during further development of the BCA after completion of BIOCOMES to reduce possible negative economic impacts. Strategic options and recommendations are discussed for each of the BCAs to minimize possible weaknesses and to avoid market threats.
Potential Impact:
BIOCOMES contributed significantly towards the expected overall impact of WP2013: (i) Reinforcing the EU’s sciences base in the bio-economy by Building a European Knowledge Based Bio-Economy based on ‘Europe 2020 – A strategy for smart, sustainable and inclusive growth’ and the Innovation Union Flagship initiative with the overall priority of bio-resource efficiency, and (ii) Providing tools to maximize the impact of research and innovation on European societies and economies consistent with the common strategic priorities of Horizon 2020: focus on societal challenges, competitiveness and excellent research. BIOCOMES also contributed significantly towards the expected specific impacts listed in the Work Programme 2013 (WP2013) in relation to the topic of KBBE.2013.1.2-5: (i) Generation of knowledge and innovative solutions for pest and disease management - targeted to new biological control products that are effective and can readily and sustainably be introduced into the market taking into account the requirements of registration under EU and national legislation including risk assessment, and (ii) Support of policy (i.e. Directive 2009/128/EC) laying down that all farmers will have to apply the general principles of integrated pest management by January, 2014.
The project contributed to the development of 11 completely new biological control agents for application in major crops and forestry and transformational improvement of 2 industrial production technologies. The project had been initiated by 6 leading European manufacturers of biocontrol products who selected the targeted new innovative solutions of BIOCOMES. Major criteria for selection were: (i) Economic sustainability on the expected future markets, (ii) Ecological sustainability with low negative impact on the environment, and (iii) High bio-resource efficiency. Major markets for plant protection products in European agriculture, horticulture and forestry were targeted. The market-driven work programme of BIOCOMES, the competence and heritage of 10 European SMEs, 3 larger enterprises, all qualified as front-runners of the industry, and the strong co-operation with 14 experienced S&T-partners guaranteed that BIOCOMES will have a strong and significant impact on European agriculture, horticulture and forestry at the economic, environmental and social level.
Economic impact of BIOCOMES
Short-term, the SMEs and INDs in the consortium had a direct economic benefit because BIOCOMES significantly increased their capacity for research and development of BCAs. In total, 64 additional researches had been recruited by project partners during the course of the project. Mid-term, the increase in production, marketing and sales of new biological control agents will strengthen the economic basis of the SMEs involved and gives them room for expansion of their commercial operations. Important aspect is that each BCA within BIOCOMES is selected by the partner (SME or IND) after thorough evaluation of commercial parameters: market potential, economic production characteristics, field efficacy, and the assessment of environmental risks. Long-term, all stakeholders, farmers, forestry industry, European biocontrol industry, citizens and governments will benefit from the market-driven approach of BIOCOMES generating a major increase in new sustainable alternatives for pest and pathogen control for the main agricultural sectors coming available under Regulation 1107/2009.
The BIOCOMES consortium consisted of a group of dedicated SMEs and larger industries with long-term experience in the development of BCAs, all active and based in the European biocontrol industry. The intensive co-operation between leading SMEs, RTD providers and recognized registration experts giving guidance with respect to regulatory issues during product development created synergies on effective product development within and beyond BIOCOMES.
Environmental impact of BIOCOMES
The innovative solutions for pest and pathogen management of BIOCOMES will (after registration) lead to a strong reduction of the use of chemical agents in the European Union. Firstly, the sustainable alternatives of BIOCOMES will decrease the amount of residues of chemical agents on agricultural and fresh produce which is beneficial to human health. A second direct effect is that the exposure of soil, (ground) water and air to residues will also be significantly reduced. In addition to these direct effects, the reduction of the use of chemical agents will induce beneficial effects on the population density of natural enemies and pollinators in support of sustainable production of agricultural products in the European Union.
Social impact of BIOCOMES
The increase in the use of BCAs and the subsequent reduction in use of pesticides projected from BIOCOMES will reduce the exposure of workers in agriculture and forestry as well as consumers to toxic chemical agents. Moreover, the generation and dissemination of broad knowledge on new sustainable solutions for pest and pathogen management based on BCAs will improve the acceptance of these products in society. This was specifically supported by the communication of BIOCOMES via animations and videos.
The average costs of the development of a BCA are estimated at $3-5 million (compared to $250 million for a new synthetic pesticide). Market entry takes approximately 3 years for a BCA in the USA (compared to 10 years for a synthetic pesticide) and, due to different registration requirements, a few years longer in the EU. BIOCOMES contributed significantly to the development and implementation of 11 new BCAs and 2 new industrial production technologies with a project duration of 4 years and total budget of 12 Mio€. S&T strategies had been chosen that led to product development close to implementation for all innovations., e.g. for two microbial BCAs registration has already been initiated by industries, two macrobial BCAs are ready for marketing and one improved production technology is already implemented by industry. Involved industries are all highly experienced in product development and launching of BCAs into markets. Depending on the progress in R&T and economic assessments, industries already invested and will further invest in product development. Follow-up research in national and pan-European IPM programs and networks on the integration of the new BCAs in control strategies of pests and diseases in major agricultural and horticultural crops and different forest systems will ensure the implementation of the results of BIOCOMES.
Support of policy to apply the general principles of integrated pest management (Directive 2009/128/EC)
The project contributed to the development of 11 new BCAs to the stage close to implementation. BCAs and targeted diseases and pests had been selected by the industrial participants (SME or IND) after thorough evaluation of commercial parameters including market potential. Main targeted production areas were arable crops, field-grown vegetable crops, fruit production and forestry in all European climatic zones. The new BCAs will partly substitute the use of major groups of insecticides and fungicides. In particular, EPN will substitute the use of soil insecticides (WPs 1, 2 and 3). The microbial insecticides (WPs 4 and 9) and macrobials (WPs 10 and 11) in BIOCOMES will replace canopy treatments with insecticides. This will have a major impact especially on the use of neonicotinoid insecticides which are suspected to contribute to the colony collapse syndrome in bees and are on the list of compounds to be phased out. Furthermore, the use of pyrethroids, which have major negative impacts on soil life (which is why they are only authorized for use every three years) will partly be substituted. Biological fungicides will replace synthetic fungicides used for seed and seedling treatments (WPs 3, 5 and 6). Foliage treatments with microbial BCAs will replace synthetic fungicides in major fruit (WP7) and arable crops (WP8). This will have a major impact on the currently strong dependency on and broad use of fungicides, including endocrine-disrupting azole fungicides which are also listed to be phased out.
At the end of BIOCOMES, new BCAs reached the stage for implementation in IPM programs which already exist or are under development in various national and international IPM initiatives.
BIOCOMES pro-actively linked project activities with IPM networks. Two members of the External Advisory Board (EAB) are international senior experts in implementation of IPM solutions in agriculture and forestry. They continuously evaluated project progress and advised BIOCOMES participants to ensure that strong links with IPM networks were established during the project period. The ambition is to provide IPM networks with prototype formulations of the BIOCOMES BCAs soon after the project. In this way, many stakeholders will be reached early in the pre-implementation phase. Stakeholders as ‘problem owners’ such as farmers’ unions, NGOs, governmental bodies, traders, retailers or water boards were all important members of IPM knowledge dissemination networks.
Building a European Knowledge Based Bio-Economy
The European biological control manufacturers are operating on a market with particular growth potential. The BCA market at present represents 4.0% of the worldwide overall pesticide market. Sales of BCAs in Europe represent 14% of the BCA world market. Sales within the EU in 2008 are estimated at 79 Mio€ for beneficial insects, 12 Mio€ for beneficial nematodes and 52 Mio€ for microbial biocontrol agents including beneficial viruses, bacteria and fungi. The global biopesticides market has grown exponentially in recent years. Key drivers for this growth are the increasing pressure on the use of synthetic pesticides because of their toxicity, maximum residue levels (MRLs) and negative impact on the environment in combination with the emergence of an increasing number of efficient biocontrol solutions. This in particular applies for greenhouse crops with Spanish vegetable production as a strong example. Global annual growth rates for BCA markets are estimated at 16% compared to a growth rate of 5% for the global pesticides market. BIOCOMES especially targeted the markets in open crops, including arable crops and forestry where use of BCAs is far behind compared to use in protected crops. 71% of the acreage of protected crops in Europe (148 thousand ha) is treated with BCAs whereas only 3% of field vegetables (3,060 thousand ha), 10 % of grapes (3,280 thousand ha), 5% of fruits (2,880 thousand ha), less than 1% of arable crops (108,975 thousand ha) and less than 0.1% of forestry (1,020,000 thousand ha) are treated with BCAs (Blum et al, 2011). Field crops including oilseed crops are expected to be the emerging crop segments in biopesticide markets. Considering the huge acreages grown with arable and open field vegetable crops and forests, enormous markets are still not reached by BCAs offering excellent opportunities for increasing sales of BCAs. This market-driven choice of BCAs together with the estimated growth rates for use of BCAs clearly demonstrates the significant impact that BIOCOMES had and will have on the market positions of the involved biocontrol SMEs and larger biocontrol industries as part of the European Knowledge Based Bio-Economy.
The involved bio-economy enterprises expect a significant increase in production and sales of BCAs, also resulting in an increase in employment of qualified employees. The BIOCOMES project contributed in 2 ways. Industries will jointly develop new BCAs using complementary expertise of the network of industrial and S&T participants. Examples were discovery of new BCA strains, risk assessments, and field trials. The substantial EU contribution enabled industries to invest both into major developments of new technologies and into new BCAs. This opened opportunities for transformational rather than incremental steps in business development of the involved bio-economy industries. Implementation of the new technologies also will allow a transformational rather than incremental step in the use of BCAs in European agriculture, horticulture and forestry as economically and ecologically viable alternatives to synthetic pesticides.
The expected technological breakthroughs, in combination with the general advantages of BCAs over synthetic pesticides, will foster the implementation of BCAs. Examples of such advantages are: lower costs of R&D, incremental growth of markets for IPM and organic production, residue-free production, high labor and harvest flexibility (because of short re-entry times), low environmental impact and sustainable production independent on chemistry or fossil resources. Biocontrol enterprises expect annual revenues of BCAs developed in BIOCOMES of >1 Mio€ in the years after market implementation with increasing market penetration during the next decade leading to an estimated annual turnover of 2.5 to 25 Mio€ per BCA.
The European Knowledge Based Bio-Economy represented in BIOCOMES has been an excellent network consisting of 6 enterprises active as BCA manufacturers, 7 enterprises and 14 scientific research institutions, all supporting the development of BCAs with their specific expertise. Besides the BCA manufacturers, all other bio-economy participants had benefit from their participation in BIOCOMES. GAB is the leading European consultancy partner for registration issues and risk assessment studies of BCAs. GAB had profit from the project collaboration through a large increase in knowledge on selection of microbial strains, the development of fermentation technologies and appropriate formulations. Furthermore, the contact with field research institutions gave GAB the opportunity to gain insight into the development strategies for the use of BCAs under field conditions. GAB had profit from this knowledge also by transferring it to evaluating authorities to facilitate the registration of microbial plant protection products. SEK encompassed life cycle assessment data for cradle-to-grave ecological optimization. The participation in BIOCOMES strengthened the position of SEK as consultancy partner for ecological evaluations in the broader bio-economy. Participants AGPL, ARA, COILLTE, HEFA, and PCFRUIT evaluated new crop protection products under field conditions and integrated these together with producers and growers as end users in their cropping systems. Participation in BIOCOMES strengthened their expertise in testing BCAs, and their collaboration with the biocontrol industry and the biocontrol science. The internationally well-recognized research experts in various aspects of biological control present in BIOCOMES had profit from the collaboration by deepening their expertise in their particular research area, by educating young scientists, and by the synergisms arising at many levels when different participants collaborate within WPs and across WPs. The structured Platforms (P1 to P5) across WPs enabled and stimulated all participants to achieve this goal. The focus of BIOCOMES on the development of economic sustainable industrial solutions trained scientists to develop rational scientific approaches to meet the goals of the industry. BIOCOMES contributed also in this way to the building of a European Knowledge Based Bio-Economy, thus strengthening the European research area.
Providing tools to maximize the impact of research and innovation on European societies and economies and bridging towards Horizon 2020.
BIOCOMES contributed to the three integrated elements of Horizon 2020. The project contributed to Smart growth by developing new opportunities for the biocontrol industry which directly contributed to the European bio-economy (see above). Furthermore, young scientists have been educated and trained in a dedicated program to empower the future development of a bio-economy based on knowledge and innovation. In this program young scientists have directly been involved in project activities with a total of 22 PhD positions. Additionally, 16 young scientists have been trained in a workshop in the development of economic and ecologically sustainable BCAs. This workshop had been organized by a group of industry and research participants and also included visits to two production plants of participants. BIOCOMES will make a significant contribution to Sustainable growth by a more efficient, greener and more competitive agriculture and forestry industry. BCAs as ‘green’ products have been developed with strong emphasis on economic sustainability, guaranteed by the strong guidance of the project by the BCA industry. Continuous risk assessment, life cycle assessment and assessment of bio-resource efficiency for each technology developed by highly qualified specialists (GAB, SEK) demonstrated the strong emphasis of BIOCOMES on ecological sustainability of the BCAs. BIOCOMES contributed to Inclusive growth by creating ample opportunities for new employment in growing bio-economy SMEs. Furthermore, BIOCOMES generated opportunities for the production of low or no-residue agricultural and horticultural products in all EU zones, which contributes strongly to the competitiveness of the European agricultural and forestry industry on a global scale.
The specific potential impact of the different BCAs and production technologies developed by BIOCOMES is presented below:
Production technologies for LdMNPV and EPN (WP1)
Production technologies for LdMNPV
In vitro production of baculoviruses contributes to an improved employee safety in the production companies. Whereas in insect rearings complex safety precautions have to be taken to protect employees from wing scales and stinging hairs, these issues are completely absent in cell culture production systems. Baculoviruses can be produced location-independently, because cell cultures can be established in any suitable laboratory. Extensive quarantine measures are not needed any more when working with non-native insect species. Furthermore, it would enable us to produce baculoviruses from hard-to-breed pests. The in vitro production technology of baculoviruses leads to a significantly reduced microbial contamination level in the final product due to higher hygiene standards and the absence of naturally occurring larval gut microbiota. Multiple formulations are possible, which allows easier storage and application of the product for the customers. If the productions costs can be reduced to a lower level than in vivo production, this could also open up a new field of application in terms of markets, crops and customers.
Production technologies for EPN
The market for EPN is increasing continuously, which has led to investment into larger bioreactor volumes. The establishment of the decanter technology for EPN downstream processing and the ability to store the nematodes at higher densities will enable an upscaling of the production process at much lower investment cost. This and the reduction of labour costs for the downstreaming process and reduced losses by microbial contaminations will reduce the costs for EPN products in the future. This will make EPN more competitive compared to chemical insecticides and will allow to enter the outdoor markets, like control of the corn rootworm (Diabrotica virgifera virgifera).
Genetic s for trait improvement of entomopathogenic nematodes (WP2)
The generation of genetically improved nematode strain with increase resistance to stress, prolonged longevity, field persistence and product shelf life and enhanced virulence will increase product stability, quality during transport to the user and allow the used to use less nematodes per area. This will significantly reduce the application costs and enable the use of the nematodes also in large scale out-door cropping systems. These innovative nematode products will provide farmers with alternative, environmentally friendly management tools. The results of the project will fill gaps caused by phasing out synthetic chemical pesticides, will reduce exposure of applicants to dangerous compounds and contribute to reduction of pesticide residues in food and feed. The project has enabled the SME e-nema GmbH to introduce molecular techniques to professionalise the future breeding programme and support biotechnological production also of other biocontrol agents.
BCAs for control of forest pests and diseases (WP3)
Plantation forestry provides a number of important goods and services including wood products, climate change mitigation through carbon sequestration, biodiversity benefits and recreation. Pests and diseases are a major threat to the sector. Directive 2009/128/EC limits the use of chemical pesticides to reduce their impact on human health and the environment. In addition, EU forests are subject to national laws and international commitments to ensure their sustainability. Sustainable forest management (SFM) is monitored and confirmed by certification processes, and this imposes additional requirements on forest managers to reduce the use of chemical pesticides. This project has taken major steps towards providing biological solutions to three major biotic threats to Europe’s forestry. Cypermethrin and alpha-cypermethrin are being phased out for control of pine weevil Hylobius abietis under SFM, and the proposed replacement chemicals are in the neonicotinoid class. EPN can suppress weevil populations, and EPN species produced by e-nema can be recommended for incorporation into integrated pest management programs for this pest, thus replacing the use of chemical pesticides on sites suitable for their application. Gypsy moth Lymantria dispar occurs in many types of forest and urban landscape on mainland Europe. Outbreaks cause damage in up to thousands of hectares. Stands of oak, gypsy moth’s preferred species, are often located in Natura 2000 areas, where the use of chemical insecticides is prohibited. Gypsy moth also attacks roadside trees, whose chemical protection is banned under EU law. The development of a biological formulation based on virus characterized by high pathogenicity for the host, will provide effective protection of forests threatened by the gypsy moth. Damping-off is the most important cause of losses during transplant production. The disease may cause disruption in the continuity of nursery production and losses must be supplemented by purchasing suitable material from an external source. As much manual work is involved in the production of seedlings, the economic importance is high. Many fungicides are registered for other crops but not for forest plants. This means that there is a strong need for biological control agents as alternatives for damping-off control in forest nurseries.
PhopGV for control of Tuta absoluta (tomato) and Phthorimaea operculella / Tecia solanivora (potato) (WP4)
Advanced market analyses revealed a substantial potential for a PhopGV product against the pest Tuab. For Phop, the market potential is more limited. However, the results from the field trials 2017 showed that one single isolate seems to be one of the most pathogenic and virulent one for both Phop and Tuab larvae.
More field trials will be conducted to further increase the efficacy by optimization of application rate and time, as well as the application strategy. The production process will be further optimized to reduce the production costs.
All data necessary for the registration dossier will be generated and the registration process started. For a successful implementation of the product in the market, it is important to develop an adequate marketing strategy in close collaboration with distributors. The registration dossier for the new product for Tuab control will be compiled and it is expected to be submitted as soon as possible. The new agent will extend the control options for T. absoluta by an efficient, sustainable and environmentally friendly alternative. Tomato growers will have an urgently needed tool for resistance management, as resistance to chemicals occurs more and more frequently.
Bacterial seed treatments against Brassica diseases with focus on oilseed rape (WP5)
We found that the way how plants react to the bacterial seed treatments is disease-pressure and cultivar-specific. While we found positive antifungal effect of the bacterial seed treatments on one oilseed rape cultivar, the same treatment resulted in negative emergence rate and corresponding loss of yield in another cultivar. The study of the seed microbiome of the three commercially used cultivars of oilseed rape showed strong differences in the seed microbiome structure between the cultivars. Therefore, we conclude that the structure of the seed microbiome is an important factor in the development of colonization resistance against pathogens. It also has a strong influence on the response of seedlings to biological seed treatments. These novel insights into seed microbiome structure will enable the development of next generation strategies combining both biocontrol and breeding approaches to address world agricultural challenges. On the other hand, cultivar-specific seed treatments will create new market opportunities and strengthen competitiveness and growth of SMEs.
Trichoderma harzianum DSM25764 seed treatment for control of Fusarium in cereals (WP6)
The new seed treatment product based on strain INAT11 capable of controlling F. graminearum in wheat and maize and most likely also other fungal diseases (for example, Septoria sp. in wheat) can constitute a sustainable plant protection product, in compliance with Dir. 2009/128/EC. Fusarium control in maize is currently based on Good Agricultural Practices (GAP), but with partial control only; on wheat, instead, fungicide treatments (DeMethylation Inhibitors and Strobilurin analogues) are used. Once placed on the market, strain INAT11 could become an additional environmentally friendly disease control option for wheat growers and a completely novel control tool for maize growers, which could help to improve food and feed security due to reduction in mycotoxin contamination. However, notwithstanding the promising results obtained against F. gramineaum on wheat and maize and Septoria sp., further field testing with strain INAT11 must be performed against Fusarium sp., and also against other pathogens in cereals, in order to collect more information on the market potential of strain INAT11. A detailed Data Gap analysis for the preparation of the dossier for the Annex I inclusion of strain INAT11 and the registration of the wettable powder formulation has been carried out, and the results obtained and the knowledge gained within BIOCOMES can be exploited for dossier preparation. However, these data must be completed with additional information to fulfil all data requirements. Once all the information will be available, an advanced market analyses will be performed to evaluate whether the introduction on the market of strain INAT11 could actually be cost effective.
Penicillium frequentans 909 and Bacillus subtilis CPA-8 for Monilinia control in stone fruit (WP7)
WP7 has provided two new biocontrol formulated products based on the bacterium Bacillus amyloliquefaciens CPA-8 and the filamentous fungus Penicillium frequentans Pf909 ready to be registered and commercialized. These new tools are effective to control brown rot on stone fruit and can be easily implemented in a standard commercial strategy. They will allow growers to produce high quality stone fruits covering consumers demand for safer and environment friendly products. The impact of these foregrounds will positively affect all the end-users related with stone fruits: growers, retailers and consumers.
Otherwise, the completely defined optimized production and formulation processes for both BCAs, the quality control protocols that can be easily implemented to other BCAs and the data set of all field trials in different producing countries that can be used for the registration documentation, are other foregrounds achieved in this WP.
Opennatur is looking for partners to share registration of CPA-8, Bayer Crop Science has declined to be involved in the Pf909 registration process, and other companies are in negotiation in order to go ahead with the registration process for developed products. Only final toxicological studies and last economic evaluations are needed to compile documentation necessary in both microorganisms.
Fungal BCA for control of powdery mildew in cereals (WP8)
Wheat, barley, rye and triticale are grown on 44,146,000 ha in the EU. Powdery mildew is a key fungal disease in these crops. Powdery mildew is regularly controlled with single or multiple applications of specific mildew or broad-spectrum fungicides across Europe. In WP8 potential BCAs for spray applications for control of powdery mildew in wheat have been selected. Selected isolates of Tilletiopsis pallescens have shown significant effects for lower powdery mildew severity on wheat in bioassays and in (small scale) field trials. Once mass production, formulation and shelf life of these isolates meet the industrial standards for production and storage, these isolates have a good potential to become a BCA of powdery mildew in cereals.
Isaria fumosorosea for pest insect control in vegetables (WP9)
The fungal entomopathogen Isaria fumosorosea (formally Paecilomocyces fumosoroseus) was first described by Wize in 1904 and subject of many scientific studies. The objective of this project was to modify the existing prototype product based on I. fumosorosea from one project partner. Specifically an extension of the applicability and performance under different and broader environmental and climatic conditions was targeted.
The characterization of host range aspects on fungal growth at different environmental conditions, the sporulation under fermentation conditions, the biological efficacy against several pest insects and beneficial insects were compared for ten different Isaria fumosorosea strain candidates at the beginning of the project. After an intensive screening cascade one strain candidate was selected for further development and formulated in the same way as the existing prototype product. Both candidates were evaluated under laboratory conditions prior to initiating field trials. Strength and weaknesses of the formulations were evaluated in field- and greenhouse experiments in different vegetables over a period of 2 ½ years. Three project partners responsible, performed in total 24 field trials under GEP and according to EPPO guidelines in only 2 ½-project years. The excellent cooperation of all six project partners and the expansion of the already existing experiences by dealing with the pests insect and fungal microorganisms Isaria were essential for the success of this project.
As outcome following can be stated: both Isaria fumosorosea isolates applied as formulated products on whitefly (Bemesia tabaci, Trialeurodes vaporarorium) and diamondback moth (Plutella xylostell) showed partially statistically significant medium level control effects in cash crops (tomato and sweet pepper), which is a good result for a biocontrol agent. The reliability in appropriate climatic conditions is acceptable, whereas climatic influences on the efficacy are currently still a problem. The efficacy data ascertained in this project is a comprehensive base for the further development of a microbial biocontrol product for the market.
Parasitoids against aphids in fruit tree crops (WP10)
A new strategy for the biocontrol of aphids in fruit tree orchards was developed by the WP10 within the Biocomes project. This strategy is based on several successive releases of aphid parasitoids carried out in a preventive manner early in the spring. Field trials showed that the strategy is efficient for aphid control and that aphid infestations can be significantly reduced with the use of parasitoids. This biocontrol strategy allows an overall reduction in the number of insecticides applied in orchards concerned by the parasitoid releases. Releases can be performed easily and safely by the end-users and represent therefore a true alternative to the use of pesticides for the control of aphids. The reduction in the number of insecticides used in the orchards also induces an increase in aphid predator populations which work in a synergic manner with released parasitoids. Negative impacts of chemical treatments on the natural ecosystems and water resources can therefore be limited and fruits are also less susceptible to be contaminated with harmful pesticide residues.
Telenomus laeviceps for control of Mamestra brassicae in brassica crops (WP11)
During the project, we could demonstrate the potential benefits by using T. laeviceps in the control of the cabbage moth. The density of released parasitoids needs to be adjusted, in order to reach the desired effects. This parasitoid is also well suited to be integrated in conservation biocontrol programs, with low input plant protection regimes. Field trials showed that this parasitoid could benefit from nectar resources provided through habitat management. Releases of T. laeviceps in more intensively cultivated brassica fields need to be further explored since harmful effects of the standard insecticides may be limiting for usage of the parasitoid.
Implementation and ensuring exploitation and impact of production technologies and 11 BCAs developed (WP12)
Risk assessment
Data that are required for the regulatory approval of the microorganisms and their products were generated and collected during the project. These data represent the basis of the dossier that needs to be submitted to regulatory authorities. In additions, recommendations were given how to address data requirements that are not covered yet. This will allow to compile the dossier in a relatively short time and to accelerate the access to the market. It was demonstrated that no unacceptable risk is anticipated for the use of these products in crop protection. The products are expected to replace products which potentially have a higher impact on human health, the environment or non-target organisms.
Ecological sustainability
Detailed ecological pressures along the whole production life cycle for 11 BCAs have been elaborated using SPIonWeb (http://spionweb.tugraz.at/) and measures to lower the ecological impact within the production phase have been proposed. It is shown that the used fossil based energy is the main driver for the overall footprint in most of the production processes. In some exceptional cases, also materials like polystyrene and substances for disinfectant can cause relevant ecological pressures. Furthermore, the positive impact of using renewable energy within the production of some producers is presented.
As final step, application scenarios for specific agricultural products and specific diseases/pests were created and comparisons of BCA to conventional alternatives based on applications to these plants were drawn. It is shown that in most of these comparisons the BCAs perform ecological better than their conventional competitors do. Additionally it is presented that in some scenarios the application to the fields (in particular the machine use) is a considerable contributor to the ecological impact of pest control.
Economic sustainability
Analysis of the economic sustainability has been performed in a standardized way by means of a strategic SWOT analysis for each of the BCAs developed in BIOCOMES. Main market threats and bottlenecks for development and marketing are described for each BCA. Generally speaking, analysis of the BCAs under development shows a reasonable to good economic sustainability, the strengths of the BCAs in BIOCOMES fit well with the market requirements and opportunities. Several BCAs are already in an advanced stage of development and could go already into application for active ingredient approval mid 2018 or in 2019. Other BCAs for specific niche markets are also particularly suited for purpose and meet the needs of market and society. Common conditions to promote the adoption of the BCAs in Integrated Pest Management (IPM) in this evaluation of the economic sustainability are: (1) selectivity of / compatibility with chemical active substances, (2) production costs of the BCA and (3) regulatory hurdles of which the costs of preparation of the registration dossier and uncertainties in the timeline for evaluation are the main issues. If the target disease/pest of the BCA is the key disease/pest in a crop, crop management can be adapted for the BCA which will promote its efficacy and commercial development. If market threats are either avoidable or can be influenced, counteractive actions are described to reduce the impact of threats on the development of the BCA.
Main dissemination activities and exploitation of results
The new biological opportunities and project results of BIOCOMES were communicated with IPM networks and stakeholders developing new IPM methods and technologies through various communication channels. Awareness of the different new biocontrol solutions, which were under development in BIOCOMES, and of further steps needed for each product and their potential for use in future IPM has been raised. It is expected that various BIOCOMES BCAs will directly be included in upcoming IPM projects on the development of new IPM strategies.
End users and influences of end users have been informed about the contribution of the BIOCOMES products and the important role of biological pest control in IPM via our website, the newsletters and the slideshare presentations. The BIOCOMES website fulfilled an important and successful role in this which is underpinned by the number of visits that has been increased ever since the launch of the website. During the project period, the BIOCOMES website had 154.233 page views by 75.878 visitors. The website will be active until end of 2020.
In our 13 newsletters we were able to focus on specific research updates and other relevant items concerning biological control developments and obstacles which enabled the dissemination of the project results. This was very much appreciated by the subscribers, resulting in a substantial and regular grow of the number of subscribers to 477 subscribers for the final newsletter.
The 3 animated videos explaining what is biological control and how the BIOCOMES BCAs are tackling several diseases and pests and have contributed 1) to explain why the use of biological control products is important and how it works and 2) how specific BIOCOMES products are working and thus controlling pests and diseases.
The 14 flyers with the final results of the developed BCAs and technologies show their advantages in integrated pest management and in economic value and environmental effects in agriculture and forestry. These flyers are interesting for the scientific public including IPM networks, but also for the biological control industry and regulatory and certification authorities organizations and policy makers.
A video on the public-private partnership for new biocontrol products and 16 videos on specific BIOCOMES results are important dissemination tools of BIOCOMES. The videos are in a relatively easy to understand way informing end users and their influencers and the general public about the final results. The videos can be accessed via the BIOCOMES website and the YouTube channel of BIOCOMES.
Individual BIOCOMES partners were very active in dissemination of project objectives, progress and results. In total, there were 384 dissemination activities during the course of the project . Additionally, 24 peer reviewed publications, 52 papers in proceedings of conferences, 2 articles in edited books, and 26 thesis had been finalized at the end of the project and a substantial number of further publications are expected during the year after project termination.
Links to BIOCOMES dissemination materials
Website www.biocomes.eu (all other dissemination materials can be reached via this website)
Newsletters (13): Newsletter 1 (February 2015), Newsletter 2, (April 2015), Newsletter 3 (July 2015), Newsletter 4 (October 2015), Newsletter 5 (January 2016), Newsletter 6 (April 2016), Newsletter 7 (August 2016), Newsletter 8 (November 2016), Newsletter 9 (February 2017), Newsletter 10 (April 2017), Newsletter 11 (July 2017), Newsletter 12 (October 2017), Newsletter 13 (December 2017)
Slideshares (9): 11 new biological control products; Gypsy moth: field experiences; Fusarium; Control of aphids with parasitoids; Cabbage moth; Powdery mildew; Gypsy moth; Seed treatment development for the control of Verticillium wilt; Brown rot on stone fruits.
Animations (3): What is biological control?; Biological control of diseases; Biological control of pests
Flyers (14): Monilinia control in stone fruit; Fungal root diseases; Seed treatment for control of Fusarium in cereals; New antagonists for powdery mildew control in cereals; Bacterial seed treatments against Brassica diseases; Parasitoids against aphids in fruit tree crops; Control of Mamestra brassicae in brassica crops; Lymantria dispar for control of forest pests and diseases; EPN for pine weevil control in reforestation; Control of tomato leaf miner and potato moths; Isaria fumosorosea for pest insect control in vegetables; Production technologies for LdMNPV; Genetics for trait improvement of EPN.
Videos (16): YouTube channel of BIOCOMES with:
BIOCOMES: A public-private partnership for new biocontrol products;
Animations: What is biological control?; Biological control of diseases; Biological control of pests
Results on: Economic stability, Ecological sustainability and Risk assessment of the BCAs
Results on: BIOCOMES research on biological control of tomato leaf miner and potato moths; BIOCOMES research on biological control of Verticillium wilt; BIOCOMES research on biological control of large pine weevil in forests; BIOCOMES research on biological control of aphids in fruit orchards; BIOCOMES research on biological control of whitefly in greenhouses; BIOCOMES research on biological control of brown rot in stone fruit; BIOCOMES research on biological control of fusarium in cereals; BIOCOMES research on biological control of fungal root diseases; BIOCOMES research on biological control of gypsy moth; BIOCOMES research on biological control of cabbage moth; BIOCOMES research on entomopathogenic nematodes; BIOCOMES research on entomopathogenic viruses; BIOCOMES research on biological control of powdery mildew in cereals.
Exploitation of results
The BIOCOMES project has shown that it is possible to boost the development of new biological control products in just four years. A large international public-private partnership with industries and scientific institutes made this possible by successfully combining the needed scientific and commercial expertise.
Two new biological control products are on the way to registration. One against Tuta absoluta in tomato and one against Fusarium in cereals. Another biological control agent based on nematodes – for which no registration is needed – has considerable commercial potential to be further developed to control pests in maize and forestry. The entomopathogenic nematodes have significantly been improved by marker assisted breeding and their production technology has significantly been improved to fulfill demands on the growing markets. Parasitoid-based products to control aphids in fruit orchards and a cabbage pest have been developed. Here too, commercial production of this BIOCOMES development is almost certain. Also for most other biocontrol products under development, perspectives are positive and the partners of BIOCOMES are committed to proceed with the development and market introduction of the new biological control products and production technologies.
Another very important outcome of BIOCOMES might be termed ‘process knowledge’. This resulted in bringing all fourteen research institutes and thirteen companies to the table in a constructive way and being able to avoid potential conflicts regarding intellectual property rights. The BIOCOMES project has been able to close the gap between the needs for research (research and technology development for good scientific publications) and the needs for industry (high probability for a substantial improvement or a new product).
List of Websites:
Public website: www.biocomes.eu
Contact details:
Dr. Jürgen Köhl
Wageningen University & Research
(former Dienst Landbouwkundig Onderzoek - DLO)
Droevendaalsesteeg 1
P.O. Box 16
6700 AA Wageningen
The Netherlands
jurgen.kohl@wur.nl
