Final Report Summary - INBIOSOIL (Innovative biological products for soil pest control)
Executive Summary:
Many herbivorous insect pests (more than 70%) have soil dwelling developmental stages. Control of these pests is difficult to achieve due to the discrete habitats of these species and the limited control strategies available. As of October 2009 the directive 2009/128/EC of the European Parliament and Council established a framework for a community action to achieve a sustainable use of pesticides. This directive demands that each Member State adopts a National Action Plan to set “quantitative objectives, targets, measures and timetables to reduce risks and impacts of pesticide use on human health and the environment”. Since many year demand for nature-based biopesticides has been steadily increasing worldwide. However, integrated pest management options for belowground herbivores are not available for many species; this problem being even more aggravated in organic farming systems. Therefore, INBISOIL aimed at developing innovative control strategies, based on an “attract and kill” approach, both for organic and conventional farmers, by exploring synergistic effects between entomopathogenic fungi (EPFs), entomopathogenic nematodes (EPNs), combined with semiochemicals, making use of strategies derived from nature. These novel formulations aimed at overcoming drawbacks typical encountered when applying biological control agents like EPFs, e.g. low efficacy or shelf life, consequently, increasing costs for their application. The INBIOSOIL project developed a novel capsule type based on bio-polymers and sustainable raw materials combined with specific isolates of entomopathogenic fungi. When applied into the soil these capsules produce carbondioxide, a cue used by most soil dwelling larval stages to locate their host plant roots. When coming into contact with these capsules fungal spores attach to the cuticle of the larvae and kill them by invading the insect body. The capsule type developed by INBIOSOIL efficiently controls wireworms in potato production systems; an insect pest complex which causes severe damage to tubers in autumn, entailing serious economic losses for farmers. These capsules (brand name ATTRACAP) have been registered for use in potato according to regulation Article 53 of Regulation No 1107/2009, and two companies are now involved in the production and regular registration of this INBIOSOIL product. Specific capsule types have also been designed for other herbivorous pest species, such as western corn rootworm, black vine weevil or fungus gnat larvae; however these strategies are not yet commercially effective. Additionally, INBIOSOIL has generated data on non-target effects of these capsules on aboveground and belowground predators, and has developed tools which allow identifying the fate of the fungal spores in the soil over the growing season. Protocols for a risk assessment of entomopathogenic fungi (RAFBCA-Rebeca) have been updated and analytical tools have been improved to analyze the fate of mycotoxins, eventually by these fungi, in the food chain. Finally the socio-economic risks and benefits of implementing biological control strategies have been evaluated and a novel approach has been proposed taking into account direct and indirect costs of the application of biocontrol agents versus synthetic insecticidal compounds. These risk assessment data, generated by INBIOSOIL, will promote an accelerated registration of biological control agents and will help policy makers and stakeholders in decision making processes based on the safety of these environmentally friendly agents and strategies.
Project Context and Objectives:
Many herbivorous insect pests (more than 70%) have soil dwelling developmental stages. Control of these pests is difficult to achieve due to the discrete habitats of these species and the limited control strategies available. As of October 2009 the directive 2009/128/EC of the European Parliament and Council established a framework for a community action to achieve a sustainable use of pesticides. This directive demands that each Member State adopts a National Action Plan to set “quantitative objectives, targets, measures and timetables to reduce risks and impacts of pesticide use on human health and the environment. With the increased environmental awareness of the public, and the pollution potential and health hazards from many of the conventional pesticides, the demand for nature-based biopesticides has been increasing steadily worldwide. However, integrated pest management options for belowground herbivores are still limited; this is specifically true for organic farming systems. The recently introduced 2-year-moratorium on neonicotinoid compounds, widely applied as seed coatings, even aggravated the problem of limited access to diversified control options for these pests.
INBISOIL aimed at developing new biological control products, both for organic and conventional farmers, by exploring synergistic effects between entomopathogenic fungi (EPFs) and efficacy enhancing agents making use of strategies derived from nature. Because soil dwelling larvae of many herbivorous insect species orientate towards their host plant roots using carbon dioxide serving as an orientation cue, and which is released by growing roots, the INBIOSOIL starting idea for an innovative control strategy was to combine CO2 releasing capsules with a killing component. When applied to the soil, these capsules would attract the larvae when searching for food, and by contact with the killing agent, the populations of the pest insects would be reduced.
These novel capsule formulations include encapsulated and dried entomopathogenic fungi (EPFs) as well as innovative co-formulations based on these EPFs, or a Bacillus thuringiensis strain in combination with efficacy enhancing agents. All formulations were processed in a way that they could be dried and stored for an extended shelf period; end-products were made available for a scaled up production process. Different co-formulations were evaluated for different strategies, named “attract and kill”, “stress and kill”, or “confuse and kill”, based on efficacy enhancing agents such as CO2 or botanicals.
To allow implementation of these capsules in control strategies in the field, additional data need to be compiled for a registration process. Therefore INBIOSOIL developed protocols to determine the cytotoxicity and genotoxicity of crude extracts and purified metabolites of entomopathogenic fungi (Metarhizium brunneum isolates) in plants and insects, and the effects on aquatic organisms. A major challenge for INBIOSOIL was to develop protocols and guidelines that effectively differentiate between microorganisms that can be safely used for biocontrol strategies and organisms that by producing toxic compounds at concentrations which potentially posing a risk to human health and/or the environment. Analytical methods for destruxins (dtxs) quantification, based on capillary electrophoresis and UHPLC-MS/MS were developed for the qualitative and quantitative assessment of the most important mycotoxins potentially produced by Metarhizium spp. isolates. Isolation of the most relevant destruxin analytes from strawberry and maize plants, which were used as proxies for relevant crop plants, demonstrated that these metabolites are not entering the food chain, and therefore should not pose a risk to consumer and animal health.
INBIOSOIL also assessed the fade of these Metarhizium isolates when applied under field conditions. Specific molecular genetic tools for the detection of applied Metarhizium spp. isolates were developed. These markers were successfully used to monitor M. brunneum isolates over time in both pot and field experiments. Moreover, a “second generation sequencing” approach was implemented to assess potential effects of the application of these EPFs on microbial communities, when applied to the soil. The application of spore suspensions resulted in short-term changes in the fungal communities, but these effects mostly decreased or disappeared by the end of the experiments after approx. 4 months.
To assess the influence of Metarhizium isolates on non-target organisms, four non-target predator species were selected and these species were tested in standardized pot experiments, based on a protocol developed during the INBIOSOIL project phase as well. When high EPF concentrations were applied moderate effects on mortality, fecundity and longevity were found in the lab, yet responses varied and depended on predator susceptibility. Given the specific administration of the EPF spores via capsule formulations the risk on non-target organisms is regard much lower than in previous control strategies, where high densities of spore suspensions need to be applied for the pest control becoming effective.
In parallel to the tests of Metarhizium formulations in the lab on non-target organisms INBIOSOIL also tested potential side effects of various biocontrol treatments (targeting Diabrotica virgifera virgifera), including different formulations of Metarhizium brunneum (F52, BIBESCO5) on non-target arthropods on four experimental fields in Austria. To assess the diversity and population level of species of the epigeous fauna the most economical way is the use of pitfall traps. In addition, the “sentinel prey method” was used to determine the predation activity of specific arthropod taxa in these experimental plots by offering a known amount of static prey items placed in the investigated area. For a certain time period the removal of the prey can then be evaluated by counting the remaining portion that is left behind (not yet consumed). The data compiled during these field epxeriments do not show any significant differences with regard to predator activity, specifically not in Metarhizium treatments compared to controls. Pitfalls trapping as well as sentinel prey evaluations are relevant methods in risk assessment under field situations, and the risk of field applications of Metarhizium spp. formulations non-target arthropods can be regarded negligible, based on the data compiled by the INBIOSOIL project.
Following the efficacy evaluation of different isolates of Metarhizium brunneum against larvae of the western corn rootworm, wireworms, vine weevils, or fungus gnat larvae, different strains of the entomopathogenic fungus were selected. These isolates were tested in lab and field experiments in different crops (maize, potato, strawberry, garden soils). Whereas in first year trials an application of capsules containing spores and capsules releasing CO2 were tested separately (co-application), follow up trials allowed to test the co-formulated capsules (spores contained in CO2-releasing capsules). For wireworm control in potato INBIOSOIL was able to demonstrate that the innovative approach of a “lure & kill” strategy can be effective. These prototype capsules were, however, a first test and it became evident that capsules needed to be improved for achieving a more pronounced damage reduction on potato tubers. The refined capsule types used in the last year of the project (2015) were not only tested by the partners of the INBIOSOIL project, but also by stakeholders in Germany. At certain conditions, depending on the population level of the wireworms, and abiotic conditions in the soil, the “lure & kill” strategy controlled wireworms by on average 50%. The INBIOSOIL capsule type developed within the project phase of INBIOSOIL thus resulted in a product (brand name ATTRACAP), which has now been registered according to regulation Article 53 of Regulation No 1107/2009 of the European parliament and of the council (emergency situations in plant protection) in Germany for 2016.
The “lure & kill” approach did, however, not work for wireworm control in maize fields. Application of the capsules did not significantly reduce wireworm damage on seedlings. Although the build-up of CFUs in the soils could be documented, the overall efficacy was limited, probably because the entomopathogenic fungus needs more that 3 weeks to kill a wireworm larvae; a time interval where damage to the root systems of the maize plants is continuously progressing.
For the control of western corn rootworms (WCR) a combined application of entomopathogenic nematodes and fungi was tested. In all years the lowest number of adults hatching from the plots was achieved when combing both biocontrol agents with an insecticidal seed treatment. Overall, both greenhouse and field experiments did not provide data showing a significant reduction of WCR larval populations. Therefore capsules types so far developed under the INBIOSOIL project cannot be recommended for WCR control.
Several experiments proved that fungus gnat larvae cannot be controlled by EPFs; thus a different approach needed to be adopted for controlling this pest species in greenhouse potting soils. A “lure & kill” approach was developed using Bacillus thuringiensis spores encapsulated in beads releasing CO2. Although these capsules where highly attractive to the larvae of this pest, the efficacy is not sufficient yet to proceed with the commercialization of these capsules.
For vine weevil control a different approach within the INBIOSOIL project has been developed. Previous studies have demonstrated that some plants are highly preferential to a pest and can thus be used to divert them from the main crop. Therefore several plants have been tested for their ovipositional attractiveness and one plant (Euonymus sp.) elicited a strong ovipositional preference in female vine weevils and could therefore be used as a trap crop. In combination with a lure INBIOSOIL results established the basis for a push-pull strategy.
The best biological control strategies are useless in case they are not adopted by farmers. The most important reason for not adopting these strategies is the higher costs per application unit. INBIOSOIL therefore reviewed the socio-economic impacts of controlling pests, such as the western corn root worm or wireworms, with various strategies in maize and potato, drawing on European as well as North American experiences. Specifically, the potential of biological control agents, such as entomopathogenic nematodes or fungi, perceived to a large extent as alternatives to chemical control, also due to increasing legislation on pesticide restriction, were analysed. Given the current level of knowledge on biological control options and data collected during the INBIOSOIL project allowed to conclude that under certain conditions biological control agents can provide an economical viable alternative for the control of the western corn rootworms and wireworms and a market for such kind of control strategies exists. From a social welfare perspective uncertainty over benefits and costs, irreversibility effects and externalities need to be considered as well. INBIOSOIL introduced an approach, the maximum incremental social tolerable irreversible costs (MISTICs), I*, as a tool for such consideration. Only when the incremental benefits of an IPM strategy outweigh possible irreversible costs should an IPM strategy be introduced. Given higher MISTICs values for wireworm control in potato the introduction of a biological control strategy is more likely to be economically feasible compared to a biological control strategy of western corn rootworms in maize. Based on these data compilations an online platform for the assessment of a business plan has also been developed by the INBIOSOIL project, allowing an investor to make an informed decision about the project’s profitability, viability and risk profile.
Based on these data INBIOSOIL assessed the cost/benefit for the innovative capsules targeting wireworms in potato. The evaluation resulted in a price for the granules, at an application rate of 30 kg/ha, of about 200 €/ha. The optimization of the production process of the capsules in combination with a higher efficacy would allow to commercialize the ATTRACAP capsules at a price of about 300 €/ha. The regulatory steps and associated costs for obtaining a regulatory approval for the biological control agent, the innovative ATTRACAP capsules, have been identified, according to the relevant regulatory framework currently applicable in Europe. Given the analysis INBIOSOIL concluded that regulatory and registration uncertainties remain high and that future approval of the product largely depends on pending registration processes for other biological control agents, serving as a proxy for ATTRACAP capsules as well.
Project Results:
Workpackage 1
Deliverables contained in this workpackage report on regular meetings (including the kick-off meeting) and the final international symposium organized at the end of the INBIOSOIL project. The regular meetings (including Skype conferences) have been used to update partners about the progress made during the preceding weeks, to discuss problems and to coordinate the next tasks.
Workpackage 2
INBIOSOIL developed a screening protocol for testing semiochemicals against wireworms (Deliverable 2.1). Several compounds provided by P 12 were identified as being repellent; however, application under field conditions would be too expensive and is therefore not regarded a suitable strategy for diverting wireworms away from the potato tubers. P6 tested turmeric oil and garlic for its repellent properties against wireworms and identified garlic as the more effective one. Based on the results of dose response tests carried out in Petri dishes and terraria experiments P4 calculated a dose between 20 and 70 kg of the specific compound per hectare to be an appropriate dose for synergy studies. The garlic compound was also tested for potential synergies with a fungal strain, identified as being most effective against wireworms. However, the results could not confirm any synergistic activities of these two agents (Deliverable 2.1). Potential synergistic effects of Turmeric and a formulated Metarhizium brunneum (strain BIPESCO5) product were also tested for their efficacy to control western corn rootworm larvae. However, a synergy between these agents could not be confirmed in the experiments.
an enhanced infection of Western corn rootworm larvae in a laboratory study
Tests using fungus gnat larvae revealed that larvae were attracted to CO2-releasing capsules and were significantly repelled by turmeric oil or spearmint in a dose-response relationship (P4; P1). Vine weevil larvae were also attracted by CO2-releasing capsules, but were not arrested at these capsules (P4). Synergistic activities of biocontrol agents were also evaluated. The application of the BIPESCO5 (Metarhizium brunneum) capsules alone and in synergy with entomopathogenic nematodes (EPN) did not result in a reduced adult emergence. BIPESCO5 is therefore not suitable for fungus gnat control. The entomopathogenic nematode Steinernema feltiae, widely used as a biocontrol measure, also failed to control fungus gnat larvae (Deliverable 2.2).
Turmeric oil was identified as a behaviour modifying substance for western corn rootworm larvae with repellent properties (P1). Results from the dose response tests showed that a concentration of 12.5 μL turmeric oil/40g soil (= 0.31 μL/g soil) is currently the lowest concentration for an effective repellent effect for 1st instar WCR larvae and 25 μL turmeric oil/40g (= 0.63 μL/g soil) was the lowest concentration for an effective repellent effect for 2nd instar WCR larvae, respectively.
P1 and P2 screened polymers and additives and minimum doses of active ingredients with efficiency enhancing agents (Deliverable 2.2). Although the finalization of this deliverable was delayed, results of these screening tests were constantly used to update the formulation of the capsules constituents. Several biopolymers were tested as matrix materials for capsule systems; at the same time different encapsulation methods were evaluated. Theses capsules were simultaneously tested for their mechanical stability and their persistence in different soils types. Given the results of these tests P2 focused on alginate based formulations, using the added value of gelatine as a nutrient for the entomopathogenic fungus and chitosan as a protective coating due to its antimicrobial properties.
Based on previous results with capsules releasing CO2, P2 developed co-formulations using entomopathogenic fungi (EPF) with efficacy enhancing agents (EEAs) (Deliverable 2.3). Spore concentrations were varied and the influence of temperature regimes on fungal growth was also tested. Additional technology developments were needed for the production of a capsule type which allowed spores of the entomopathogenic fungal strain to germinate and grow out of the capsules, and which could be technically dried to enhance shelf life and application of the capsules by commercially available granule spreaders. P2 also performed a series of studies on the development of the fungus within different beads, on increasing of the sporulation, the influence of drying on germination and persistence in soil as well as the optimization of these interactive processes.
The prototype of the capsule, which already showed promising features with regard to the requirements for field application, was then tested in parallel or in next year experiments by different partners of INBIOSOIL. For example P1 tested different capsule types in rhizotron experiments for their attractiveness for wireworms and western corn rootworm larvae (Deliverable 2.5). The results of these experiments were promising and demonstrated the feasibility of the “attract & kill” approach for control of wireworms in potato.
P2 also developed a capsule type, releasing CO2 and containing spores of Bacillus thuringiensis as the killing agent. These capsules were tested by P1 and P13 for their efficacy in controlling fungus gnats in different soil media. The “attract and kill” co-formulation containing baker’s yeast as an attractant resulted in the best performance with regard to fungus gnat control (Deliverable 2.5). However, it became evident that different media influenced the efficacy of the co-formulations, being also attractive for the larvae. This indicates that co-formulations employing the “attract & kill” strategy need to be further tested in different types of soilless growing media to ensure effective fungus gnat control under varying soil conditions. In summary, the potential of CO2 releasing capsules in combination with Bt showed promising results and should be developed further.
Capsules have also been tested for a “confuse and kill” approach based on entomopathogenic fungi (EPF) as the “kill”-component and semiochemicals/plant extracts as the “confuse”-component (Deliverable 2.5). Different treatments were set-up to test this approach for wireworm control on potato. Although the encapsulated EPF could significantly reduce the wireworm damage, no synergy with encapsulated garlic could be observed. Reduced germination rates of spores from garlic treated substrates even suggest some level of antagonism between these agents.
Based on the results reported in Deliverable 2.1 - to 2.5 and on the lab, greenhouse, and field experiments P2, in cooperation with P1 and P11 finally evaluated the granules produced by extrusion and subsequent crosslinking and drying in fluidized bed dryer. Granules produced by these techniques were tested in large scale field trials in 2015. A comparison of the properties of alginate beads and granules was done by P1. Briefly, the differences between the two granule types were marginal and both showed CO2-emission and outgrowth of mycelium. In summary the novel formulations (capsules or granules) systems with encapsulated spores are suitable for mass production; however, there is room for optimizing this process.
Workpackage 3
Formulations with encapsulated entomopathogenic fungi may were shown to improve the efficacy against target pests. However, prior to registration these novel formulations need to be carefully screen for potential side-effects on beneficial organisms. P5 developed a protocol with a decision tree based on different exposure scenarios. The test organisms were selected to represent different orders of beneficial predatory arthropods and species with different degrees of contact with soil, and hence risk of exposure to entomopathogenic fungi. The protocol (Deliverable 3.1) describes in detail the set-up of the trials and the analytical procedures. An important prerequisite for these tests is the use of same aged non-target insects, reared under standardized conditions. P14 has established lab colonies of the test organisms (part of Deliverable 3.1) which allowed for the experiments described in detail in the Deliverable 3.9. This laboratory study was conducted to assess the effects of the entomopathogenic fungi Metarhizium brunneum strain BIPESCO 5 (used in field experiments) on four non-target beneficial predatory arthropods which represent different levels of contact with soil. The commercially available predators evaluated were: Dalotia coriaria (Kraatz); Orius majusculus (Reuter); Gaeolaelaps aculeifer (Canestrini) and Aphidoletes aphidimyza Rondani. The studies demonstrated that O. majusculus was the most affected predator with the highest mortality, achieving 96% and reduced fecundity when compared to the control group. G. aculeifer was only marginally affected; the relative risk after exposition to fungal spores was almost null, only 1.2 % higher comparing with the control and no effect on fecundity was observed. D. coriaria showed significant effects on longevity and fecundity; however, 92.7% survived after the fungal exposition. In these experiments, the insects were exposed to soil mixed with a high concentration of fungal spores. This set-up could be regarded as a worst case scenario, because the innovative capsules developed by INBIOSOIL are aimed at reducing the spore load in the soil by containing them in the beads. When the fungus is growing out of the capsules the non-target organisms might come into contact with the spores as well; however, the incidence of these contact events should be substantially be reduced compared to a situation as given in the experiments described above.
Additional pot trials were set-up to assess the interactions between the predatory gall midge, Aphidoletes aphidimyza, and the entomopathogenic fungus Metarhizium brunneum (Deliverable 3.9). The study was conducted in greenhouse conditions, sweet corn (Zea mays var. saccharata) seeds were grown in pots filled with the natural soil inoculated with M. brunneum. This soil type was used, instead of artificial soils as, for example, recommended by OECD guidelines, because during screening tests it became obvious that these artificial soils do not mimic the interactions between the capsules in the soil and the outgrowth of the fungus. Thus soils from natural settings were used instead throughout the experiments. The plants were infested with Rhopalosiphum padi and A. aphidimyza pupae were introduced in the soil. At the end of the experiment Metarhizium was isolated from the leave surfaces and PCR amplifications were performed for one representative isolate of each multilocus genotype. When both natural enemies were combined in one treatment, the effect was less than additive, but the control of aphids by A. aphidimyza was only slightly affected. Thus under field conditions with more pest species being present entomopathogenic fungi could supplement other control agents. P5 also tested the capsules containing M. brunneum (Bipesco 5) for side effects (mortality, fecundity) on the 3 model arthropods (Dalotia coriaria, Orius majusculus, and A. aphidimyza) provided by P14. No significant difference was found with regard to mortality and longevity between treatments for any of the three species. However, a significantly lower fecundity was reported for O. majusculus when exposed to the M. brunneum capsules treatment. Based on the results of this investigation a very low risk for beneficial arthropods can be expected when capsules are applied in the soil of agricultural fields.
A pilot study was also set up by P5 to test various combinations of Metarhizium brunneum capsules and garlic extract capsules on O. majusculus. Garlic capsules had a negative effect on the outgrowth/ conidia production of Metarhizium and its infectivity on the predatory species. Based on these results a combination of these two actors in a “stress and kill” approach cannot be recommended (Deliverable 3.9).
To assess the abundance and survival of the fungal strain spores following the application to the soil over time it is essential to have specific markers which allow to discriminate between fungal strains already present in the soil and the ones which have been added. Therefore P6 established a reference strain collection of 65 fungal isolates representing 11 known Metarhizium species. The second goal was to develop an efficient genotyping tool that allowed discrimination of isolates of the different Metarhizium spp. (Deliverable 3.3). Different numbers of markers were amplified for the different fungal species. PCR protocols for amplification of the respective target loci from the four Metarhizium species were developed and optimized. Specificity of the designed primers was confirmed first by performing blast similarity searches in the non-redundant nucleotide database of GenBank and second by performing PCR amplifications with all the 65 isolates of the isolate collection. A strategy for genotyping Metarhizium field isolates has been proposed.
In the European Union (EU) microbial biological control agents (MBCAs) used in plant protection are regulated according to the EU Council Directive 91/414/EEC which has been replaced by the new Regulation (EC) 1107/2009. This regulation lays down rules for the authorisation of plant protection products in commercial form and for their placing on the market. In Annex II, 5.3.2 (methods of analysis) methods of residue analysis for the active substance and relevant metabolites in plant, animal and environmental matrices and drinking water, as appropriate, are asked to be validated.
Evaluating potential effects of biological control agents (BCA) on non-target organisms is a crucial component in the risk assessment process required for registration of products. Beneficial arthropods represent the prime focus in non-target studies, however, with the implementation of EU regulation (EC) No 1107/2009 studies on potential effects on microorganisms, e.g. bacteria and fungi, have to be included.
A risk assessment on metabolites, produced by fungal biocontrol agents using the RAFBCA-REBECA decision scheme (Deliverable 3.6) was developed on crude extracts and to individual metabolites from Metarhizium brunneum and Beauveria brongniartii production strains. It was obvious to INBIOSOIL experts that the potential risks related with the effect of fungal metabolites needed much more scientific investigation to come up with an appropriate, validated test system. Therefore, the objective of INBIOSIOL was to set up protocols and to screen for cytotoxic and mutagenic fungal secondary metabolites produced by the production strains which meet the requisite of maximum expression and detection of potential toxicity of extracts. INBIOSOIL partners set up standard procedures for the production of crude extracts (cultivation method, extraction protocol, bioassays) because these extracts usually contain toxins in high concentration as they come from pure cultures of the fungus grown in the best condition for metabolite production.
Three promising INBIOSOIL Metarhizium strains (BIPESCO 5, EAMa 01/58-Su, ART 2825) were grown in a bioreactor system in nitrogen rich complex media, in a semi complex media and also in a synthetic minimal medium (BIPESCO 5) aimed at encouraging secretion of a wide range of metabolites. RAFBCA protocols (developed by P3) were used to determine the metabolite profile of these three isolates and key metabolites were purified to homogeneity for use as standards and in subsequent studies (Deliverable 3.4). A novel sample preparation protocol for Destruxin (the major toxin compound class produced by this fungal species) purification and enrichment and a UHPLC-DAD-QTOF-MS/MS method was established which allowed evaluating qualitative and quantitative metabolite production. This novel validated assay achieves decent assay sensitivity, precision, and accuracy and can be considered as stable and reproducible for the quantification of Destruxin metabolites in fungal culture samples. INBIOSOIL partner P7 developed another method, the Supercritical Fluid Chromatography as an alternative tool for the qualitative and quantitative analysis of Metarhizium brunneum metabolites from culture broth. Although the UHPLC-method showed a better analytical performance in terms of achievable lower quantification limits, the established UHPSFC-method represents a good alternative technique for the qualitative and quantitative analysis of Destruxins in fungal culture broth. If a more sensitive quantitation is needed there is still the option to replace the PDA detector with the triple-quadrupole mass spectrometer, which is one of the most sensitive detection systems.
Using the latter method P7 was able to demonstrate that the different strains of Metarhizium produced different Destruxin metabolites in different cultivation media. Using High Speed Counter Current Chromatography (HSCCC) it was possible to isolate dtxA, dtxB, dtxE and dtxE-diol with purities ranging between 65 and 95%. These were further purified by column chromatography over a Sephadex LH-20 material combined with flash chromatography.
P3 developed validated protocols for isolation of metabolites from crops, which were used to assess whether metabolites could enter the food chain. Fruit samples were harvested from semi-field and field studies set-up by P3 in Austria. With regard to risk assessment and registration purposes protocols for isolating, qualitative verification and quantitative determination of fungal metabolites (specifically Destruxins) from different matrices, such as culture media and crops, have been developed by INBIOSOIL. To detect Destruxin traces in fruits, such as strawberry and maize, a QuEChERS-based sample preparation protocol was established which allowed for the first time extraction of Destruxin traces from crop matrices. The assay showed good sensitivity, precision and accuracy and can be considered as a new method for routine risk assessment allowing the detection of destruxin traces. This new analytical method shall become a routine method for answering the question whether Destruxins might be able to enter the food chain. The QuEChERS-based extraction protocol was also combined with a fast and selective UHPLC-QTOF-MS assay for the detection and quantification of Metarhizium brunneum metabolites from honey samples (Deliverable 3.4). The assay showed satisfying sensitivity, precision and accuracy and can be considered as stable and reproducible. The suitability of the assay was further proven by analysing real samples from a field study using M. brunneum to control the ectoparasitic mite Varroa destructor in bee colonies. Destruxins were not detected in any of the samples which were tested proving that the honey was residue free. In conclusion INBIOSOIL partners have validated procedures for the isolation of the most relevant Destruxin analytes from strawberry and maize and were able to demonstrate that these metabolites did not enter the food chain. Given the application of the ATTRACAP capsules, containing Metarhizium, the results demonstrate that this biocontrol agent should not pose a risk to consumer and animal health. Based on these published protocols INBIOSOIL partners, stakeholders, and SMEs are able to generate data important for registration purposes of entomopathogenic fungi (e.g. Beauveria and Metarhizium). This outcome of INBIOSOIL will also have high value for regulatory authorities in Europe and OECD countries.
Details regarding the analytical methods described above can be found in:
Taibon, J and Strasser H (in press) Analytical methods for secondary metabolite detection; in: Glare T. & Moran-Diez M (Eds.) Methods in Molecular Biology series on Microbial-Based Biopesticides - Methods and Protocols. Spinger, Dordrecht.
Taibon J, Sturm S, Seger C, Parth M, Strasser H, Stuppner H (2014) Development of a fast and selective UHPLC-DAD-QTOF-MS/MS method for the qualitative and quantitative assessment of Destruxin profiles. Analytical and Bioanalytical Chemistry 406, 7623–7632.
Carpio, A., Arroyo-Manzanares, N., Ríos-Moreno, A., Garrido-Jurado, I., Gámiz-Gracia, L., García-Campaña, A.M. Quesada-Moraga, E., Arce, L. (2016) Development of a QuEChERS-based extraction method for the determination of destruxins in potato plants by UHPLC-MS/MS; Talanta, 146, 815-822.
Entomopathogenic fungi (EPF) are known to produce a wide array of metabolites some which have been characterised while others are still being discovered. To conduct a risk assessment of each compound would be a lengthy and costly process which would prohibit development of these agents for pest control. The RAFBCA-REBECA decision scheme suggests that risk assessment should be done not just on selected metabolites but also concentrated crude preparations of the metabolites.
Available protocols for testing cytotoxicity and genotoxicity of specific compounds are design for chemical pesticides; at present there are no guidelines on the risk assessment of fungal biological control agents. INBIOSOIL partner P7 therefore aimed at aim developing robust, internationally approved protocols, which are tailored towards evaluating entomopathogenic fungi (Deliverable 3.5). Several aquatic invertebrates have been used in the current protocol, and Anopheles mosquito together with Daphnia spp. and Artemia salina have been demonstrated to be excellent and complementary, aquatic ecotox indicator species of fungal biocontrol agents. A. salina and more notably D. magna proved to be extremely sensitive test organisms and are suitable model organisms for rapid and inexpensive screening of toxicity of BCAs at an early stage of product development.
Details regarding the testing protocols described above can be found in:
Garrido-Jurado I, Alkhaibari A, Williams SR, Oatley-Radcliffe DL, Quesada-Moraga E, Butt TM (2015). Toxicity testing of Metarhizium conidia and toxins against aquatic invertebrates. Journal of Pest Science; doi 10.1007/s10340-015-0700-0.
Non-target effects of BCA applications on soil microbial communities were assessed in two different pot trials, set up by P6, one to control Agriotes and one to control Diabrotica to test efficacies of the new formulations. For assessment of fungal and prokaryotic (including archaea and bacteria) communities genomic DNA was extracted from the soil of each soil sample (one sample per pot). Ribosomal target regions, i.e. for fungi ITS2 and for prokaryota V3-V4 of 16S DNA, were PCR amplified and sequencing reads were subjected to a bioinformatics pipeline, which compromised quality filtering, trimming of primer pairs and grouping sequences into operational taxonomic units (OTUs) at 97% sequence similarity (Deliverable 3.7). Fungal and prokaryotic community structures reacted differently to treatments. Effects were detected for treatments containing the active compounds M. brunneum, nematodes and/or garlic; however, detected effects mostly decreased or disappeared by the end of the experiment (15 or 18 weeks post application). The study revealed that fungal biocontrol agent applications represent a “stress” to which microbial communities react. Under field conditions fungal biocontrol agent applications may thus induce changes in microbial communities, but, if at all, they have a temporal character only, i.e. communities are resilient, particularly once applications have been dismissed.
Workpackage 4
Entomopathogenic fungal isolates are known to significantly differ with regard to their efficacy against target pest species. Therefore INBIOSOIL aimed at testing the efficacy of various isolates against several pest species as these would be more commercially viable than strains with a narrow host range (Deliverable 4.1). Based on these results different isolates were identified for different pest species, namely M. brunneum ART2825 for wireworms and M. brunneum BIPESCO 5 for western corn rootworms.
First year experiments in the field by P1 used prototypes of capsules, produced by P2, either emitting CO2 or containing spores of ART2825. These capsules were applied separately or in combination. Controls used either a spore suspension of ART2825 or no capsules (Deliverable 4.2). CO2 emissions were measured at several days past inoculation, proving that the capsules increase CO2 levels significantly above the background level. The data collected during the field season 2013 indicated that the “lure and kill” approach allows a direct control of wireworms in potato fields. However, results were variable, depending on the mode of application and the time. These data have now been analysed and a manuscript has been submitted (Brandl MA, Schumann M, Przyklenk M, Patel A, Vidal S. Wireworm damage reduction in potatoes with an attract-and-kill strategy using Metarhizium brunneum; submitted to Entomological Science).
For western corn rootworm (WCR) control greenhouse experiments were established by P1 testing different capsule types produced by P2 (Deliverable 4.3). The results identified a specific capsule type with a good, though not outstanding, performance with regard to larval suppression. Based on these results a BIESCO 5 capsule type II was recommended for field trials by P 3.
The “confuse & kill” strategy against western corn rootworm (WCR) larvae was tested in a large scale greenhouse set up in small plastic containers (Deliverable 4.4). Based on previous work the synergy components consisted of the botanical Turmeric (Curcuma longa) (provided by P12), evaluated as a repellent substance as part of deliverable 2.1 and 2.2 and the encapsulated M. brunneum strain BIPESCO5 (provided by P3), screened as the most effective entomopathogenic fungal (EPF) strain. On one hand the results of this experiment demonstrated the repellent action of Turmeric towards WCR larvae. However, the application of capsules containing the entomopathogenic fungus only or in combination with Turmeric failed to control WCR larvae. A synergistic action between Turmeric and M. brunneum could therefore not be measured, making an implementation of a “Confuse & Kill” approach not feasible as a control option for WCR larvae.
Additionally to the field trials initiated in 2013 and continued in 2014 and 2015 (Deliverable 4.4) pot trials were carried out by P 3 to test the robustness and efficacy of the nematode Heterorhabditis bacteriophora, BIPESCO 5, in comparison to a synthetic insecticide (Clothianidin) for WCR larval control. This complex experiment was conducted in cooperation with P6, who assessed establishment of the applied strain in the experimental plot and determined the effect of the applied agents on non-target organisms (see Deliverable 3.7). The experiment consisted of eight treatments, each replicated 6 times. No statistically significant differences were identified between the treatments with regard to the number of adults recovered or root damage. However, genotype analyses using SSR markers revealed that the increase of Metarhizium density in the pots treated with the capsules was due to an increase of the density of the applied strain. Thus, although the entomopathogenic fungus established in the soil the efficacy of the biological control strategy was much less compared to a chemical control option.
These treatments (plus one additional chemical control option) were also tested by P3 in field experiments in 2014 and 2015 (Deliverable 4.4). Again, no significant differences were found between the treatments with regard to the number of WCR adults recovered. However, the lowest number of WCR adults per plant was found in plot with an IPM strategy, i.e. in treatments, where the biological control agents have been combined with a synthetic insecticide. Due to the high variability of the data effects of the treatments on yields did also not differ significantly. Metarhizium spp. density in plots treated with BIPESCO 5 increased slightly in 2013, but significantly in 2014 and 2015. It remains to be tested whether these higher densities of colony forming units (CFUs) in the fields are maintained when a crop rotation would be implemented.
The persistence of both the biological control agent Heterorhabditis bacteriophora and M. brunneum isolate BIPESCO 5 were also investigated in naturally black vine weevil infested strawberry fields in South Tyrol over a period of three years by P3 (Deliverable 4.4). The nematode was found in up to 80% of the soil samples taken one month after application. At least some nematodes survived through the winter and were detected after the winter months and in the following autumn. No clear evidence of a negative or positive impact of the application of the entomopathogenic fungus on the number of the nematode was found. Metarhizium spore densities generally increased after the application of fungus; however, neither a negative nor a positive impact of the incidence of the nematode on the fungal biocontrol agent could be established. Overall these experiments, together with the ones described above, indicate that an application of the fungus, either in form of capsules or as GranMet™ into the soil, does not result in persistent and high densities of the fungal isolates in next season crops. Significantly fewer larvae of the black vine weevil were found in plots treated with a combination of the nematode and the fungus applied as a spore suspension compared to the combination where the fungus was applied as active ingredient in granules. The results of these experiments elucidate the synergistic potential of entomopathogenic nematodes and fungi, when applied together; however, the data so far do not allow recommending a specific strategy for the application of these biocontrol agents in different cropping systems.
Synergistic effects between botanically derived fumigants and nematodes for the control of sciarid flies were tested by P4. Different plant compounds were tested with diethyl maleate and eucalyptol the mostly likely to exhibit synergy with Heterorhabditis downesi, although linalool could also potentially synergise with the two other Heterorhabditis species tested. Four chemicals identified during the experiments proved to be effective fumigants in their own right for sciarid control. In the experiments very low doses were used, so 100% mortality was rarely achieved. No obvious synergy between botanicals and the nematode Steinernema feltiae was found; this species were chosen being the primary commercial nematode to control sciarid flies. In conclusion, these experiments proven the potential of synergistic activities of plant derived compounds and nematodes; however, the results do not provide a clear strategy as how to proceed with an commercial implementation.
Based on the preliminary experiments with different prototypes of the “attract & kill capsules”, the integrated capsule type was repeatedly tested in field experiments in 2014 and 2015 for wireworm control in potato and maize (Deliverable 4.5) by P1. The co-formulated A&K capsules differed with regard to virulence (lower rate of mycosis), CO2 emission (earlier CO2 emission with dry co-formulated product) and establishment of the fungus in the soil (lower CFU count) compared to an AK co-application (capsules producing CO2 and capsules containing spores of the fungus). However, the efficacy of the co-formulated compared to the co-applied capsules in the field with regard to the reduction in wireworm damage on tubers during harvest was not significantly different. The level of efficiency was, however, very variable; compared to the insecticide treatment “Goldor Bait” the effectiveness was lower. Field experiments of stakeholders in Lower Saxony and Rhineland-Palatinate, using the integrated final dry capsule type, reported variable results as well, but also demonstrated a better potential of this control strategy compared to other non-chemical control options. Abiotic environmental conditions of the soil seem to play an important role for the ATTRACAP strategy to become effective; parameters which will be evaluated in follow-up projects in detail.
The “attract & kill” strategy was also tested by P1 in maize fields, targeting the wireworms as well. Results from 2013 to 2015 demonstrated that wireworms were not effectively controlled, probably because the fungus needs several weeks to kill a wireworm following the infection of the larvae (Deliverable 4.5). During this period the wireworms, however, are still able to cause damage on the maize plants. I may be speculated that a control would act on the population level of the pest in the field, thus being visible only as a long-term effect. This hypothesis needs to be tested in further experiments. The capsules were also tested for western corn rootworm control in maize fields in Croatia.
P4 tested a strategy targeting both adults and juveniles of the black vine weevil control (Deliverable 4.5). The goal of the study was to demonstrate that some plants are highly preferential to a pest and can be used to divert them from the main crop. A glasshouse experiments demonstrated a strong ovipositional preference of female black vine weevils for a specific Euonymus species compared to strawberries, an important food plant often infested with weevils. This plant might thus be used as a trap plant for adults in combination with a control strategy for the larvae as exemplified above (Deliverable 4.4).
In conclusion the co-formulation of M. brunneum and CO2 offers a highly innovative and ecologically friendly product for pest control, specifically for wireworm control. Recommendations for the use of this “attract & kill” strategy are discussed in (Deliverable 4.6). Currently the A&K control approach is most feasible for wireworm control in potato and the dry A&K capsules can be applied with any standard granulator at a rate of 30 kg/ha. They mean efficacy achieved in field experiments was a 30% reduction in tuber damage at this application rate; these capsules work best at low wireworm pressure and wet soil conditions because the dry A&K capsules need to re-swell, significantly hampered under dry soil conditions.
The details regarding the efficacy of semiochemical repellents (Deliverable 4.7) have been subsumed in different deliverable above. An additional field experiment was set-up by P6 to test the effects of semiochemicals in combination with the fungus containing capsules and fungus colonized barley kernels (FCKBs) in Switzerland. The colony forming units within the plots increased following the application of fungus containing material; however, 18 weeks post application the numbers were generally at the level of pre-application. Unfortunately, the density of wireworms was too low to generate reliable date on the efficacy of the different treatments. The main conclusion derived from these experiments indicates that the fungal isolate and the plant extract may not interact synergistically; thus a co-application of these agents would not increase the overall protection level of the potato plants.
Deliverable 4.8 lists the different capsule formulations tested during the INBIOSOIL project by P1, P6, and P6. In conclusion different capsule formulations exhibited different characteristics with regard to the target organisms tested. Parameters influencing the efficacy of these capsules were identified and can be addressed in forthcoming technical upscaling processes and application techniques. Whereas the development of the “attract & kill” strategy for control of the most urgent pending problem of wireworm damage in potato production systems has gone far beyond the early stage of development, this strategy needs still to be improved for western corn rootworm and vine weevil control.
The costs for the implementation of a biological control strategy against black vine weevils have been exemplified and evaluated for strawberry production systems (Deliverable 4.9). The strawberry sector is of high economic importance in some European countries and suffers from damage by black vine weevils. Several studies have already tested the benefits of applying different biological control agents in combination with chemical compounds; however the underlying costs for this strategy were regarded too high to be of interest for the farmers. Developing a specific model P9 was able to calculate the costs for the different application strategies. The cost benefit analysis shows that the adoption of a combined application of entomopathogenic fungi and nematodes as biological control agents for this pest results in a higher net present value that the use of the same agents alone. Thus farmers could generate higher profits depending on the wholesale or producer price for strawberries if they decide to change their production system from insecticide control-based systems to BCA control-based systems.
Workpackage 5
P9 calculated the economic feasibility of a biological control strategy using western corn rootworm or wireworms in comparison to chemical control options (Deliverable 5.1). The appropriate model specification for the productivity assessment of pest damage control inputs, such as synthetic pesticides, insect-resistant crops, or biocontrol agents, respectively is subject for an ongoing debate in agricultural economics. Pest damage control inputs do not necessarily directly increase yield, but rather, increase the share of potential output that is realized by reducing damage. Moreover, heterogeneity among farms and farmers, caused by differences in farm and farmer characteristics, are reasons for relative differences in profitability of different pest control strategies. The model calculations are becoming even more complicated because control strategies within a cropping season may change depending on pest pressure and micro climatic conditions. Because of these uncertainties and irreversible costs are difficult to calculate P8 adopted a threshold value call the maximal incremental social tolerable irreversible cost (MISTIC), which stipulates the maximum irreversible cost an individual or society is willing to accept due to the introduction of a certain technology or innovation. Although developed for the application of a new transgenic crop in Europe, this methodology can be applied to biological control strategies as well.
For western corn rootworm (WCR) and wireworm control strategies in maize production systems chemical, transgenic, and biological strategies were compared. The calculated gross farm-level benefit of a chemical control proved to be 2.3 times the costs, which implies an economic gain due to pesticide control of WCR at farm level. The cost-benefit ration for adopting a transgenic strategy (maize expressing a Bacillus thuringiensis toxin), depending on the WCR infestation levels, indicated a net return for the farmers. Crop rotation is currently regarded the most effective control strategy for WCR in Europe. For the application of entomopathogenic nematodes or fungi the farmer’s net benefit for applying these biocontrol agents would still results in positive revenue. However, additional producer benefits (chemical pesticide reductions and lower residues; host specificity of the control agents; low to zero resistance issues) would add to a conservative estimation of the benefit: costs ratio for biological control to be at 2.5 - 20:1. Given the current level of knowledge allowed concluding that under certain conditions biological control agents would provide an economical viable alternative for the control of the WCR and wireworms and a market for these control strategies should exist.
The economic analysis of applying biocontrol against WCR larvae and wireworms in maize and potato using a real option model approach took into account irreversible cost of introducing such technology. This made a threshold for decision-making called the MISTICs, I*. This economic analysis takes the long-term effect of biocontrol products in pest management into consideration. The MISTICs captures the uncertainty, flexibility and irreversibility associated with the introduction of biocontrol in Germany, France, Austria, Spain and Italy (Deliverable 5.4). At the farm-level, results suggest that an immediate introduction of biocontrol against WCR and wireworms in maize and potato is economically feasible and justifiable. However, in the case of maize, which has a considerable lower MISTIC compared to potatoes, postponing the decision to introduce the technology may be easier justified than for potatoes.
INBIOSOIL aimed at developing an innovative control strategy for soil dwelling insects. The ATTRACAP capsules developed during the project are now subjected to a registration for commercial use. The regulatory steps and associated costs for obtaining regulatory approval for this capsule have been listed, according to the relevant regulatory framework applicable in Europe (Deliverable 5.2). Relevant human health, environmental and ecotoxicological testing as well as risk assessment requirements are evaluated. On this basis, associated costs can now be calculated and missing endpoints have been identified by P9.
To be of interest for a company adopting this new control strategy the overall costs need to be computable. P15 therefore developed the components of a business plan (Deliverable 5.3) which allows a financial management and calculation of biotech products or projects. The business plan valuation (Deliverable 5.5) is accessible via the link: http://www.stolos.net/Valuation/
Potential Impact:
Potential impact of the INBIOSOIL project
INBIOSOIL aimed at developing an innovative control strategy for soil dwelling pests. The project achieved this ambitious proposition only partly. Whereas a product (ATTRACAP) based on the results and achievements of the project, is now registered in Germany for use in potato production (both conventional and organic; see below), the results regarding the efficacy of control of western corn rootworm larvae by an “attract and kill” or “confuse and kill” strategy are not at a level where commercialization would be of interest for a company.
The problem of wireworm damage in potato production systems continuously aggravated since now more than 10 years. Farmers may experience economic losses of more than 10.000 /ha each year. Since organic farmers specifically experience these drastic economic challenges several farmers already abandoned potato planting. The decision of the EU to reduce the acceptable threshold level of Fipronil (a Phenylpyrazole) contained in the product “Goldor Bait®” resulted in a withdrawal of the 2016 proposal by BASF, based on the emergency article. Thus, conventional farmers virtually have currently no alternatives for wireworm control in potato as well. This specific situation definitely offered as specific chance for the ATTRACAP product and simplified entering the market.
Within the project phase of 42 months INBIOSOIL developed a product for wireworm control in potato, fully based on biological products, which can be regarded safe to the environment and in line with the directive 2009/128/EC of the European Parliament and of the Council of 21 October 2009, which aimed at establishing a framework for community action to achieve the sustainable use of pesticides. Specifically, by developing the ATTRACAP product, INBIOSOIL addressed Clause 19 and Article14 stating that member states are obliged to implement principles of IPM with priority being given wherever possible to non-chemical methods of plant protection and pest and crop management.
The registration in Germany is based on the SANCO/10087/2013 document (Working document on emergency situations according to article 53 of regulation (EC) No 1107/2009) with several specifications (Annex I). The application is restricted to 1.000 ha between March 1st and June 28th 2016 (Notification Bundesamt für Verbraucherschutz und Lebensmittelsicherheit, AZ. 200.21320.0.140817). However, the challenge now is to apply for a registration of the ATTRACAP product on a regular basis. In this regard several problems need to be addressed: The isolate of M. brunneum used for the product is not yet listed in VO 540/2011; thus organic farmers are not allowed to use the ATTRACAP product on a regular basis. However, M. anisopliae var. anisopliae is listed in Annex I, which, due to a recent revision of the genus Metarhizium based on molecular tools, is in fact a M. brunneum strain (see Bischoff et al. 2009; Mycologia 101, 512-530). A decision is needed rapidly updating the Annex I list in this regard. The peer review of the European food safety authority on this isolate (Bipesco 5/F52), published in the EFSA Journal 2012: 10, addresses several end points, where additional safety data are requested because not yet available. In case these data need to be provided for each isolate of, for example, an entomopathogenic fungus, the registration costs will rapidly exceed the benefits for a single company, because these products are typically not produced in large quantities. The promotion and implementation of biological control options, as anticipated by the directive 2009/128/EC, may therefore recede into the distance.
The economic analysis of applying a biocontrol product against wireworms in potato using a real option model approach, developed by INBIOSOIL, will provide plant protection organizations or decision making bodies with a scheme which provides baselines necessary for evaluating the socio-economic benefit of biological compared to conventional control options.
Dissemination activities of the INBIOSOIL project
All partners of the INBIOSOIL consortium actively took part in dissemination activities (see Deliverable 6.3). These activities followed a tiered approach, with poster and lecture presentations by the scientific members at national and international conferences, workshops, and meetings with stakeholders. These dissemination activities mainly aimed at introducing the concept of the “attract and kill” strategy to the scientific community and stakeholder organizations, allowing to discuss potential problems not envisaged during this early phase of this idea. From 2014 onwards these dissemination activities concentrated on stakeholder meetings from the potato production sector (including both conventional and organic farmer organisations) because it became evident that there was an increasing demand for the development of an innovative control strategy for wireworms in potato production systems, because the registration of an effective synthetic control option (a.i. Fipronil) was suspended in several EU member states and only temporarily available based on Regulation (EC) No 1107/2009 of the European Parliament and of the Council of 21 October 2009 concerning the placing of plant protection products on the market and repealing Council Directives 79/117/EEC and 91/414/EEC. Specifically, organic potato farmers faced increasing problems year by year by wireworm damage and several farms with a high proportion of potato in their cropping systems can be regarded on the brink of being able to maintain their potato production for the next year due to the economic losses experienced each year
Based on recommendations of the coordinator, several meetings (for example: IOBC/WPRS working group “Insect pathogens and entomoparasitic nematodes” Zagreb and Riga; 47th Annual Meeting of the Society for Invertebrate Pathology; International Congress on Invertebrate Pathology and Microbial Control; Meetings of the German Scientific Society for Plant Protection and Plant Health; 22th and 23rd International Conference on Bioencapsulation; 10th European Congress of Entomology, York) were used to present the results of the INBIOSOIL consortium in specific sessions.
Two international conferences were organized by members of the INBIOSOIL consortium; one by P4 in September 2015 entitled “International Symposium “Biopesticides-Innovative Technologies for Pest Control” which aimed at bringing together stakeholders, companies producing biocontrol agents, and organisations promoting biological control. Another conferences, as fixed in the DoW was organized by P1 at the end of the project in December 2015 in Goettingen, were invited keynote speakers and the INBIOSOIL consortium were discussing specifically the progress made during the project phase regarding the “attract and kill” strategy.
The 10th Annual Biocontrol Industry Meeting (ABIM) in Basel, Switzerland, has been used to promote the “attract and kill” strategy to stakeholders and companies producing biocontrol agents. Also regular meetings of farmer organisations (Bioland-Fachtagung; Biokartoffelbautagung FiBL; Réunion phytosanitaire Grandes Cultures, Changins; 14th Potato day, National Agricultural Facility, Lippstadt; Wissenschaftstagung für den Ökologischen Landbau, Bonn and Eberswalde; etc.) were used to implement the idea of the new biocontrol strategy.
Stakeholders (Association of the potato industry Germany; German Farmers’ Association; working group potato) have been specifically contacted and results of the INBIOSOIL field experiments, demonstrating the effectiveness of the capsules for controlling wireworm damage in potato, were discussed with regard to a potential registration of the product ATTRACAP. Workshops with these stakeholders were also organised during the last year of the project in Switzerland, Austria, and Germany, respectively, to discuss the implementation of the innovative control strategy with farmer organizations. Based on these discussion and recommendations based on expert knowledge from the Julius Kuehn Institute - Federal Research Centre for Cultivated Plants (JKI) and the Bavarian State Research Center for Agriculture the INBIOSOIL consortium decided to give the development of the ATTRACAP capsules for wireworm control in potato top priority. To promote registration, independent agricultural research institutes were asked to test the INBIOSOIL capsules under field conditions, based on EPPO recommendations for field testing in potato. These organisations (Landwirtschaftskammer Niedersachsen; Dienstleistungszentrum Ländlicher Raum Rheinland-Pfalz; Berner Fachhochschule Hochschule für Agrar-, Forst- und Lebens¬mittel-wissenschaften) set-up field trials in 2015 and, based on their results, recommended to register the „attract and kill“ capsule product ATTRACAP as a regular product for wireworm control.
Overall, due to the pressing problem of wireworm control in potato and based on a support letter by the stakeholder organization UNIKA, the coordinator started to negotiate the regular production with a company located in Lower Saxony (BIOCARE GmbH), known to have experience in alginate capsule production processes. This company applied for the temporary registration of the product based on Article 53 (Emergency situations in plant protection) of regulation (EC) No 1107/2009 of the European Parliament and of the Council of 21 October 2009 concerning the placing of plant protection products on the market and repealing Council Directives 79/117/EEC and 91/414/EEC. Intellectual property rights, as discussed and documented in an internal INBIOSOIL agreement, for this product are hold by the company and P2. To facilitate the registration of the product in Germany, a strain of the entomopathogenic fungus M. brunneum, originating from an arable field near Goettingen, and owned by P1, is used for the ATTRACAP product, instead of the isolate ART 2825 which was originally isolated in Switzerland. The spore production for the ATTRACAP product is maintained by the company e-nema GmbH (P10), because during the INBIOSOIL project the company was already involved in upscaling of the production process.
List of Websites:
http://inbiosoil.uni-goettingen.de/
Georg-August-Univeristy of Goettingen
Department for Crop Sciences
Section of Agricultural Entomology
Prof. Dr. Stefan Vidal
Grisebachstr. 6
37077 Goettingen
Germany
Many herbivorous insect pests (more than 70%) have soil dwelling developmental stages. Control of these pests is difficult to achieve due to the discrete habitats of these species and the limited control strategies available. As of October 2009 the directive 2009/128/EC of the European Parliament and Council established a framework for a community action to achieve a sustainable use of pesticides. This directive demands that each Member State adopts a National Action Plan to set “quantitative objectives, targets, measures and timetables to reduce risks and impacts of pesticide use on human health and the environment”. Since many year demand for nature-based biopesticides has been steadily increasing worldwide. However, integrated pest management options for belowground herbivores are not available for many species; this problem being even more aggravated in organic farming systems. Therefore, INBISOIL aimed at developing innovative control strategies, based on an “attract and kill” approach, both for organic and conventional farmers, by exploring synergistic effects between entomopathogenic fungi (EPFs), entomopathogenic nematodes (EPNs), combined with semiochemicals, making use of strategies derived from nature. These novel formulations aimed at overcoming drawbacks typical encountered when applying biological control agents like EPFs, e.g. low efficacy or shelf life, consequently, increasing costs for their application. The INBIOSOIL project developed a novel capsule type based on bio-polymers and sustainable raw materials combined with specific isolates of entomopathogenic fungi. When applied into the soil these capsules produce carbondioxide, a cue used by most soil dwelling larval stages to locate their host plant roots. When coming into contact with these capsules fungal spores attach to the cuticle of the larvae and kill them by invading the insect body. The capsule type developed by INBIOSOIL efficiently controls wireworms in potato production systems; an insect pest complex which causes severe damage to tubers in autumn, entailing serious economic losses for farmers. These capsules (brand name ATTRACAP) have been registered for use in potato according to regulation Article 53 of Regulation No 1107/2009, and two companies are now involved in the production and regular registration of this INBIOSOIL product. Specific capsule types have also been designed for other herbivorous pest species, such as western corn rootworm, black vine weevil or fungus gnat larvae; however these strategies are not yet commercially effective. Additionally, INBIOSOIL has generated data on non-target effects of these capsules on aboveground and belowground predators, and has developed tools which allow identifying the fate of the fungal spores in the soil over the growing season. Protocols for a risk assessment of entomopathogenic fungi (RAFBCA-Rebeca) have been updated and analytical tools have been improved to analyze the fate of mycotoxins, eventually by these fungi, in the food chain. Finally the socio-economic risks and benefits of implementing biological control strategies have been evaluated and a novel approach has been proposed taking into account direct and indirect costs of the application of biocontrol agents versus synthetic insecticidal compounds. These risk assessment data, generated by INBIOSOIL, will promote an accelerated registration of biological control agents and will help policy makers and stakeholders in decision making processes based on the safety of these environmentally friendly agents and strategies.
Project Context and Objectives:
Many herbivorous insect pests (more than 70%) have soil dwelling developmental stages. Control of these pests is difficult to achieve due to the discrete habitats of these species and the limited control strategies available. As of October 2009 the directive 2009/128/EC of the European Parliament and Council established a framework for a community action to achieve a sustainable use of pesticides. This directive demands that each Member State adopts a National Action Plan to set “quantitative objectives, targets, measures and timetables to reduce risks and impacts of pesticide use on human health and the environment. With the increased environmental awareness of the public, and the pollution potential and health hazards from many of the conventional pesticides, the demand for nature-based biopesticides has been increasing steadily worldwide. However, integrated pest management options for belowground herbivores are still limited; this is specifically true for organic farming systems. The recently introduced 2-year-moratorium on neonicotinoid compounds, widely applied as seed coatings, even aggravated the problem of limited access to diversified control options for these pests.
INBISOIL aimed at developing new biological control products, both for organic and conventional farmers, by exploring synergistic effects between entomopathogenic fungi (EPFs) and efficacy enhancing agents making use of strategies derived from nature. Because soil dwelling larvae of many herbivorous insect species orientate towards their host plant roots using carbon dioxide serving as an orientation cue, and which is released by growing roots, the INBIOSOIL starting idea for an innovative control strategy was to combine CO2 releasing capsules with a killing component. When applied to the soil, these capsules would attract the larvae when searching for food, and by contact with the killing agent, the populations of the pest insects would be reduced.
These novel capsule formulations include encapsulated and dried entomopathogenic fungi (EPFs) as well as innovative co-formulations based on these EPFs, or a Bacillus thuringiensis strain in combination with efficacy enhancing agents. All formulations were processed in a way that they could be dried and stored for an extended shelf period; end-products were made available for a scaled up production process. Different co-formulations were evaluated for different strategies, named “attract and kill”, “stress and kill”, or “confuse and kill”, based on efficacy enhancing agents such as CO2 or botanicals.
To allow implementation of these capsules in control strategies in the field, additional data need to be compiled for a registration process. Therefore INBIOSOIL developed protocols to determine the cytotoxicity and genotoxicity of crude extracts and purified metabolites of entomopathogenic fungi (Metarhizium brunneum isolates) in plants and insects, and the effects on aquatic organisms. A major challenge for INBIOSOIL was to develop protocols and guidelines that effectively differentiate between microorganisms that can be safely used for biocontrol strategies and organisms that by producing toxic compounds at concentrations which potentially posing a risk to human health and/or the environment. Analytical methods for destruxins (dtxs) quantification, based on capillary electrophoresis and UHPLC-MS/MS were developed for the qualitative and quantitative assessment of the most important mycotoxins potentially produced by Metarhizium spp. isolates. Isolation of the most relevant destruxin analytes from strawberry and maize plants, which were used as proxies for relevant crop plants, demonstrated that these metabolites are not entering the food chain, and therefore should not pose a risk to consumer and animal health.
INBIOSOIL also assessed the fade of these Metarhizium isolates when applied under field conditions. Specific molecular genetic tools for the detection of applied Metarhizium spp. isolates were developed. These markers were successfully used to monitor M. brunneum isolates over time in both pot and field experiments. Moreover, a “second generation sequencing” approach was implemented to assess potential effects of the application of these EPFs on microbial communities, when applied to the soil. The application of spore suspensions resulted in short-term changes in the fungal communities, but these effects mostly decreased or disappeared by the end of the experiments after approx. 4 months.
To assess the influence of Metarhizium isolates on non-target organisms, four non-target predator species were selected and these species were tested in standardized pot experiments, based on a protocol developed during the INBIOSOIL project phase as well. When high EPF concentrations were applied moderate effects on mortality, fecundity and longevity were found in the lab, yet responses varied and depended on predator susceptibility. Given the specific administration of the EPF spores via capsule formulations the risk on non-target organisms is regard much lower than in previous control strategies, where high densities of spore suspensions need to be applied for the pest control becoming effective.
In parallel to the tests of Metarhizium formulations in the lab on non-target organisms INBIOSOIL also tested potential side effects of various biocontrol treatments (targeting Diabrotica virgifera virgifera), including different formulations of Metarhizium brunneum (F52, BIBESCO5) on non-target arthropods on four experimental fields in Austria. To assess the diversity and population level of species of the epigeous fauna the most economical way is the use of pitfall traps. In addition, the “sentinel prey method” was used to determine the predation activity of specific arthropod taxa in these experimental plots by offering a known amount of static prey items placed in the investigated area. For a certain time period the removal of the prey can then be evaluated by counting the remaining portion that is left behind (not yet consumed). The data compiled during these field epxeriments do not show any significant differences with regard to predator activity, specifically not in Metarhizium treatments compared to controls. Pitfalls trapping as well as sentinel prey evaluations are relevant methods in risk assessment under field situations, and the risk of field applications of Metarhizium spp. formulations non-target arthropods can be regarded negligible, based on the data compiled by the INBIOSOIL project.
Following the efficacy evaluation of different isolates of Metarhizium brunneum against larvae of the western corn rootworm, wireworms, vine weevils, or fungus gnat larvae, different strains of the entomopathogenic fungus were selected. These isolates were tested in lab and field experiments in different crops (maize, potato, strawberry, garden soils). Whereas in first year trials an application of capsules containing spores and capsules releasing CO2 were tested separately (co-application), follow up trials allowed to test the co-formulated capsules (spores contained in CO2-releasing capsules). For wireworm control in potato INBIOSOIL was able to demonstrate that the innovative approach of a “lure & kill” strategy can be effective. These prototype capsules were, however, a first test and it became evident that capsules needed to be improved for achieving a more pronounced damage reduction on potato tubers. The refined capsule types used in the last year of the project (2015) were not only tested by the partners of the INBIOSOIL project, but also by stakeholders in Germany. At certain conditions, depending on the population level of the wireworms, and abiotic conditions in the soil, the “lure & kill” strategy controlled wireworms by on average 50%. The INBIOSOIL capsule type developed within the project phase of INBIOSOIL thus resulted in a product (brand name ATTRACAP), which has now been registered according to regulation Article 53 of Regulation No 1107/2009 of the European parliament and of the council (emergency situations in plant protection) in Germany for 2016.
The “lure & kill” approach did, however, not work for wireworm control in maize fields. Application of the capsules did not significantly reduce wireworm damage on seedlings. Although the build-up of CFUs in the soils could be documented, the overall efficacy was limited, probably because the entomopathogenic fungus needs more that 3 weeks to kill a wireworm larvae; a time interval where damage to the root systems of the maize plants is continuously progressing.
For the control of western corn rootworms (WCR) a combined application of entomopathogenic nematodes and fungi was tested. In all years the lowest number of adults hatching from the plots was achieved when combing both biocontrol agents with an insecticidal seed treatment. Overall, both greenhouse and field experiments did not provide data showing a significant reduction of WCR larval populations. Therefore capsules types so far developed under the INBIOSOIL project cannot be recommended for WCR control.
Several experiments proved that fungus gnat larvae cannot be controlled by EPFs; thus a different approach needed to be adopted for controlling this pest species in greenhouse potting soils. A “lure & kill” approach was developed using Bacillus thuringiensis spores encapsulated in beads releasing CO2. Although these capsules where highly attractive to the larvae of this pest, the efficacy is not sufficient yet to proceed with the commercialization of these capsules.
For vine weevil control a different approach within the INBIOSOIL project has been developed. Previous studies have demonstrated that some plants are highly preferential to a pest and can thus be used to divert them from the main crop. Therefore several plants have been tested for their ovipositional attractiveness and one plant (Euonymus sp.) elicited a strong ovipositional preference in female vine weevils and could therefore be used as a trap crop. In combination with a lure INBIOSOIL results established the basis for a push-pull strategy.
The best biological control strategies are useless in case they are not adopted by farmers. The most important reason for not adopting these strategies is the higher costs per application unit. INBIOSOIL therefore reviewed the socio-economic impacts of controlling pests, such as the western corn root worm or wireworms, with various strategies in maize and potato, drawing on European as well as North American experiences. Specifically, the potential of biological control agents, such as entomopathogenic nematodes or fungi, perceived to a large extent as alternatives to chemical control, also due to increasing legislation on pesticide restriction, were analysed. Given the current level of knowledge on biological control options and data collected during the INBIOSOIL project allowed to conclude that under certain conditions biological control agents can provide an economical viable alternative for the control of the western corn rootworms and wireworms and a market for such kind of control strategies exists. From a social welfare perspective uncertainty over benefits and costs, irreversibility effects and externalities need to be considered as well. INBIOSOIL introduced an approach, the maximum incremental social tolerable irreversible costs (MISTICs), I*, as a tool for such consideration. Only when the incremental benefits of an IPM strategy outweigh possible irreversible costs should an IPM strategy be introduced. Given higher MISTICs values for wireworm control in potato the introduction of a biological control strategy is more likely to be economically feasible compared to a biological control strategy of western corn rootworms in maize. Based on these data compilations an online platform for the assessment of a business plan has also been developed by the INBIOSOIL project, allowing an investor to make an informed decision about the project’s profitability, viability and risk profile.
Based on these data INBIOSOIL assessed the cost/benefit for the innovative capsules targeting wireworms in potato. The evaluation resulted in a price for the granules, at an application rate of 30 kg/ha, of about 200 €/ha. The optimization of the production process of the capsules in combination with a higher efficacy would allow to commercialize the ATTRACAP capsules at a price of about 300 €/ha. The regulatory steps and associated costs for obtaining a regulatory approval for the biological control agent, the innovative ATTRACAP capsules, have been identified, according to the relevant regulatory framework currently applicable in Europe. Given the analysis INBIOSOIL concluded that regulatory and registration uncertainties remain high and that future approval of the product largely depends on pending registration processes for other biological control agents, serving as a proxy for ATTRACAP capsules as well.
Project Results:
Workpackage 1
Deliverables contained in this workpackage report on regular meetings (including the kick-off meeting) and the final international symposium organized at the end of the INBIOSOIL project. The regular meetings (including Skype conferences) have been used to update partners about the progress made during the preceding weeks, to discuss problems and to coordinate the next tasks.
Workpackage 2
INBIOSOIL developed a screening protocol for testing semiochemicals against wireworms (Deliverable 2.1). Several compounds provided by P 12 were identified as being repellent; however, application under field conditions would be too expensive and is therefore not regarded a suitable strategy for diverting wireworms away from the potato tubers. P6 tested turmeric oil and garlic for its repellent properties against wireworms and identified garlic as the more effective one. Based on the results of dose response tests carried out in Petri dishes and terraria experiments P4 calculated a dose between 20 and 70 kg of the specific compound per hectare to be an appropriate dose for synergy studies. The garlic compound was also tested for potential synergies with a fungal strain, identified as being most effective against wireworms. However, the results could not confirm any synergistic activities of these two agents (Deliverable 2.1). Potential synergistic effects of Turmeric and a formulated Metarhizium brunneum (strain BIPESCO5) product were also tested for their efficacy to control western corn rootworm larvae. However, a synergy between these agents could not be confirmed in the experiments.
an enhanced infection of Western corn rootworm larvae in a laboratory study
Tests using fungus gnat larvae revealed that larvae were attracted to CO2-releasing capsules and were significantly repelled by turmeric oil or spearmint in a dose-response relationship (P4; P1). Vine weevil larvae were also attracted by CO2-releasing capsules, but were not arrested at these capsules (P4). Synergistic activities of biocontrol agents were also evaluated. The application of the BIPESCO5 (Metarhizium brunneum) capsules alone and in synergy with entomopathogenic nematodes (EPN) did not result in a reduced adult emergence. BIPESCO5 is therefore not suitable for fungus gnat control. The entomopathogenic nematode Steinernema feltiae, widely used as a biocontrol measure, also failed to control fungus gnat larvae (Deliverable 2.2).
Turmeric oil was identified as a behaviour modifying substance for western corn rootworm larvae with repellent properties (P1). Results from the dose response tests showed that a concentration of 12.5 μL turmeric oil/40g soil (= 0.31 μL/g soil) is currently the lowest concentration for an effective repellent effect for 1st instar WCR larvae and 25 μL turmeric oil/40g (= 0.63 μL/g soil) was the lowest concentration for an effective repellent effect for 2nd instar WCR larvae, respectively.
P1 and P2 screened polymers and additives and minimum doses of active ingredients with efficiency enhancing agents (Deliverable 2.2). Although the finalization of this deliverable was delayed, results of these screening tests were constantly used to update the formulation of the capsules constituents. Several biopolymers were tested as matrix materials for capsule systems; at the same time different encapsulation methods were evaluated. Theses capsules were simultaneously tested for their mechanical stability and their persistence in different soils types. Given the results of these tests P2 focused on alginate based formulations, using the added value of gelatine as a nutrient for the entomopathogenic fungus and chitosan as a protective coating due to its antimicrobial properties.
Based on previous results with capsules releasing CO2, P2 developed co-formulations using entomopathogenic fungi (EPF) with efficacy enhancing agents (EEAs) (Deliverable 2.3). Spore concentrations were varied and the influence of temperature regimes on fungal growth was also tested. Additional technology developments were needed for the production of a capsule type which allowed spores of the entomopathogenic fungal strain to germinate and grow out of the capsules, and which could be technically dried to enhance shelf life and application of the capsules by commercially available granule spreaders. P2 also performed a series of studies on the development of the fungus within different beads, on increasing of the sporulation, the influence of drying on germination and persistence in soil as well as the optimization of these interactive processes.
The prototype of the capsule, which already showed promising features with regard to the requirements for field application, was then tested in parallel or in next year experiments by different partners of INBIOSOIL. For example P1 tested different capsule types in rhizotron experiments for their attractiveness for wireworms and western corn rootworm larvae (Deliverable 2.5). The results of these experiments were promising and demonstrated the feasibility of the “attract & kill” approach for control of wireworms in potato.
P2 also developed a capsule type, releasing CO2 and containing spores of Bacillus thuringiensis as the killing agent. These capsules were tested by P1 and P13 for their efficacy in controlling fungus gnats in different soil media. The “attract and kill” co-formulation containing baker’s yeast as an attractant resulted in the best performance with regard to fungus gnat control (Deliverable 2.5). However, it became evident that different media influenced the efficacy of the co-formulations, being also attractive for the larvae. This indicates that co-formulations employing the “attract & kill” strategy need to be further tested in different types of soilless growing media to ensure effective fungus gnat control under varying soil conditions. In summary, the potential of CO2 releasing capsules in combination with Bt showed promising results and should be developed further.
Capsules have also been tested for a “confuse and kill” approach based on entomopathogenic fungi (EPF) as the “kill”-component and semiochemicals/plant extracts as the “confuse”-component (Deliverable 2.5). Different treatments were set-up to test this approach for wireworm control on potato. Although the encapsulated EPF could significantly reduce the wireworm damage, no synergy with encapsulated garlic could be observed. Reduced germination rates of spores from garlic treated substrates even suggest some level of antagonism between these agents.
Based on the results reported in Deliverable 2.1 - to 2.5 and on the lab, greenhouse, and field experiments P2, in cooperation with P1 and P11 finally evaluated the granules produced by extrusion and subsequent crosslinking and drying in fluidized bed dryer. Granules produced by these techniques were tested in large scale field trials in 2015. A comparison of the properties of alginate beads and granules was done by P1. Briefly, the differences between the two granule types were marginal and both showed CO2-emission and outgrowth of mycelium. In summary the novel formulations (capsules or granules) systems with encapsulated spores are suitable for mass production; however, there is room for optimizing this process.
Workpackage 3
Formulations with encapsulated entomopathogenic fungi may were shown to improve the efficacy against target pests. However, prior to registration these novel formulations need to be carefully screen for potential side-effects on beneficial organisms. P5 developed a protocol with a decision tree based on different exposure scenarios. The test organisms were selected to represent different orders of beneficial predatory arthropods and species with different degrees of contact with soil, and hence risk of exposure to entomopathogenic fungi. The protocol (Deliverable 3.1) describes in detail the set-up of the trials and the analytical procedures. An important prerequisite for these tests is the use of same aged non-target insects, reared under standardized conditions. P14 has established lab colonies of the test organisms (part of Deliverable 3.1) which allowed for the experiments described in detail in the Deliverable 3.9. This laboratory study was conducted to assess the effects of the entomopathogenic fungi Metarhizium brunneum strain BIPESCO 5 (used in field experiments) on four non-target beneficial predatory arthropods which represent different levels of contact with soil. The commercially available predators evaluated were: Dalotia coriaria (Kraatz); Orius majusculus (Reuter); Gaeolaelaps aculeifer (Canestrini) and Aphidoletes aphidimyza Rondani. The studies demonstrated that O. majusculus was the most affected predator with the highest mortality, achieving 96% and reduced fecundity when compared to the control group. G. aculeifer was only marginally affected; the relative risk after exposition to fungal spores was almost null, only 1.2 % higher comparing with the control and no effect on fecundity was observed. D. coriaria showed significant effects on longevity and fecundity; however, 92.7% survived after the fungal exposition. In these experiments, the insects were exposed to soil mixed with a high concentration of fungal spores. This set-up could be regarded as a worst case scenario, because the innovative capsules developed by INBIOSOIL are aimed at reducing the spore load in the soil by containing them in the beads. When the fungus is growing out of the capsules the non-target organisms might come into contact with the spores as well; however, the incidence of these contact events should be substantially be reduced compared to a situation as given in the experiments described above.
Additional pot trials were set-up to assess the interactions between the predatory gall midge, Aphidoletes aphidimyza, and the entomopathogenic fungus Metarhizium brunneum (Deliverable 3.9). The study was conducted in greenhouse conditions, sweet corn (Zea mays var. saccharata) seeds were grown in pots filled with the natural soil inoculated with M. brunneum. This soil type was used, instead of artificial soils as, for example, recommended by OECD guidelines, because during screening tests it became obvious that these artificial soils do not mimic the interactions between the capsules in the soil and the outgrowth of the fungus. Thus soils from natural settings were used instead throughout the experiments. The plants were infested with Rhopalosiphum padi and A. aphidimyza pupae were introduced in the soil. At the end of the experiment Metarhizium was isolated from the leave surfaces and PCR amplifications were performed for one representative isolate of each multilocus genotype. When both natural enemies were combined in one treatment, the effect was less than additive, but the control of aphids by A. aphidimyza was only slightly affected. Thus under field conditions with more pest species being present entomopathogenic fungi could supplement other control agents. P5 also tested the capsules containing M. brunneum (Bipesco 5) for side effects (mortality, fecundity) on the 3 model arthropods (Dalotia coriaria, Orius majusculus, and A. aphidimyza) provided by P14. No significant difference was found with regard to mortality and longevity between treatments for any of the three species. However, a significantly lower fecundity was reported for O. majusculus when exposed to the M. brunneum capsules treatment. Based on the results of this investigation a very low risk for beneficial arthropods can be expected when capsules are applied in the soil of agricultural fields.
A pilot study was also set up by P5 to test various combinations of Metarhizium brunneum capsules and garlic extract capsules on O. majusculus. Garlic capsules had a negative effect on the outgrowth/ conidia production of Metarhizium and its infectivity on the predatory species. Based on these results a combination of these two actors in a “stress and kill” approach cannot be recommended (Deliverable 3.9).
To assess the abundance and survival of the fungal strain spores following the application to the soil over time it is essential to have specific markers which allow to discriminate between fungal strains already present in the soil and the ones which have been added. Therefore P6 established a reference strain collection of 65 fungal isolates representing 11 known Metarhizium species. The second goal was to develop an efficient genotyping tool that allowed discrimination of isolates of the different Metarhizium spp. (Deliverable 3.3). Different numbers of markers were amplified for the different fungal species. PCR protocols for amplification of the respective target loci from the four Metarhizium species were developed and optimized. Specificity of the designed primers was confirmed first by performing blast similarity searches in the non-redundant nucleotide database of GenBank and second by performing PCR amplifications with all the 65 isolates of the isolate collection. A strategy for genotyping Metarhizium field isolates has been proposed.
In the European Union (EU) microbial biological control agents (MBCAs) used in plant protection are regulated according to the EU Council Directive 91/414/EEC which has been replaced by the new Regulation (EC) 1107/2009. This regulation lays down rules for the authorisation of plant protection products in commercial form and for their placing on the market. In Annex II, 5.3.2 (methods of analysis) methods of residue analysis for the active substance and relevant metabolites in plant, animal and environmental matrices and drinking water, as appropriate, are asked to be validated.
Evaluating potential effects of biological control agents (BCA) on non-target organisms is a crucial component in the risk assessment process required for registration of products. Beneficial arthropods represent the prime focus in non-target studies, however, with the implementation of EU regulation (EC) No 1107/2009 studies on potential effects on microorganisms, e.g. bacteria and fungi, have to be included.
A risk assessment on metabolites, produced by fungal biocontrol agents using the RAFBCA-REBECA decision scheme (Deliverable 3.6) was developed on crude extracts and to individual metabolites from Metarhizium brunneum and Beauveria brongniartii production strains. It was obvious to INBIOSOIL experts that the potential risks related with the effect of fungal metabolites needed much more scientific investigation to come up with an appropriate, validated test system. Therefore, the objective of INBIOSIOL was to set up protocols and to screen for cytotoxic and mutagenic fungal secondary metabolites produced by the production strains which meet the requisite of maximum expression and detection of potential toxicity of extracts. INBIOSOIL partners set up standard procedures for the production of crude extracts (cultivation method, extraction protocol, bioassays) because these extracts usually contain toxins in high concentration as they come from pure cultures of the fungus grown in the best condition for metabolite production.
Three promising INBIOSOIL Metarhizium strains (BIPESCO 5, EAMa 01/58-Su, ART 2825) were grown in a bioreactor system in nitrogen rich complex media, in a semi complex media and also in a synthetic minimal medium (BIPESCO 5) aimed at encouraging secretion of a wide range of metabolites. RAFBCA protocols (developed by P3) were used to determine the metabolite profile of these three isolates and key metabolites were purified to homogeneity for use as standards and in subsequent studies (Deliverable 3.4). A novel sample preparation protocol for Destruxin (the major toxin compound class produced by this fungal species) purification and enrichment and a UHPLC-DAD-QTOF-MS/MS method was established which allowed evaluating qualitative and quantitative metabolite production. This novel validated assay achieves decent assay sensitivity, precision, and accuracy and can be considered as stable and reproducible for the quantification of Destruxin metabolites in fungal culture samples. INBIOSOIL partner P7 developed another method, the Supercritical Fluid Chromatography as an alternative tool for the qualitative and quantitative analysis of Metarhizium brunneum metabolites from culture broth. Although the UHPLC-method showed a better analytical performance in terms of achievable lower quantification limits, the established UHPSFC-method represents a good alternative technique for the qualitative and quantitative analysis of Destruxins in fungal culture broth. If a more sensitive quantitation is needed there is still the option to replace the PDA detector with the triple-quadrupole mass spectrometer, which is one of the most sensitive detection systems.
Using the latter method P7 was able to demonstrate that the different strains of Metarhizium produced different Destruxin metabolites in different cultivation media. Using High Speed Counter Current Chromatography (HSCCC) it was possible to isolate dtxA, dtxB, dtxE and dtxE-diol with purities ranging between 65 and 95%. These were further purified by column chromatography over a Sephadex LH-20 material combined with flash chromatography.
P3 developed validated protocols for isolation of metabolites from crops, which were used to assess whether metabolites could enter the food chain. Fruit samples were harvested from semi-field and field studies set-up by P3 in Austria. With regard to risk assessment and registration purposes protocols for isolating, qualitative verification and quantitative determination of fungal metabolites (specifically Destruxins) from different matrices, such as culture media and crops, have been developed by INBIOSOIL. To detect Destruxin traces in fruits, such as strawberry and maize, a QuEChERS-based sample preparation protocol was established which allowed for the first time extraction of Destruxin traces from crop matrices. The assay showed good sensitivity, precision and accuracy and can be considered as a new method for routine risk assessment allowing the detection of destruxin traces. This new analytical method shall become a routine method for answering the question whether Destruxins might be able to enter the food chain. The QuEChERS-based extraction protocol was also combined with a fast and selective UHPLC-QTOF-MS assay for the detection and quantification of Metarhizium brunneum metabolites from honey samples (Deliverable 3.4). The assay showed satisfying sensitivity, precision and accuracy and can be considered as stable and reproducible. The suitability of the assay was further proven by analysing real samples from a field study using M. brunneum to control the ectoparasitic mite Varroa destructor in bee colonies. Destruxins were not detected in any of the samples which were tested proving that the honey was residue free. In conclusion INBIOSOIL partners have validated procedures for the isolation of the most relevant Destruxin analytes from strawberry and maize and were able to demonstrate that these metabolites did not enter the food chain. Given the application of the ATTRACAP capsules, containing Metarhizium, the results demonstrate that this biocontrol agent should not pose a risk to consumer and animal health. Based on these published protocols INBIOSOIL partners, stakeholders, and SMEs are able to generate data important for registration purposes of entomopathogenic fungi (e.g. Beauveria and Metarhizium). This outcome of INBIOSOIL will also have high value for regulatory authorities in Europe and OECD countries.
Details regarding the analytical methods described above can be found in:
Taibon, J and Strasser H (in press) Analytical methods for secondary metabolite detection; in: Glare T. & Moran-Diez M (Eds.) Methods in Molecular Biology series on Microbial-Based Biopesticides - Methods and Protocols. Spinger, Dordrecht.
Taibon J, Sturm S, Seger C, Parth M, Strasser H, Stuppner H (2014) Development of a fast and selective UHPLC-DAD-QTOF-MS/MS method for the qualitative and quantitative assessment of Destruxin profiles. Analytical and Bioanalytical Chemistry 406, 7623–7632.
Carpio, A., Arroyo-Manzanares, N., Ríos-Moreno, A., Garrido-Jurado, I., Gámiz-Gracia, L., García-Campaña, A.M. Quesada-Moraga, E., Arce, L. (2016) Development of a QuEChERS-based extraction method for the determination of destruxins in potato plants by UHPLC-MS/MS; Talanta, 146, 815-822.
Entomopathogenic fungi (EPF) are known to produce a wide array of metabolites some which have been characterised while others are still being discovered. To conduct a risk assessment of each compound would be a lengthy and costly process which would prohibit development of these agents for pest control. The RAFBCA-REBECA decision scheme suggests that risk assessment should be done not just on selected metabolites but also concentrated crude preparations of the metabolites.
Available protocols for testing cytotoxicity and genotoxicity of specific compounds are design for chemical pesticides; at present there are no guidelines on the risk assessment of fungal biological control agents. INBIOSOIL partner P7 therefore aimed at aim developing robust, internationally approved protocols, which are tailored towards evaluating entomopathogenic fungi (Deliverable 3.5). Several aquatic invertebrates have been used in the current protocol, and Anopheles mosquito together with Daphnia spp. and Artemia salina have been demonstrated to be excellent and complementary, aquatic ecotox indicator species of fungal biocontrol agents. A. salina and more notably D. magna proved to be extremely sensitive test organisms and are suitable model organisms for rapid and inexpensive screening of toxicity of BCAs at an early stage of product development.
Details regarding the testing protocols described above can be found in:
Garrido-Jurado I, Alkhaibari A, Williams SR, Oatley-Radcliffe DL, Quesada-Moraga E, Butt TM (2015). Toxicity testing of Metarhizium conidia and toxins against aquatic invertebrates. Journal of Pest Science; doi 10.1007/s10340-015-0700-0.
Non-target effects of BCA applications on soil microbial communities were assessed in two different pot trials, set up by P6, one to control Agriotes and one to control Diabrotica to test efficacies of the new formulations. For assessment of fungal and prokaryotic (including archaea and bacteria) communities genomic DNA was extracted from the soil of each soil sample (one sample per pot). Ribosomal target regions, i.e. for fungi ITS2 and for prokaryota V3-V4 of 16S DNA, were PCR amplified and sequencing reads were subjected to a bioinformatics pipeline, which compromised quality filtering, trimming of primer pairs and grouping sequences into operational taxonomic units (OTUs) at 97% sequence similarity (Deliverable 3.7). Fungal and prokaryotic community structures reacted differently to treatments. Effects were detected for treatments containing the active compounds M. brunneum, nematodes and/or garlic; however, detected effects mostly decreased or disappeared by the end of the experiment (15 or 18 weeks post application). The study revealed that fungal biocontrol agent applications represent a “stress” to which microbial communities react. Under field conditions fungal biocontrol agent applications may thus induce changes in microbial communities, but, if at all, they have a temporal character only, i.e. communities are resilient, particularly once applications have been dismissed.
Workpackage 4
Entomopathogenic fungal isolates are known to significantly differ with regard to their efficacy against target pest species. Therefore INBIOSOIL aimed at testing the efficacy of various isolates against several pest species as these would be more commercially viable than strains with a narrow host range (Deliverable 4.1). Based on these results different isolates were identified for different pest species, namely M. brunneum ART2825 for wireworms and M. brunneum BIPESCO 5 for western corn rootworms.
First year experiments in the field by P1 used prototypes of capsules, produced by P2, either emitting CO2 or containing spores of ART2825. These capsules were applied separately or in combination. Controls used either a spore suspension of ART2825 or no capsules (Deliverable 4.2). CO2 emissions were measured at several days past inoculation, proving that the capsules increase CO2 levels significantly above the background level. The data collected during the field season 2013 indicated that the “lure and kill” approach allows a direct control of wireworms in potato fields. However, results were variable, depending on the mode of application and the time. These data have now been analysed and a manuscript has been submitted (Brandl MA, Schumann M, Przyklenk M, Patel A, Vidal S. Wireworm damage reduction in potatoes with an attract-and-kill strategy using Metarhizium brunneum; submitted to Entomological Science).
For western corn rootworm (WCR) control greenhouse experiments were established by P1 testing different capsule types produced by P2 (Deliverable 4.3). The results identified a specific capsule type with a good, though not outstanding, performance with regard to larval suppression. Based on these results a BIESCO 5 capsule type II was recommended for field trials by P 3.
The “confuse & kill” strategy against western corn rootworm (WCR) larvae was tested in a large scale greenhouse set up in small plastic containers (Deliverable 4.4). Based on previous work the synergy components consisted of the botanical Turmeric (Curcuma longa) (provided by P12), evaluated as a repellent substance as part of deliverable 2.1 and 2.2 and the encapsulated M. brunneum strain BIPESCO5 (provided by P3), screened as the most effective entomopathogenic fungal (EPF) strain. On one hand the results of this experiment demonstrated the repellent action of Turmeric towards WCR larvae. However, the application of capsules containing the entomopathogenic fungus only or in combination with Turmeric failed to control WCR larvae. A synergistic action between Turmeric and M. brunneum could therefore not be measured, making an implementation of a “Confuse & Kill” approach not feasible as a control option for WCR larvae.
Additionally to the field trials initiated in 2013 and continued in 2014 and 2015 (Deliverable 4.4) pot trials were carried out by P 3 to test the robustness and efficacy of the nematode Heterorhabditis bacteriophora, BIPESCO 5, in comparison to a synthetic insecticide (Clothianidin) for WCR larval control. This complex experiment was conducted in cooperation with P6, who assessed establishment of the applied strain in the experimental plot and determined the effect of the applied agents on non-target organisms (see Deliverable 3.7). The experiment consisted of eight treatments, each replicated 6 times. No statistically significant differences were identified between the treatments with regard to the number of adults recovered or root damage. However, genotype analyses using SSR markers revealed that the increase of Metarhizium density in the pots treated with the capsules was due to an increase of the density of the applied strain. Thus, although the entomopathogenic fungus established in the soil the efficacy of the biological control strategy was much less compared to a chemical control option.
These treatments (plus one additional chemical control option) were also tested by P3 in field experiments in 2014 and 2015 (Deliverable 4.4). Again, no significant differences were found between the treatments with regard to the number of WCR adults recovered. However, the lowest number of WCR adults per plant was found in plot with an IPM strategy, i.e. in treatments, where the biological control agents have been combined with a synthetic insecticide. Due to the high variability of the data effects of the treatments on yields did also not differ significantly. Metarhizium spp. density in plots treated with BIPESCO 5 increased slightly in 2013, but significantly in 2014 and 2015. It remains to be tested whether these higher densities of colony forming units (CFUs) in the fields are maintained when a crop rotation would be implemented.
The persistence of both the biological control agent Heterorhabditis bacteriophora and M. brunneum isolate BIPESCO 5 were also investigated in naturally black vine weevil infested strawberry fields in South Tyrol over a period of three years by P3 (Deliverable 4.4). The nematode was found in up to 80% of the soil samples taken one month after application. At least some nematodes survived through the winter and were detected after the winter months and in the following autumn. No clear evidence of a negative or positive impact of the application of the entomopathogenic fungus on the number of the nematode was found. Metarhizium spore densities generally increased after the application of fungus; however, neither a negative nor a positive impact of the incidence of the nematode on the fungal biocontrol agent could be established. Overall these experiments, together with the ones described above, indicate that an application of the fungus, either in form of capsules or as GranMet™ into the soil, does not result in persistent and high densities of the fungal isolates in next season crops. Significantly fewer larvae of the black vine weevil were found in plots treated with a combination of the nematode and the fungus applied as a spore suspension compared to the combination where the fungus was applied as active ingredient in granules. The results of these experiments elucidate the synergistic potential of entomopathogenic nematodes and fungi, when applied together; however, the data so far do not allow recommending a specific strategy for the application of these biocontrol agents in different cropping systems.
Synergistic effects between botanically derived fumigants and nematodes for the control of sciarid flies were tested by P4. Different plant compounds were tested with diethyl maleate and eucalyptol the mostly likely to exhibit synergy with Heterorhabditis downesi, although linalool could also potentially synergise with the two other Heterorhabditis species tested. Four chemicals identified during the experiments proved to be effective fumigants in their own right for sciarid control. In the experiments very low doses were used, so 100% mortality was rarely achieved. No obvious synergy between botanicals and the nematode Steinernema feltiae was found; this species were chosen being the primary commercial nematode to control sciarid flies. In conclusion, these experiments proven the potential of synergistic activities of plant derived compounds and nematodes; however, the results do not provide a clear strategy as how to proceed with an commercial implementation.
Based on the preliminary experiments with different prototypes of the “attract & kill capsules”, the integrated capsule type was repeatedly tested in field experiments in 2014 and 2015 for wireworm control in potato and maize (Deliverable 4.5) by P1. The co-formulated A&K capsules differed with regard to virulence (lower rate of mycosis), CO2 emission (earlier CO2 emission with dry co-formulated product) and establishment of the fungus in the soil (lower CFU count) compared to an AK co-application (capsules producing CO2 and capsules containing spores of the fungus). However, the efficacy of the co-formulated compared to the co-applied capsules in the field with regard to the reduction in wireworm damage on tubers during harvest was not significantly different. The level of efficiency was, however, very variable; compared to the insecticide treatment “Goldor Bait” the effectiveness was lower. Field experiments of stakeholders in Lower Saxony and Rhineland-Palatinate, using the integrated final dry capsule type, reported variable results as well, but also demonstrated a better potential of this control strategy compared to other non-chemical control options. Abiotic environmental conditions of the soil seem to play an important role for the ATTRACAP strategy to become effective; parameters which will be evaluated in follow-up projects in detail.
The “attract & kill” strategy was also tested by P1 in maize fields, targeting the wireworms as well. Results from 2013 to 2015 demonstrated that wireworms were not effectively controlled, probably because the fungus needs several weeks to kill a wireworm following the infection of the larvae (Deliverable 4.5). During this period the wireworms, however, are still able to cause damage on the maize plants. I may be speculated that a control would act on the population level of the pest in the field, thus being visible only as a long-term effect. This hypothesis needs to be tested in further experiments. The capsules were also tested for western corn rootworm control in maize fields in Croatia.
P4 tested a strategy targeting both adults and juveniles of the black vine weevil control (Deliverable 4.5). The goal of the study was to demonstrate that some plants are highly preferential to a pest and can be used to divert them from the main crop. A glasshouse experiments demonstrated a strong ovipositional preference of female black vine weevils for a specific Euonymus species compared to strawberries, an important food plant often infested with weevils. This plant might thus be used as a trap plant for adults in combination with a control strategy for the larvae as exemplified above (Deliverable 4.4).
In conclusion the co-formulation of M. brunneum and CO2 offers a highly innovative and ecologically friendly product for pest control, specifically for wireworm control. Recommendations for the use of this “attract & kill” strategy are discussed in (Deliverable 4.6). Currently the A&K control approach is most feasible for wireworm control in potato and the dry A&K capsules can be applied with any standard granulator at a rate of 30 kg/ha. They mean efficacy achieved in field experiments was a 30% reduction in tuber damage at this application rate; these capsules work best at low wireworm pressure and wet soil conditions because the dry A&K capsules need to re-swell, significantly hampered under dry soil conditions.
The details regarding the efficacy of semiochemical repellents (Deliverable 4.7) have been subsumed in different deliverable above. An additional field experiment was set-up by P6 to test the effects of semiochemicals in combination with the fungus containing capsules and fungus colonized barley kernels (FCKBs) in Switzerland. The colony forming units within the plots increased following the application of fungus containing material; however, 18 weeks post application the numbers were generally at the level of pre-application. Unfortunately, the density of wireworms was too low to generate reliable date on the efficacy of the different treatments. The main conclusion derived from these experiments indicates that the fungal isolate and the plant extract may not interact synergistically; thus a co-application of these agents would not increase the overall protection level of the potato plants.
Deliverable 4.8 lists the different capsule formulations tested during the INBIOSOIL project by P1, P6, and P6. In conclusion different capsule formulations exhibited different characteristics with regard to the target organisms tested. Parameters influencing the efficacy of these capsules were identified and can be addressed in forthcoming technical upscaling processes and application techniques. Whereas the development of the “attract & kill” strategy for control of the most urgent pending problem of wireworm damage in potato production systems has gone far beyond the early stage of development, this strategy needs still to be improved for western corn rootworm and vine weevil control.
The costs for the implementation of a biological control strategy against black vine weevils have been exemplified and evaluated for strawberry production systems (Deliverable 4.9). The strawberry sector is of high economic importance in some European countries and suffers from damage by black vine weevils. Several studies have already tested the benefits of applying different biological control agents in combination with chemical compounds; however the underlying costs for this strategy were regarded too high to be of interest for the farmers. Developing a specific model P9 was able to calculate the costs for the different application strategies. The cost benefit analysis shows that the adoption of a combined application of entomopathogenic fungi and nematodes as biological control agents for this pest results in a higher net present value that the use of the same agents alone. Thus farmers could generate higher profits depending on the wholesale or producer price for strawberries if they decide to change their production system from insecticide control-based systems to BCA control-based systems.
Workpackage 5
P9 calculated the economic feasibility of a biological control strategy using western corn rootworm or wireworms in comparison to chemical control options (Deliverable 5.1). The appropriate model specification for the productivity assessment of pest damage control inputs, such as synthetic pesticides, insect-resistant crops, or biocontrol agents, respectively is subject for an ongoing debate in agricultural economics. Pest damage control inputs do not necessarily directly increase yield, but rather, increase the share of potential output that is realized by reducing damage. Moreover, heterogeneity among farms and farmers, caused by differences in farm and farmer characteristics, are reasons for relative differences in profitability of different pest control strategies. The model calculations are becoming even more complicated because control strategies within a cropping season may change depending on pest pressure and micro climatic conditions. Because of these uncertainties and irreversible costs are difficult to calculate P8 adopted a threshold value call the maximal incremental social tolerable irreversible cost (MISTIC), which stipulates the maximum irreversible cost an individual or society is willing to accept due to the introduction of a certain technology or innovation. Although developed for the application of a new transgenic crop in Europe, this methodology can be applied to biological control strategies as well.
For western corn rootworm (WCR) and wireworm control strategies in maize production systems chemical, transgenic, and biological strategies were compared. The calculated gross farm-level benefit of a chemical control proved to be 2.3 times the costs, which implies an economic gain due to pesticide control of WCR at farm level. The cost-benefit ration for adopting a transgenic strategy (maize expressing a Bacillus thuringiensis toxin), depending on the WCR infestation levels, indicated a net return for the farmers. Crop rotation is currently regarded the most effective control strategy for WCR in Europe. For the application of entomopathogenic nematodes or fungi the farmer’s net benefit for applying these biocontrol agents would still results in positive revenue. However, additional producer benefits (chemical pesticide reductions and lower residues; host specificity of the control agents; low to zero resistance issues) would add to a conservative estimation of the benefit: costs ratio for biological control to be at 2.5 - 20:1. Given the current level of knowledge allowed concluding that under certain conditions biological control agents would provide an economical viable alternative for the control of the WCR and wireworms and a market for these control strategies should exist.
The economic analysis of applying biocontrol against WCR larvae and wireworms in maize and potato using a real option model approach took into account irreversible cost of introducing such technology. This made a threshold for decision-making called the MISTICs, I*. This economic analysis takes the long-term effect of biocontrol products in pest management into consideration. The MISTICs captures the uncertainty, flexibility and irreversibility associated with the introduction of biocontrol in Germany, France, Austria, Spain and Italy (Deliverable 5.4). At the farm-level, results suggest that an immediate introduction of biocontrol against WCR and wireworms in maize and potato is economically feasible and justifiable. However, in the case of maize, which has a considerable lower MISTIC compared to potatoes, postponing the decision to introduce the technology may be easier justified than for potatoes.
INBIOSOIL aimed at developing an innovative control strategy for soil dwelling insects. The ATTRACAP capsules developed during the project are now subjected to a registration for commercial use. The regulatory steps and associated costs for obtaining regulatory approval for this capsule have been listed, according to the relevant regulatory framework applicable in Europe (Deliverable 5.2). Relevant human health, environmental and ecotoxicological testing as well as risk assessment requirements are evaluated. On this basis, associated costs can now be calculated and missing endpoints have been identified by P9.
To be of interest for a company adopting this new control strategy the overall costs need to be computable. P15 therefore developed the components of a business plan (Deliverable 5.3) which allows a financial management and calculation of biotech products or projects. The business plan valuation (Deliverable 5.5) is accessible via the link: http://www.stolos.net/Valuation/
Potential Impact:
Potential impact of the INBIOSOIL project
INBIOSOIL aimed at developing an innovative control strategy for soil dwelling pests. The project achieved this ambitious proposition only partly. Whereas a product (ATTRACAP) based on the results and achievements of the project, is now registered in Germany for use in potato production (both conventional and organic; see below), the results regarding the efficacy of control of western corn rootworm larvae by an “attract and kill” or “confuse and kill” strategy are not at a level where commercialization would be of interest for a company.
The problem of wireworm damage in potato production systems continuously aggravated since now more than 10 years. Farmers may experience economic losses of more than 10.000 /ha each year. Since organic farmers specifically experience these drastic economic challenges several farmers already abandoned potato planting. The decision of the EU to reduce the acceptable threshold level of Fipronil (a Phenylpyrazole) contained in the product “Goldor Bait®” resulted in a withdrawal of the 2016 proposal by BASF, based on the emergency article. Thus, conventional farmers virtually have currently no alternatives for wireworm control in potato as well. This specific situation definitely offered as specific chance for the ATTRACAP product and simplified entering the market.
Within the project phase of 42 months INBIOSOIL developed a product for wireworm control in potato, fully based on biological products, which can be regarded safe to the environment and in line with the directive 2009/128/EC of the European Parliament and of the Council of 21 October 2009, which aimed at establishing a framework for community action to achieve the sustainable use of pesticides. Specifically, by developing the ATTRACAP product, INBIOSOIL addressed Clause 19 and Article14 stating that member states are obliged to implement principles of IPM with priority being given wherever possible to non-chemical methods of plant protection and pest and crop management.
The registration in Germany is based on the SANCO/10087/2013 document (Working document on emergency situations according to article 53 of regulation (EC) No 1107/2009) with several specifications (Annex I). The application is restricted to 1.000 ha between March 1st and June 28th 2016 (Notification Bundesamt für Verbraucherschutz und Lebensmittelsicherheit, AZ. 200.21320.0.140817). However, the challenge now is to apply for a registration of the ATTRACAP product on a regular basis. In this regard several problems need to be addressed: The isolate of M. brunneum used for the product is not yet listed in VO 540/2011; thus organic farmers are not allowed to use the ATTRACAP product on a regular basis. However, M. anisopliae var. anisopliae is listed in Annex I, which, due to a recent revision of the genus Metarhizium based on molecular tools, is in fact a M. brunneum strain (see Bischoff et al. 2009; Mycologia 101, 512-530). A decision is needed rapidly updating the Annex I list in this regard. The peer review of the European food safety authority on this isolate (Bipesco 5/F52), published in the EFSA Journal 2012: 10, addresses several end points, where additional safety data are requested because not yet available. In case these data need to be provided for each isolate of, for example, an entomopathogenic fungus, the registration costs will rapidly exceed the benefits for a single company, because these products are typically not produced in large quantities. The promotion and implementation of biological control options, as anticipated by the directive 2009/128/EC, may therefore recede into the distance.
The economic analysis of applying a biocontrol product against wireworms in potato using a real option model approach, developed by INBIOSOIL, will provide plant protection organizations or decision making bodies with a scheme which provides baselines necessary for evaluating the socio-economic benefit of biological compared to conventional control options.
Dissemination activities of the INBIOSOIL project
All partners of the INBIOSOIL consortium actively took part in dissemination activities (see Deliverable 6.3). These activities followed a tiered approach, with poster and lecture presentations by the scientific members at national and international conferences, workshops, and meetings with stakeholders. These dissemination activities mainly aimed at introducing the concept of the “attract and kill” strategy to the scientific community and stakeholder organizations, allowing to discuss potential problems not envisaged during this early phase of this idea. From 2014 onwards these dissemination activities concentrated on stakeholder meetings from the potato production sector (including both conventional and organic farmer organisations) because it became evident that there was an increasing demand for the development of an innovative control strategy for wireworms in potato production systems, because the registration of an effective synthetic control option (a.i. Fipronil) was suspended in several EU member states and only temporarily available based on Regulation (EC) No 1107/2009 of the European Parliament and of the Council of 21 October 2009 concerning the placing of plant protection products on the market and repealing Council Directives 79/117/EEC and 91/414/EEC. Specifically, organic potato farmers faced increasing problems year by year by wireworm damage and several farms with a high proportion of potato in their cropping systems can be regarded on the brink of being able to maintain their potato production for the next year due to the economic losses experienced each year
Based on recommendations of the coordinator, several meetings (for example: IOBC/WPRS working group “Insect pathogens and entomoparasitic nematodes” Zagreb and Riga; 47th Annual Meeting of the Society for Invertebrate Pathology; International Congress on Invertebrate Pathology and Microbial Control; Meetings of the German Scientific Society for Plant Protection and Plant Health; 22th and 23rd International Conference on Bioencapsulation; 10th European Congress of Entomology, York) were used to present the results of the INBIOSOIL consortium in specific sessions.
Two international conferences were organized by members of the INBIOSOIL consortium; one by P4 in September 2015 entitled “International Symposium “Biopesticides-Innovative Technologies for Pest Control” which aimed at bringing together stakeholders, companies producing biocontrol agents, and organisations promoting biological control. Another conferences, as fixed in the DoW was organized by P1 at the end of the project in December 2015 in Goettingen, were invited keynote speakers and the INBIOSOIL consortium were discussing specifically the progress made during the project phase regarding the “attract and kill” strategy.
The 10th Annual Biocontrol Industry Meeting (ABIM) in Basel, Switzerland, has been used to promote the “attract and kill” strategy to stakeholders and companies producing biocontrol agents. Also regular meetings of farmer organisations (Bioland-Fachtagung; Biokartoffelbautagung FiBL; Réunion phytosanitaire Grandes Cultures, Changins; 14th Potato day, National Agricultural Facility, Lippstadt; Wissenschaftstagung für den Ökologischen Landbau, Bonn and Eberswalde; etc.) were used to implement the idea of the new biocontrol strategy.
Stakeholders (Association of the potato industry Germany; German Farmers’ Association; working group potato) have been specifically contacted and results of the INBIOSOIL field experiments, demonstrating the effectiveness of the capsules for controlling wireworm damage in potato, were discussed with regard to a potential registration of the product ATTRACAP. Workshops with these stakeholders were also organised during the last year of the project in Switzerland, Austria, and Germany, respectively, to discuss the implementation of the innovative control strategy with farmer organizations. Based on these discussion and recommendations based on expert knowledge from the Julius Kuehn Institute - Federal Research Centre for Cultivated Plants (JKI) and the Bavarian State Research Center for Agriculture the INBIOSOIL consortium decided to give the development of the ATTRACAP capsules for wireworm control in potato top priority. To promote registration, independent agricultural research institutes were asked to test the INBIOSOIL capsules under field conditions, based on EPPO recommendations for field testing in potato. These organisations (Landwirtschaftskammer Niedersachsen; Dienstleistungszentrum Ländlicher Raum Rheinland-Pfalz; Berner Fachhochschule Hochschule für Agrar-, Forst- und Lebens¬mittel-wissenschaften) set-up field trials in 2015 and, based on their results, recommended to register the „attract and kill“ capsule product ATTRACAP as a regular product for wireworm control.
Overall, due to the pressing problem of wireworm control in potato and based on a support letter by the stakeholder organization UNIKA, the coordinator started to negotiate the regular production with a company located in Lower Saxony (BIOCARE GmbH), known to have experience in alginate capsule production processes. This company applied for the temporary registration of the product based on Article 53 (Emergency situations in plant protection) of regulation (EC) No 1107/2009 of the European Parliament and of the Council of 21 October 2009 concerning the placing of plant protection products on the market and repealing Council Directives 79/117/EEC and 91/414/EEC. Intellectual property rights, as discussed and documented in an internal INBIOSOIL agreement, for this product are hold by the company and P2. To facilitate the registration of the product in Germany, a strain of the entomopathogenic fungus M. brunneum, originating from an arable field near Goettingen, and owned by P1, is used for the ATTRACAP product, instead of the isolate ART 2825 which was originally isolated in Switzerland. The spore production for the ATTRACAP product is maintained by the company e-nema GmbH (P10), because during the INBIOSOIL project the company was already involved in upscaling of the production process.
List of Websites:
http://inbiosoil.uni-goettingen.de/
Georg-August-Univeristy of Goettingen
Department for Crop Sciences
Section of Agricultural Entomology
Prof. Dr. Stefan Vidal
Grisebachstr. 6
37077 Goettingen
Germany