International Symposium and Closing Meeting of the EU-Project

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1 International Symposium and Closing Meeting of the EU-Project FAIR6 CT BIOCONTROL OF IMPORTANT SOIL DWELLING PESTS BY IMPROVING THE EFFICACY OF INSECT PATHOGENIC FUNGI 24 th January 2002 ABSTRACTS Institute of Microbiology Leopold-Franzens University Innsbruck Austria Swiss Federal Research Station of Agroecology and Agriculture Zürich, Switzerland Eric Schweizer Samen AG Research Center for Agriculture and Forestry Laimburg, Italy School of Biological Sciences University of Wales Swansea, UK National University of Athens, Greece Agrifutur s.r.l., Alfianello, Italy Royal Veterinary and Agricultural University Frederiksberg, Denmark Federal Biological Research Center for Agriculture and Forestry, Darmstadt Germany Prophyta Biologischer Pflanzenschutz GmbH Malchow, Germany Gartenhilfe GmbH, Linz Institute of Pharmaceutical Technology University of Vienna Austria

2 Symposium Location Institute of Pharmaceutical Technology and Biopharmaceutics University of Vienna Center of Pharmacy / UZA II Althanstrasse Vienna AUSTRIA Programme Welcome and opening addresses by the Local Organiser Tariq Butt (University of Wales, Swansea, UK) Fungi as Biocontrol Agents: Progress, Problems and Potentials Lene Thomsen (Ministry of Environment, Danish Forest and Nature Agency, DK) Demands in Risk Assessment of Fungal Biocontrol Agents Coffee break Hermann Strasser (BIPESCO project Co-ordinator, Leopold-Franzens-University, Innsbruck, A) BIological PEStCOntrol - an RTD-project funded by the Fourth Framework Programme of the European Union (FAIR6) Jørgen Eilenberg (Royal Veterinary and Agricultural University, Frederiksberg, DK) Melolontha melolontha, Otiorhynchus spp. and Strophosoma spp.: a pest problem in Europe? Siegfried Keller (Swiss Federal Research Station of Agroecology and Agriculture, Zürich, CH) Approaches in biocontrol of selected soil dwelling pests Jürg Enkerli (Swiss Federal Research Station of Agroecology and Agriculture, Zürich, CH) Persistence of Beauveria brongniartii in the field Susanne Vestergaard (Royal Veterinary and Agricultural University, Frederiksberg, DK) Impact of entomopathogenic fungi on non-target invertebrates and persistence of Metarhizium anisopliae Lunch Gisbert Zimmermann (Federal Biological Research Center for Agriculture and Forestry, Darmstadt, G) Economic mass production of mycoinsecticides Andrea Horaczek (Institute of Pharmaceutical Technology and Biopharmaceutics, Vienna, A) Formulation-strategies of mycoinsecticides Milton Typas (National University of Athens, GR) Is there a need to impove mycoinsecticides by genetic engineering? General discussion

3 Index Tariq M. Butt, Farooq Shah and Chengshu Wang; Fungi as Biocontrol Agents: Progress, Problems and Potential. 1 Lene Thomsen; Demands in risk assessment of fungal biocontrol agents. 2 Hermann Strasser; Biological Pest Control (BIPESCO) - an RTD project funded by the 4 th Framework Programme of the European Union (FAIR6). 3 Siegfried Keller and Jørgen Eilenberg; Approaches in microbial control of soil dwelling pests in Europe. 4 Jørgen Eilenberg, Susanne Harding, Siegfried Keller, Hermann Strasser, Roland Zelger and Gisbert Zimmermann; Melolontha melolontha, Otiorhynchus spp. and Strophosoma spp: A pest problem in Europe? 5 Jürg Enkerli, Philip Kessler and Siegfried Keller; Persistence of Beauveria brongniartii BCA strains in the field. 6 Susanne Vestergaard, Charlotte Nielsen and Siegfried Keller; Impact of entomopathogenic fungi on non-target invertebrates and persistence of Metarhizium anisopliae. 7 Gisbert Zimmermann; Economic mass production of mycoinsecticides. 8 Andrea Horaczek, Josef Raffalt and Helmut Viernstein; Formulation Strategies for Mycoinsecticides. 9 Milton A. Typas; Is there a need to improve mycoinsecticides by genetic engineering? 10

4 Fungi as Biocontrol Agents: Progress, Problems and Potential. Tariq M. Butt, Farooq Shah and Chengshu Wang School of Biological Sciences, University of Wales Swansea, Swansea, SA2 8PP, UK Increasing public sensitivity to environmental pollution and problems of pest resistance to chemical pesticides has led to a global consensus to reduce or phase out extremely noxious pesticides (e.g. methyl bromide). But this will not be possible unless a concerted effort is made to develop new, environmentally friendly crop protection strategies. At present, crop protection is trapped between the increasing number of prohibited chemical pesticides and the lack of safe, efficient alternatives. Entomogenous fungi offer an environmentally friendly alternative to chemical pesticides but little is invested in these biocontrol agents (BCAs) because of their narrow host range and inconsistency and poor performance in field trials. Although niche markets exist for fungal BCAs, considerable progress still needs to be made in the following areas: technical (production, formulation, application), agronomic (integration of BCAs into cropping systems), socioeconomic (public perception, economic feasibility) and political (improved registration procedures, reducing registration costs, more support for industry and organic growers). Fungal BCAs would be used more extensively once the obstacles within these areas were resolved. This paper focuses on specific technical problems and briefly outlines the progress made in increasing the efficacy of entomogenous fungi for pest control. The major technical hurdles include: (1) improvements in screening and quality control to ensure that only viable, virulent inoculum reaches the market; (2) development of media to increase the virulence and ecological fitness of conidia; (3) improved formulations to increase both the virulence and shelf life of the inoculum; (4) better application strategies to ensure that sufficient inoculum contacts the pest because mortality is dose-related above certain thresholds. Use of entomogenous fungi will increase once growers feel more confident in their use. The number of niche markets will also expand as new target pests are discovered. However, governments could play a more active role in the commercialisation of fungal BCAs. For example, they could strengthen extension services to accelerate technology transfer from research institutes and industry to the grower. They could also streamline policies and/or procedures to reduce product development time and/or costs. 1

5 Demands in risk assessment of fungal biocontrol agents Lene Thomsen Ministry of Environment, Danish Forest and Nature Agency, Haraldsgade 53, DK-2100 Copenhagen Ø, Denmark By the adoption of Council Directive 91/414/EEC concerning the placing of plant protection products on the market the European Community established a Community procedure for authorisation of plant protection products, including products based on microorganisms as active substances. Six annexes are connected to the Directive, and the annexes providing more specific guidance are divided in an A (chemicals) and a B (microbials) part. The information to be provided by the annexes is: Annex I - Community positive list of active substances which may be used in plant protection products; Annex IIB - data requirements concerning new active microorganisms; Annex IIIB - data requirements concerning the authorisation of plant protection products; Annex IVB - risk phrases; Annex VB - safety phrases; Annex VIB - uniform principles for the evaluation and authorisation of plant protection products. The development of these annexes is a still ongoing work. When a specific strain of an active organism has been included in Annex I, the national authorities in each country regulate the authorisations of different formulations based on that strain. Annex VIB, the uniform principles, providing guidelines ensuring that national authorities use uniform criteria for the evaluation and authorisation of plant protection products based on organisms included in Annex I is right now under development. A microbial plant protection product can only be approved on condition that it has; i) no unacceptable risk to human and animal health; ii) no unacceptable influence on the environment; iii) adequate efficiency. In order to have a new organism included in Annex I, a dossier must be submitted to the national authorities (to be chosen freely by the applicant) in a Member State (Rapporteur). The Rapporteur makes a risk assessment based on the submitted information in the dossier and a recommendation to the Commission on whether the active organism can/cannot be included in Annex I of the Directive. A dossier must contain information on both the active organism at the strain level (Annex IIB) and a plant protection product based on this strain (Annex IIIB). Commission Directive 2001/36/EC has recently amended these two annexes. The national authorities are obliged to implement these new, more specific data requirements in their national legislation not later than 1 st May Selected parts of the new data requirements will be addressed more specifically. 2

6 Biological Pest Control (BIPESCO) - an RTD project funded by the 4th Framework Programme of the European Union (FAIR6) Hermann Strasser (Coordinator of BIPESCO project) Institute of Microbiology, Leopold-Franzens University Innsbruck, Technikerstraße 25, A-6020 Innsbruck, Austria, Hermann.Strasser@uibk.ac.at; The aim of the BIPESCO project was to study and develop entomogenous fungi for the control of subterranean insect pests like scarabs and weevils. Leading experts from seven different European countries including four industrial partners participated in this multidisciplinary, multifaceted project. Particular attention focussed on the widespread, soil-borne pathogens, Beauveria brongniartii and Metarhizium anisopliae. Strains of these fungi were developed for use in integrated pest management programmes to help replace or reduce the input of chemical pesticides in European agriculture, forestry and horticulture in accord with the reformed common agriculture policy guidelines. The specific objectives of this project were to (1) control scarabs and weevils with virulent, ecologically competent strains of insect-pathogenic fungi (2) improve production and formulation technologies, (3) develop biochemical methods to monitor fungal virulence and nutritional (carbon) requirements, (4) use molecular techniques to characterise strains to monitor the pathogen in the field (spatial-temporal distribution, genetic stability, interactions with autochthonous, conspecific strains) and for the detection of instability factors (e.g. transposons, mycoviruses), (5) test new application systems for effective targeting of the pathogen, (6) study the impact of the pathogens on target and non-target insects, (7) conduct field trials to demonstrate/evaluate the efficacy of the fungal biological control agents, and (8) address some of the criteria for the registration of insect pathogenic fungi. The BIPESCO team have achieved the main goals of this project and have generated new products based on these fungi. The participants of this project will present the findings of this project. Demonstration trials in the future should help validate the efficacy and safety of these fungi for the control of chafers and weevil pests. 3

7 Approaches in microbial control of soil dwelling pests in Europe Siegfried Keller & Jørgen Eilenberg* Federal Research Station for Agroecology and Agriculture, Reckenholzstrasse 191, CH-8046 Zürich. *Department of Ecology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frb. C, Copenhagen. The most important soil dwelling pests in central Europe are the larvae of some scarabaeid beetles, especially Melolontha melolontha, M. hippocastani, Amphimallon solstitiale, Phylloperta horticola and Hoplia spp. (Scarabaeidae), but also the larvae of some curculionid beetles (Curculionidae) and of clickbeetles (Elateridae). Larvae of Diptera and Lepidoptera as well can cause damage but are less important and so far no biological control programme was initiated. Soil dwelling pests are attacked by all types of pathogens: viruses, bacteria, protozoa, fungi and nematodes. Dominant pathogens are the mitosporic fungi Metahrhizium anisopliae, Beauveria bassiana and B. brongniartii. They sometimes cause epizootics among their host, thus proving their high virulence. In contrast to most other pathogens they have the advantage to grow and multiply in artificial media. These characteristics make them very potent candidates for the use as biocontrol agents. Indeed, the specific B. brongniartii has been developed as a mycoinsecticide for the control of Melolontha spp. and there are programmes to use the less specific M. anisopliae for the control of other scarabaeids, of curculionids and wireworms. Several aspects of the use of fungi for the control of soil dwelling pests are presented and discussed. 4

8 Melolontha melolontha, Otiorhynchus spp. and Strophosoma spp: A pest problem in Europe? Jørgen Eilenberg 1, Susanne Harding 1, Siegfried Keller 2, Hermann Strasser 3, Roland Zelger 4 and Gisbert Zimmermann 5 1 Dept. Ecology, Royal Vet. Agric. Univ., Copenhagen, Denmark; 2 Swiss Fed. Res. Stat. Agroecol. Agric., Zürich, Switzerland; 3 Inst. Microbiol., Univ. Innsbruck, Innsbruck, Austria; 4 Fed. Biol. Res. Center Agric. Forestry, Darmstadt, Germany; 5: Res. Center Agric. Forestry, Laimburg, Italy. JEI@KVL.dk The European cockchafer (Melolontha melolontha, Coleoptera: Scarabaeidae) is a severe pest insect throughout Europe. The beetle has a 3-4 year life cycle, during which the larva stays in the soil. During this period, the larva feeds on the roots of herbs and woody plants. The crops where the most severe damages occur differ, however, due to climatic conditions and cropping systems. In Central Europe (Switzerland, Northern Italy, Austria), M. melolontha is present in dense populations and the larvae are primarily harmful to grass pastures. Other places in Europe, M. melolontha may be detrimental to fruit orchards by pruning the roots of even older trees. Being formerly considered an agricultural pest, today the main damage of the European cockchafer in Denmark occurs in Christmas tree plantations. Both with respect to fruit orchards and Christmas trees, M. melolontha may damage even at low population levels. Other damaging species from Scarabaeidae include Melolontha hippocastani, Amphimallon spp. and Phyllopertha horticola. These pest organisms cause considerable damage to amenity areas (e.g. golf courses), pastures (for grazing), orchards, forests and agricultural crops. The genus Otiorhynchus (Coleoptera: Curculionidae) includes several damaging species. Otiorhynchus weevils are injurious as larvae as well as in the adult stage. O. sulcatus is an important and heavily controlled pest in glasshouses; other species like eg. O. ovatus feed on the roots of several outdoor crops and may be a serious problem in nurseries and vegetable crops as well as in afforestation areas. In forestry, also the adults of O. singularis cause damage to newly planted trees by feeding on buds, leaves and bark. Also within Curculionidae, pest insects occur in the genus Strophosoma, which includes the species S. melanogrammum and S. capitatum. These species are well-known pest insects in forestry, where the adult beetles feed on the foliage of coniferous and broadleaved trees in reforestation areas. They also cause injury to high value crops, damaging Abies plantations for decoration greenery and Christmas trees. 5

9 Persistence of Beauveria brongniartii BCA strains in the field Jürg Enkerli, Philip Kessler and Siegfried Keller Swiss Federal Research Station for Agroecology and Agriculture, Reckenholzstrasse 191, 8046 Zürich, Switzerland Persistence of a biocontrol agent (BCA) in the field is crucial for successful and reliable pest control. We have addressed this issue with the BCA Beauveria brongniartii the fungus used to control Melolontha melolontha. In 1998/1999 we collected soil samples from 29 test sites treated either in 1985, 1988 or 1991 with B. brongniartii BCA strains. The samples were analysed for presence, density and genetic identity of B. brongniartii. In 87% of the treated sites B. brongniartii was detected whereas in untreated sites the fungus was not found. To investigate the genetic identity of the fungal isolates a total of 81 B. brongniartii isolates originating from 9 treated sites were analysed using 6 microsatellite markers (Enkerli et al. 2001, Mycol. Res., 105: 1079). Data revealed that 81% of these isolates were identical to the BCA strains applied. At some sites B. brongniartii populations consisted exclusively of the BCA strains whereas at other sites indigenous strains were detected in addition to the BCA strains. This study demonstrates that B. brongniartii BCA strains can persist in soil at least for 14 years. To determine factors that influence growth and survival of B. brongniartii BCA strains in soil we investigated the influence of various biotic and abiotic soil parameters as well as the presence or absence of the pest. Results indicate that temperature, clay content and catalase activity are crucial factors for the initial growth of the BCA in the soil environment. Survival of B. brongniartii BCA strains in the soil appears to be dependent on the presence of the pest. Our findings may help to develop improved application strategies for B. brongniartii BCA strains. 6

10 Impact of entomopathogenic fungi on non-target invertebrates and persistence of Metarhizium anisopliae Susanne Vestergaard, Charlotte Nielsen & Siegfried Keller* Department of Ecology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK Frb. C, Denmark.* Swiss Federal Research Station of Agroecology and Agriculture, Reckenholz, Rechenholzstrasse 191, 8046 Zürich, Switzerland Non-target invertebrates were collected from fields treated with either Beauveria brongniartii or Metarhizium anisopliae to control the European cockchafer, Melolontha melolontha and the nut leaf weevil, Strophosoma melanogrammum, respectively. B. brongniartii was applied as colonised barley kernels in the soil at 10 cm depth, while M. anisopliae was sprayed as a conidial suspension on the soil surface. Mainly non-target from Coleoptera, Hemiptera, Psocoptera and Lumbricidae (earthworms) were collected, but also Ixodidae (ticks) were collected. No effect on non-targets was observed in field trials where B. brongniartii was applied, but some effect of M. anisopliae was observed for several species of Hemiptera and for ticks. However, the effect was only present within the first weeks after application and then the effect decreased drastically. A 1000 time decrease of M. anisopliae colony forming units within a year was found in the field and practically no horizontal spread of the fungus occurred. Vertical movement of the fungus was found down to at least 25 cm depth. In the laboratory different strains of B. brongniartii and M. anisopliae were tested against two species of collembolans (Folsomia fimitaria and Proisotoma minuta). P. minuta, was not susceptible to the tested strains even at high concentrations, but some effect was observed for F. fimitaria, whereas no effect was seen at a low concentration for neither species. For earthworms (Lumbricus terrestris) no negative effect from application of B. brongniartii or M. anisopliae were found either on weight or mortality. Twenty Carabidae species and Cicindela campestris (Cincindelidae) were tested in bioassays. Adult insects were immerged in a 10 7 conidia suspension. Four of the Carabidae species were infected with B. brongniartii namely Clivina fossor, Harpalus affinis, Nebria brevicollis and Pterostichus versicolor. It was also possible to infect C. campestris, but the mortality was low (17%). However, it has to be kept in mind that the adult ground beetles live on the soil surface and that the inoculum normally is placed into 10 cm depth in order to control M. melolontha, which makes it unlikely that the insects get in contact with the fungus. For M. anisopliae the situation is different because it was sprayed onto the ground and more insects will potentially be exposed to the fungus. Several insects from many orders are known to be susceptible to M. anisopliae, examples will be given. 7

11 Economic mass production of mycoinsecticides Gisbert Zimmermann Federal Biological Research Center for Agriculture and Forestry, Institute for Biological Control, Heinrichstr. 243, D Darmstadt, Germany A successful commercialisation and use of entomopathogenic fungi in practice mainly depends on economic mass production, efficient formulation and a suitable application technique. However, the final costs of a commercial product are directly affected by the production technology. During the past years, an increase in commercialisation efforts using entomopathogenic fungi has been noticed. Also within the BIPESCO project, the development of an economic production and commercialization of Beauveria brongniartii and Metarhizium anisopliae was one of the main goals. In principle, the method used for mass production of an entomopathogenic fungus mainly depends on (1) the growth characteristics of a fungus or strain, i.e. the ability to produce conidia or submerged spores, (2) the available equipment, (3) the intended use and the end product desired and (4) on economical calculations. Furthermore, besides the choice for an economic mass production system, also post-fermentation processes have to be considered, such as (1) separation of spores (e.g. centrifugation, filtration), (2) formulation and drying, and (3) packaging, in order to get a cheap, stable and effective product. The fundamental question for economic, commercial production of a mycoinsecticide is whether enough infectious propagules (conidia, blastospores etc.) can be produced at a reasonable price to effectively control a target insect over a certain area. Therefore, the main question is: How many spores can be produced per kg/l or m² substrate or medium and how many spores are necessary to control effectively a pest insect on one hectare? Generally, effective rates of entomopathogenic fungi per hectare are between , mostly about 1 x This amount has to be produced at a cost competitive with the per hectare costs of chemical insecticides. For economic calculation of the mass production technique, first of all, the capital and recurrent costs have to be considered. These are: Equipment, personal costs, consumables, substrate, electricity, gas and water, royalties, rent and overheads. Additionally, also a time factor should be included in any economical calculation, i.e. the main fermentation parameter that characterizes the efficiency of a production unit is expressed by grams of product per kg/l per hour (g product kg/l -1 h -1 ) or number of propagules kg/l -1 h -1 ). For economic mass production of mycoinsecticides, various solid and liquid fermentation technologies are available. They are presently used to produce fungus kernels, conidia and submerged spores, including blastospores or biomass. Today, most of the commercial products available from entomopathogenic fungi are based on conidia. Only PreFeral (Paecilomyces fumosoroseus) and Vertalec (Verticillium lecanii) contain blastospores. B. brongniartii is commercially produced as fungus kernels and, newly, also as pure conidia, M. anisopliae mostly as conidia growing superficially on cereals. 8

12 Formulation Strategies for Mycoinsecticides A. Horaczek 1, J. Raffalt 2 and H. Viernstein 1 1 Institute of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Centre of Pharmacy, Althanstrasse 14, A-1090 Vienna, Austria; 2 F. Joh. Kwizda GmbH, Agricultural Division, Dr. Karl Lueger Ring 6, A-1010 Vienna, Austria Mycoinsecticides are regarded as a safe and efficient alternative to chemical control agents that put the environment and human life at hazardous risks. In many cases, however, great difficulties are encountered in making available suitable microorganism preparations having a standardised activity and prolonged shelf life. Some biocontrol agents have been very effective in the laboratory but fail to act in the field. Common cause is poor stability of active ingredient during storage prior to application. Furthermore, an insufficient amount of active material actually reaches the target. Another cause is the rapid degradation of active material on the target. Formulation technology is a powerful tool to solve these problems. Adequate formulation should be capable of satisfying the requirements of efficacy, shelf life and safe and easy handling. But formulation is probably also the most significant technical hurdle to be overcome in the development of biocontrol agents due to the sensitive, delicate nature of the microorganisms. Their formulation requirements are more stringent than those of chemical products. Therefore, from the great variety of technically feasible methods only a few formulation technologies are suitable for the production of microorganism containing preparations. During the formulation process no harsh chemical or physical conditions should occur that have an adverse effect on the activity of the active ingredient. Formulation always has an impact on the microorganism. The extent of impact varies depending on the formulation technology used. With the help of some different types of granulation methods it was possible to process the active ingredient - fungal material of Metarhizium anisopliae and Beauveria brongniartii. They were entrapped or encapsulated in solid, biodegradable matrices to protect them from rapid desiccation, cell injuries and other adverse environmental factors. The implementation of a range of formulation technologies like wet granulation, pelletization, tablet processing and spraydrying resulted in information about the behaviour of Metarhizium anisopliae and Beauveria brongniartii exposed to mechanical, physical and thermal stress. Due to those technologies standardized formulations with the accuracy of dosage, that are easy and safe to handle for the end user were achieved. The ultimate aim for the formulator is to create formulation that protects the active ingredient, guarantees controllable release and resistance to losses possibly caused by environmental conditions. Furthermore, a safe handling and cost-efficiency are highly necessary to get products to market. 9

13 Is there a need to improve mycoinsecticides by genetic engineering? Milton A. Typas Department of Genetics and Biotechnology, Faculty of Biology, University of Athens, Athens , Greece Biological control is an exciting and rapidly developing research area, with an increasing interest in the exploitation of fungi for the control of invertebrate pests. As a result of this interest a considerable number of commercial products is available or under development. However, several problems arise even today in the development and implementation of alternative crop protection programmes, as re-education of growers, improvement of production, formulation and application technologies. The narrow host range and the inconsistency shown in field controls due to environmental factors, like solar radiation, temperature, humidity etc. are certainly handicaps in the effort for replacing chemical pesticides. Considerable research has been focused lately on the elucidation of the mechanisms of pathogenicity, as well as deciphering the potential risk on toxicity to beneficial invertebrates and other organisms, as well as on allergenicity to humans. Improvements of pathogens have been attempted through parasexual crossing and protoplast fusion or by conventional mutagenesis, but genetic engineering by direct addition of one or more genes coding for pathogenicity determinants promises the most targeted and flexible approach to altering the physiology of pathogenic fungi. Such an approach will allow the production of transgenic fungi with improved pathogenic qualities. Genetically altered organisms are unique in that they have never existed in nature in their altered form and consequently their potential hazards can only be definitively evaluated once the pathogen has been released into the environment. A wise precaution to construct isolates containing two or more different marker/reporter genes in their genomes are prerequisite, as transgenic fungi must be well monitored in the environment and multiple markers are rarely lost at once if gene transfer is to be occurred. In this aspect, their ability to persist in nature will be hampered and the possibility of causing potential damage through natural recombination with other fungi is minimized. Whatever the case, well before releasing GMOs into natural environment, a careful study on the understanding of pathogenicity mechanisms/elements is absolutely necessary. Thus, genetic engineering may help in improving mycoinsecticides and their efficacy, but in order to be released into the environment, all hazards must be assessed, step by step concerning all potential dangers to non-target organisms. 10

14 Support and Sponsoring The project "Biocontrol of Important Soil Dwelling Pests by Improving the Efficacy of Insect Pathogenic Fungi - FAIR6 CT " was funded by the European Union. The authors and their team thank the EU for funding the BIPESCO project. This meeting was sponsored by: Institute of Pharmaceutical Technology, University of Vienna, Austria. F. Joh. Kwizda GmbH, Wien, Austria. Eric Schweizer Samen AG, Switzerland. Research Center for Agriculture and Forestry Laimburg, Italy. Agrifutur s.r.l., Alfianello, Italy. Institute of Microbiology, University of Innsbruck, Austria.