Onderzoek naar en ontwikkeling van een nieuw bacterieel biopesticide voor de biologische bestrijding van trips en spint

In dit project worden enkele veelbelovende bacteriestammen gescreend op hun parasitaire werking tegen trips (Frankliniella occidentalis) en spint (Tetranychus urticae). In de loop van het onderzoek zijn verschillende bacterie-isolaten onderzocht en steeds is getoetst of deze isolaten qua werking en veiligheid voldoende perspectief boden als mogelijk nieuw bestrijdingsmiddel. Uiteindelijk zijn de onderzochte bacterie-isolaten om verschillende redenen allemaal afgevallen. Wat deze studie wel heeft opgeleverd is het inzicht dat verschillende soorten bacteriën die in of op de plant kunnen voorkomen een dodend effect op insecten en mijten kunnen hebben. Hoewel het hier beschreven onderzoek niet geresulteerd heeft in een nieuw bestrijdingsmiddel heeft het wel laten zien dat bacteriën in en op de plant in potentie grote effecten op gewasbelagers kunnen hebben

The development of microbial pest control products for control of arthropods: a critical evaluation and a roadmap to success

Microbial pesticides have been developed for a hundred years, but many of these biological crop protection products have not been successful in the market. This is illustrated in chapter 1 by the history of microbial pest control products and the biopesticide companies producing those. In this thesis I recognize the need for a model that would facilitate the development and commercialization of biopesticides based on entomopathogenic bacteria, fungi, viruses, and nematodes. The aim of this thesis was to develop a rational and structured approach that will increase the chances of achieving success with microbial pest control products for control of arthropods. The initial step is finding a microbial pest control agent which has the potential to control the pest (chapter 2). The search for a novel agent is directed by an elaborate description of the pest problem. The first level of selection is the type of entomopathogen: bacteria, fungi, viruses, protozoa, and entomopathogenic nematodes. The second level is at the species and strain level. This study identified three decisive selection criteria for a commercial microbial insecticide: mortality, production efficiency, and safety to humans and the environment. The consecutive steps in the screening process have been identified as the collection of isolates, laboratory screening on efficacy in well-standardized bio-assays, and on production efficiency, assessment of mode of action and toxicological properties, and efficacy in small glasshouse trials. This selection process should deliver determinative information on which one or at the most three to four strains are chosen for further development. The next phase is the investigation of the feasibility of economic mass production of the selected strain(s) and the development of a stable product (chapter 3). Two phases are distinguished, the development of the production process, including medium development and downstream processing, and the development of the product, including formulation, packaging and field testing. Mass production is preferably an in vitro process because that offers more control than an in vivo process. Bacteria, fungi and entomopathogenic nematodes are generally produced in vitro, whereas baculoviruses must be produced in vivo. The critical technical and economic factors are identified and evaluated for these four types of pathogens. The goal is to produce the greatest number of infective propagules for the lowest cost. A stable product requires a formulation. The four main objectives in formulating the infective propagules are: to stabilize the propagules for reasons of packaging, shelf-life and shipping; to create a user-friendly product that can be effectively delivered to the target; to protect the propagule, once applied, to improve its persistence at the target site; and to minimize risks of exposure to the applicator. Formulation considerations and recommendations are presented per formulation function as well as per type of pathogen. Field testing links all steps in the developmental process. It provides information on the efficacy of the selected strain, on the quality of the produced propagules, on the formulation, and on the optimal application strategy. Results from field tests provide a continuous circle of feedback that allows improvement of each of the steps of the entire developmental process. The price of a product is an essential element and a cost price model for biopesticides is presented. The model provides a perspective on the makeup of the end-user’s price. Economy of scale, full use of the production capacity, and capacity planning are pivotal factors to keep the costs low. Key elements to successful biopesticides are both production efficiency and product efficacy. Quality control (chapter 4) provides feedback on the production and formulation processes, and on the final product. The continuous process of improvements will ultimately decrease costs and improve performance of the production system and the product. Products must meet product specifications. Parameters checked per batch are the number of effective propagules, microbial purity, presence of toxins, technical properties and efficacy. Standardization and comparison with a reference product are prerequisites for proper quality control. Quality control is also required for registration, but standard methods and criteria are lacking. Therefore, guidance documents need to be developed. Biocontrol companies should ensure that product quality is maintained through the whole distribution chain and that end-users receive high quality products. I showed that in that way, both the biocontrol industry and its customers benefit from proper quality control. In chapter 5 regulations for microorganisms are reviewed. Microorganisms, except nematodes, need to be registered as plant protection products for crop protection. Registration is perceived as the main hurdle to the development of a biopesticide. The procedures in the EU are presented and difficulties discussed. The issues relate to inappropriate data requirements, lack of guidance for applicants and regulators, testing methods for microbials, lack of experience in regulators, national registration procedures, and the inexperienced small biopesticide companies. Registration is expensive and takes many years. I presented registration cost estimates for each type of entomopathogenic product. Initiatives for improvements from the EU-REBECA project, from the OECD BioPesticides Steering Group, and some national projects are presented. I also provided recommendations for improvements for data requirements and regulatory procedures. New regulations may offer improved procedures in the near future. Various import and export regulations affect the use of microorganisms, and the need for harmonization is emphasized. The Convention of Biodiversity may, through Access and Benefit Sharing, create a further impediment for biocontrol. The patentability of an entomopathogen is discussed as well as the criteria for granting a patent: novelty, inventive step, and industrial applicability. I also discussed costs and other considerations whether to apply for a patent for a biopesticide. The implementation strategy of the product in an IPM programme is a basic element of the use of any microbial pest control product (chapter 6). Three phases are distinguished: the optimal application strategy of the product, the incorporation of the microbial pest control product in an IPM system, and a carefully designed adoption strategy. Determinative parameters for each phase, and for each type of product are identified. For instance, for a successful use, the compatibility with chemical pesticides and with natural enemies and pollinators needs to be investigated. Furthermore, knowledge transfer and training are pivotal elements. All stakeholders need to participate in this process. These phases require a considerable amount of research which should be conducted before market launch. Recommendations are provided for a tiered approach which results in reliable information for commercial conditions. Many companies underestimated or even neglected this part of product development. In my opinion, these phases are paramount for good market introduction. I reported the most relevant requirements for successful use of a microbial pest control product. Successful implementation of a microbial pest control product depends on how well relevant interactions are studied and translated into practical recommendations for the grower. This phase continues after market introduction. It requires a continuous effort from producer, distributor and customer to ensure that product adoption will increase and satisfied customers will remain using the new product in their IPM system. In chapter 7, I noted that commercialization is the final and most difficult step in the development and the market introduction of a microbial pest control product. The factors that determine success or failure are identified for a company as well as for a product, and recommendations are provided that will facilitate success. Figures on the global biopesticide market are reviewed. The European market is estimated to be €57 million at end-user level, and the market in the Netherlands at €5-6 million. The European biopesticide market comprises less than 1% of the total European crop protection market. Biopesticides are predominantly used in protected crops and in orchards. Companies which contemplate the development and commercialization of a biopesticide need realistic data on five key aspects to make their decision: market demand, market size, profit margin, time to market, and time to volume. The biggest mistake companies still make today is a misjudgement of the potential market size and the expected market adoption rate. I proposed the use of a stage-gate process with objective, quantifiable, and transparent tools in decision-making. Examples of scorecards are presented to quantify decisions. The business model that performs best at present seems to be a small company which follows an incremental and manageable growth of the organization. Total developmental costs and time to market are significant factors of a company’s success. Costs amount to € 10-15 million for a company that still needs to be built; while in an existing company, costs may reach € 5-10 million for a biopesticide project. Time to market including registration is five to seven years. I have identified five determinants for successful commercialization: 1) acceptable expenses and time to market; 2) a high quality product; 3) a sufficiently large market; 4) a profit margin that allows expansion in new markets and products; and 5) the appropriate business approach. A new product development project is extensive and it is difficult to oversee. In chapter 8 I have made an analysis of the various phases and I highlighted the most important topics in the development and commercialization of a microbial pest control product. This study demonstrated that the development of a microbial pest control product requires a structured project plan. The building blocks of the entire process are defined and essential factors emphasized. From this, I have divided the process in phases and steps, and designed the roadmap to a successful product. Three diagrams illustrate the stepwise approach of the entire process, the selection phase, the product development phase, and the implementation phase. Registration and commercialization are processes that relate to these phases during the entire developmental process. A future perspective on the biopesticide market is presented with limiting and promotional factors and trends. The significant drivers for success are food safety concern, changes in the regulatory climate, biodiversity and environmental issues, new research and technology, and the occurrence of new invasive pests. The biopesticide industry has reached a sufficient level of maturity and critical mass to form a base for further expansion. This will allow the biopesticide market to steadily grow. The roadmap proposed in this study will assist developers of biopesticides in accomplishing their goals in a cost- and time-effective way, which will result in successful and sustainable products and expanding biocontrol companies

Microbial bioprotectants for plant disease management

This collection provides a comprehensive coverage of the recent advances in the development of more ecologically balanced biological methods to control plant diseases. Chapters review the availability and use of bacterial, fungal and viral bioprotectants, as well as the issues that arise with their development and use

Bacterie losgelaten op plagen

Plant Research International (PRI) in Wageningen deed onderzoek naar endofyten: bacteriën die van nature in planten voorkomen. Enkele bacteriesoorten bleken effectief trips en spint te doden. Een project werd opgezet met als doel de waarde van deze bacteriën te onderzoeken voor de praktijk en er vervolgens biopesticiden van te make

Ecological arguments to reconsider data requirements regarding the environmental fate of microbial biocontrol agents in the registration procedure in the European Union

Microbial biological control agents (MBCAs) against pests and diseases in crops are regarded as sustainable tools in integrated pest management.In the European Union, biological control should be preferred to conventional chemical methods if they provide satisfactory pest reduction. There is no reason to believe that all forms of biocontrol are intrinsically safe. Therefore requirements for registration to assure safety are needed. In the current registration procedure in the European Union, MBCAs are primarily treated as potentially risky organisms that not only produce toxic substances but are also dangerous because they can multiply, spread and genetically adapt. These characteristics give rise to a concern that released MBCAs will spread and become dominant in the environment, resulting in negative effects on other organisms in the natural environment or even humans. These assumption led to extensive data requirements that are a time consuming and costly hurdle for bringing MBCAs onto the market. This paper focuses on the relevance and irrelevance of the data requirements for environmental fate and persistence of MBCAs. MBCAs are naturally occurring living organisms that are numerically enhanced to reduce specific plant pathogens or pests.In contrast to chemicals, direct toxicity is not the main mode of action, but rather a variety of mechanisms is involved, including competition, parasitism and activity of secondary metabolites. Their effects and residues cannot be evaluated as done for chemical substances and their breakdown products. Populations of introduced microorganisms always decline due to the natural biological buffering of the environment to levels that are within common fluctuations and ranges without strongly affecting microbial communities. However, the currently used concept of a natural background level as a reference to which densities of MBCAs should decline is of limited value, in particular when endpoints cannot be defined from ecological theory or risk criteria. In conclusion we state that data requirements for persistence could be more freely interpreted in all cases where there is no a priori reason to assume that organisms will not be buffered by the agro ecosystem. Since information is only needed‘when relevant’, the European Union guidelines leave space for such a proportional interpretation of the data requirements on environmental fate

Dermal absorption of chlorpyrifos in human volunteers

Objective: The methods and results are described of a study on the dermal absorption of chlorpyrifos (CPF) in humans established via urinary excretion of the metabolite 3,5,6-trichloro-2-pyridinol (TCP). Methods: Two dermal, single, doses of CPF were applied in two study groups (A and B) each comprising three apparently healthy male volunteers who gave their written informed consent. The clinical part of the study was conducted in compliance with the ICH Guideline and the EC principles of good clinical practice (GCP). An approximately 0.5 ml dilution of CPF in ethanol was applied to an area of ∼100 cm2 of the volar aspect of the forearm, resulting in doses of either 5 mg (A) or 15 mg (B) of CPF per study subject. Duration of dermal exposure was 4 h, after which the non-absorbed fraction was washed off. The following samples were collected at pre-determined intervals for the determination of either CPF or its metabolite TCP: dosing solutions, wash-off fractions and urine samples collected up to 120 h after dosing. Results: A relatively large fraction of CPF (42%-67% of the applied dose) was washed off from the exposed skin area. Application of either 5 mg (A) or 15 mg CPF (B) resulted in the total urinary excretion of 131.8 μg (A) or 115.6 μg (B) of TCP 120 h after dosing. This indicated that 4.3% of the applied dose has been absorbed (A), while in group (B) no significant increase in urinary TCP (115.6 μg) was established. The latter indicates that an increase in the dermal dose at a fixed area does not increase absorption, which suggests that the percutaneous penetration rate was constant. Further, it was observed that the clearance of CPF by the body was not completed within 120 h, suggesting that CPF or TCP was retained by the skin and/or accumulated in the body. A mean elimination half-life of 41 h was established. Conc lusion: The results show that daily occupational exposure to CPF may result in accumulation of CPF and/or its metabolites, possibly resulting in adverse effects. © Springer-Verlag 2004