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Biological control of pest insects and mites with special reference to Entomophthorales

Objetivo

A. BACKGROUND

Biological control is the general term for pest control measures based on natural relations between predator (or parasite) and its prey (host). Technology for the control of pest mites by their predators in greenhouses and stores is known and sometimes used, even though not on a large scale. Biological control of pest insects is more complicated due to the great variety of species and their very different biotopes. The application of entomopathogenic fungi is a promising solution to the problem.

Among 750 species of insect-pathogenic fungi, the Entomophthorales represent the largest order with 11 genera and more than 220 species. The host range covers 32 insect families. About 30% of the species attack pest arthropods and vector insects. They cause lethal infections and regulate insect and mite populations in nature by epizootics. In agriculture or forest ecosystems, entomophthoralean pathogens are prime candidates for development as mycoinsecticides for the biological control of pest insects, since they:

- have a high host specificity (very low risk of attacking non-target organisms or beneficials)
- infect pest insects (aphids, thrips, lepidopterous larvae) which are of great concern in agriculture world-wide
- have a high potential to cause epizootics
- they occur in temperate as well as subtropical and equatorial climates.

Currently, research in Europe on this topic covers taxonomy, ecology, lifecycle and in-vitro cultivation of selected species among the Entomophthorales. Before practical and commercial application of entomopathogenic fungal systems becomes feasible, problems relating to the spread, long-term establishment and safety of introduced biological agents need to be addressed.

The intensive agricultural practice in Europe requires frequent insecticide applications, which results in the appearance of insect populations resistant against a range of pesticides. Furthermore, chemical insecticides cause environmental and occupational hazards due to prolonged use, accidental spills or washout into groundwater. Beneficial insects (e.g. bumble bees for pollination) or predators for pest control are increasingly used in greenhouse operations, which in turn demands for highly selective agents for the control of insect pests. Mycoinsecticides are fungal pathogens that are utilised for the biological control of insect pests, and they offer potential solutions to these issues. Within this context it is suggested that research on entomophthoralean fungi be promoted with respect to their utilisation in agriculture and horticulture.

Most current research on the application of fungal entomopathogens deals with a group of fungi (Mitosporic fungi: Hyphomycetes) which have been successfully applied as emulsions or oil-based formulations of spore powder; a few have been developed into commercial products. Targets are grasshoppers, locusts, thrips, aphids, plant- and leafhoppers, lepidopterans, beetles, flies, mosquitoes and mites. Generally speaking, academic research on this group of fungi is mainly in Europe and USA, and field and applied work are performed mainly in Europe, the USA and by stations in tropical countries (Brazil/EMBRAPA, Columbia/CIAT and Benin/IITA). The proposed COST-action is intended to focus on the group of Entomophthorales. However, this is not meant as an exclusive restriction, but rather as a means to strengthen a coherent action. Synergies with current work on mitosporic fungi are likely to arise, and the respective scientists will be welcome to contribute to this action. In several COST countries research groups already work on the biological control of mites in stored food. However, interactions, contacts and exchange of young scientists between these groups are almost none. The purpose of the present proposal is to create cooperation at the European level, from laboratory experiments to field applications.

B. OBJECTIVES AND BENEFITS

The main objective of the proposed COST-action is to develop strategies for the biological control of insect pests and mites with Entomophthorales.

Target pests within this collaboration are aphids, mites, flies, thrips, lepidoptera, grasshoppers and locusts in field and greenhouse environments. Further fields of application are pest insects in floriculture, nuisance pests in animal husbandry, and pests of stored commodities. In the particular case of the latter, predators could control mites occurring in stored wheat and oilseeds. Chemical insecticides have undergone impressive improvements during the last decade in terms of target specificity, reduction of toxicity to non-target insects, rates and frequency of application, biodegradability, and user safety. Nevertheless, problems relating to insect resistance and disruption of equilibria between insects and natural antagonists remain. My coinsecticides offer solutions in this respect, but they will have to compete with traditional insecticides in terms of economics as well as reliance and performance. The main objective of the proposed action is to prepare the ground for the field application of mycoinsecticides, based in particular on entomophthoralean fungi, by increasing the knowledge of their biological and ecological features and host-pathogen interactions. A benefit, which is expected from the proposed research, lies in the development of reliable formulations for the storage and application of fungal biomass, and in developing methods for field monitoring

and the evaluation of prototype formulations. Solutions proposed in the course of the proposed action need to be compatible with currently used IPM-systems (IPM: integrated pest management) in agriculture and horticulture in order to increase both acceptance by the farmer and the prospects of commercialisation.

Secondary objectives of the proposed action are:

Establishment of a European research network dedicated to Mycoinsecticides: A considerable part of research on entomophthoralean fungi, which belong to a taxonomically distinct fungal group (Zygomycetes: Entomophthorales), is performed in European countries. A coordination of these European groups will render a more efficient use of national and common research funds, and it will definitely strengthen their position within the world-wide community of fungal biocontrol science.

Establishment of a European research network dedicated to the biological control by predators: Scientists have to get acquainted with biological control of stored food mites on grain and prepare the grounds for its practical use in co-operating countries. They will elaborate main principles of the biological control of stored food mites on grain and oilseeds, test them in practice, and work out the instructions for use.

Interdisciplinary Research: Typically, scientists in this field have an entomological background and work within institutions dedicated to agriculture (crop protection), population biology or system ecology. The proposed action invites scientists from other fields (e.g. mycologists, biotechnologists, formulation specialists, computer scientists) to collaborate. Within international research programmes on the application of insect-pathogenic fungi, the proposed COST-action will allow to establish a focal point of research on Entomophthorales in Europe. The mid-term objective of this COST-cooperation is the generation of a joint research project within the EC research framework (EC-FP 5).

Development of technology and feasibility assessment: Joint research efforts within the proposed action will facilitate the translation of fundamental research into field application and shall ultimately yield control strategies and biocontrol products which are both accepted and used by farmers and growers. The proposed action will clarify the benefits and limitations of sustainable control of pest insects, in particular by utilising entomophthoralean fungi, and as such is a prerequisite for subsequent product development and commercialisation.

C. SCIENTIFIC PROGRAMME

C.1. Population Biology and Landscape Ecology

C.1.1. Epizootiology

Epizootiological studies have been undertaken in the past either in agricultural crops (e.g. Erynia neoaphidis, Zoophthora radicans) or in forest ecosystems (e.g. Zoophthora radicans, Entomophaga maimaiga). Field populations of aphids (Aphis fabae) were treated with an inoculum of Erynia neoaphidis in the form of either living or dead, infected insects. These studies demonstrated that artificial establishment of an entomophthoralean pathogen in an aphid population is feasible, however, the introduced fungus did not protect the crop from the pest insect adequately. The prevalence of E. neoaphidis was dependent on rainfall or irrigation. Besides host density or the genotype, abiotic factors (temperature, humidity, light, etc.) influence the pathogenicity of E. neoaphidis particularly. At present, knowledge from laboratory studies is available about the influence of temperature on disease development, on germination, and how relative humidity affects sporulation and germination.

The following topics will be addressed within this context:

- Are epizootics in the field caused by a prevalent, virulent strain or by consortia of strains?
- Which factors determine host specificity or host-range?
- Attenuation of fungal virulence by host biotypes (e.g. how do natural or induced infections depend on genetic variability of the host?)
- Where and how are entomophthoralean pathogens hibernating, and what are the routes leading to re-establishment in the host population during spring/early summer?

The modelling of population dynamics and epizootics is covered in section C5.

C.1.2. Landscape ecology

Antagonistic organisms can be enhanced by providing refuge areas, known as conservation field margins, which serve as a pathogen source with a close spatial and biological link to the host organism and its life cycle. The prevalence and therefore the biocontrol capacity of a fungal pathogen can therefore be improved by environmental manipulation. Research within this topic will help to answer these questions:

- What is the genetic diversity of a field population of a given pathogen?
- What is the contribution of conservation field margins to the increase of the prevalent pathogen complex (species and strain level)?
- How can pathogen and pest prevalence be influenced by changes in cropping practices?

C.2. Isolation and Identification

Insect-pathogenic fungi occupy ecological niches, and therefore knowledge on the specificity of a host-pathogen relationship as well as the fungal life cycle is mandatory for a successful isolation. Isolation media may contain complex nutrients, and the isolation technique as well as the vegetative structure, which is used to start a culture, will vary in dependence on the species. Very often, the starting point for the identification of a new isolate is the nature of the host insect from where the fungus was isolated. The distinction into species is also based on the observed host range, and microscopic and morphological features provide additional criteria. In recent years, groups of fungal isolates were characterised based on their RAPD-PCR profiles.

Activities within this workgroup are expected to cover the following areas:

- Training in strain isolation
- Training in strain identification
- Description of new pathogen species and/or new host insects
- Investigation of the systematic relationship between entomophthoralean fungal pathogens, based on non-morphological criteria
- Investigation of pathogen host range.

Some of the methods developed within section C5 will be applied within this topic.

C.3. Production, Formulation and Application

C.3.1. Production

Members of the entomophthoralean group of fungi have been cultivated in the past on solid media or in submerged culture. Within the large and diverse group of entomophthoralean pathogens, nutritional requirements vary greatly according to genus, species, and even strain, therefore generalised production methods can not be expected. The nutritional composition of the culture media is complex; they contain various protein hydrolysates, yeast extract and dextrose as carbon sources, and addition of egg-yolk or fatty acids is frequently mandatory for growth and sporulation. Production of resting spores is feasible for a number of entomophthoralean species. They often require a period of dormancy before germination, but their use could be an option in a biocontrol program. As an alternative, Entomophthorales grow as single-cell hyphal bodies or as protoplasts in submerged culture. These fungal cells sporulate readily after formation of conidiophores and have been the focus of more recent research in mass production. Production protocols for small fermenter volumes have been published for Erynia neoaphidis, Conidiobolus sp., Zoophthora sp., and Entomophaga sp. Almost all of these reports are based on media which are too complex and expensive for industrial production. For mass production, technically and economically feasible media need be developed for each fungal species individually. These investigations remain a prerequisite for any application in biocontrol.

Research will cover these aspects:

- Medium development for fastidious (e.g. obligate biotrophic) fungi
- Development of simple production media suitable for large scale fermentation
- Protocols for the mass production of Entomophthorales.

C.3.2. Formulation and Application

In contrast to the entomopathogenic Hyphomycetes, entomophthoralean fungi do not produce dry spores that could be harvested from solid or biphasic production systems as powder suitable for storage under dry conditions and field application in a water- or oil-based spray. Instead, formulation methods need be developed and evaluated, which are using fungal biomass and which conserve the capability of sporulation and virulence. Field trials in the past were based on the introduction of the pathogen in the form of infected, living aphids or sporulating cadavers. Fungal biomass, produced in submerged liquid medium, was also used to infect host insects in the field. Mycelium from fermenter production can be dried and stabilised in a so-called marcescent process. Starch-based formulations of fungal cells have been tested with other biocontrol fungi, and recently an entomophthoralean species was encapsulated in alginate prills. With respect to storage pests the effects of elevated temperatures, fumigation and inert dust application on biocontrol organisms will be determined.

The following know-how will be established:

- Shelf-life and viability of dried fungal biomass
- Development of alternative formulation methods like immobilisation on carrier material, encapsulation, spray-drying of fungal biomass
- Testing of new formulations in bioassays, greenhouse and field trials
- Establishment of storage and viability data for a range of formulations.

C.4. Performance, Risk Assessment and Registration

Among the entomopathogenic fungi, Entomophthorales are distinguished by their high host specificity. This feature is advantageous in that interference with non-target insects (e.g. beneficials or host predators) is very unlikely. As of 1998, only very few mycoinsecticides are registered in selected European countries, and the requirements of regulatory bodies in the diverse countries is highly variable, if regulatory guidelines have been established at all.

Therefore the following problems shall be clarified:

- Development of methods for the monitoring of released biocontrol fungi
- Distinction of non-native strains from indigenous populations
- Interaction of non-native strains with indigenous strains
- Does the release of biological control agents affect biological diversity and food chains? (Interaction with non-target organisms or natural antagonists)
- Determination of performance and effectiveness of isolates or formulations applied (Which criteria should a field evaluation cover?)
- What are the product registration requirements and procedures for a fungal biocontrol agent in Europe?
- Regulatory situation and applicability with respect to the biological control of mites of stored products in collaborating countries.

C.5. Methods

C.5.1. Bioassay standardisation

A bioassay is an experimental procedure to determine pathogen virulence in a quantitative way. In order to be able to compare different species, isolates or biotypes, and for the purpose of comparing data obtained in different laboratories, a bioassay protocol, which has been mutually agreed upon, is mandatory. Therefore joint research shall yield:

- A recommendation for a virulence standard (LD50, LD80, or average survival time AST)
- Minimal requirements for performing bioassays (e.g. lab, greenhouse and field assays) with Entomophthorales (number of insect, repetitions, inoculum size, statistical data treatment)
- An answer to the question under which conditions field performance can be predicted from laboratory bioassays.

Within this topic, educational workshops on statistical treatment of bioassay data can be held.

C.5.2. Modelling

System analysis with subsequent simulation will provide insight into the operation of complex biological systems as well as provide support for the integration of biocontrol agents into integrated pest management. The epizootic development has already been modelled for a few entomophthoralean fungi. Complex models taking into account the effect of environmental parameters on the host-pathogen interaction have been formulated for fungal diseases of forest defoliators, grasshoppers in rangeland ecosystems, or agricultural pests. Starting from either existing models or commercially available modelling software the following knowledge shall be established:

- Expansion of descriptive dynamic models to other relevant entomophthoralean pathogen/host-systems
• Determination of the influence of temporal, spatial and genetic factors on host-pathogen dynamics
- Determination of threshold host and pathogen densities required for outbreaks of epizootics of selected agricultural pest insects
- Identification of factors determining virulence in the field by sensitivity analysis.

C.5.3. Molecular Biology Tools

The proposed research relies in part on the integration of methods of molecular biology. The use of DNA-markers in combination with PCR-techniques allows addressing many of the topics listed in sections C1, C2 and C4. RAPD (random amplified polymorphic DNA) analysis can be used to distinguish individual strains within a given pathogen population

(e.g. testing the genetic variability), to follow the spread of a fungal pathogen upon field release, and to investigate the dynamic pattern of host-pathogen dynamics. Molecular taxonomy with entomophthoralean fungi has up to now been based on isoenzyme profiles or ELISA-tests. Molecular biology methods for taxonomic or systematic purpose, which are based on DNA-markers, have just recently been applied for the fungal group in question. These techniques are powerful and should be made available to other scientists in this area. Collaborations would also result in further development of these methods in the following respect:

- Sets of reliable and versatile DNA-primers for PCR-work
- Establishment and evaluation of methods and protocols based on DNA-primers and/or amplification by PCR
- Method application for strain isolation, strain selection, monitoring and population biology.

D. ORGANISATION AND TIMETABLE

Workgroup Organisation

The organisation of the COST collaboration in five working groups corresponds to the above mentioned research areas.

Workgroup No. I: Population Biology and Landscape Ecology
Workgroup No. II: Isolation and Identification
Workgroup No. III: Production, Formulation and Application
Workgroup No. IV: Performance, Risk Assessment and Registration
Workgroup No. V: Methods

In a target-oriented approach, four transversal topics, namely field pest, forestry and indoor (greenhouses, stables and storage houses) pests plus predators of storage pests will cross-link with the five workgroups. Crosslinking will depend on the number of participating researchers, however it is anticipated that, with one exception, each problem complex will be linked with each of the workgroups.

Interaction with related COST-actions

With a number of related COST-actions, synergies are expected and should be realised by joint workshops or publications. The following actions are likely to offer opportunities for collaboration:

COST 816: Biological control of Weeds in Europe
(presumed topic: Risk assessment of biocontrol agents)
COST 830: Microbial Inoculants in Agriculture and Environment
(presumed topic: Formulation technology)
COST 840: Bioencapsulation Innovation and Technology
(presumed topic: Formulation technology)

With COST 836 (Agriculturally Important Toxigenic Fungi) opportunities for collaboration are not foreseen, since the pathogenicity and biocontrol potential of entomophthoralean fungi is not the result of fungal toxins.

E. ECONOMIC DIMENSION

On the basis of preliminary estimates and taking into account the coordination cost to be covered by the COST budget of the European Commission, the overall cost for nationally funded research to be carried on under the Action has been estimated (at 1998 prices) at EUR 4,55 Mio. This amount equals approximately 105 man-years.

Scientific staff: 12 man-years x EUR 60 000 EUR 0,72 Mio
Technical staff: 5 man-years x EUR 40 000 EUR 0,2 Mio
Ph.D. staff: 15 man-years x EUR 25 000 EUR 0,375 Mio
Laboratory equipment and consumables EUR 0,4 Mio
Overhead cost EUR 0,2 Mio

Total estimated cost covered by national research funds EUR 1,9 Mio/year
Coordination cost covered by the EC EUR 60 000/year

This estimate is valid under the assumption that, besides the named parties who initiated this proposal, two additional parties from different member countries will join the action. Additional participants will require to increase the total cost proportionally.

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