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Bio-control Symbioses (Symbiotic Complexes for Biological Control of Pests)

Objective

A. BACKGROUND

Approximately half of global food production is lost annually before it reaches the consumer. About 40% of production is lost before harvest, mainly to pests, diseases and weeds (Oerke et al., 1994). Chemical control, even where it is available, successful and relatively benign, has become less acceptable, because of greater appreciation of its environmental impact and concerns about chemical residues in food. This has increased demand for (and allocation of funds towards) the development of alternatives. The alternative pesticidal agents are natural enemies.

Symbioses: Nematodes (Phasmarhabditis, Steinernema, Heterorhabditis) that kill slugs or insects have specialised bacteria (Xenorhabdus, Photorhabdus) that live with the nematodes and are usually essential for the nematodes to kill their prey. Other bacteria living intimately with those same nematodes or their insect hosts (e.g. Wolbachia sp.) may influence sex ratios or otherwise manipulate nematode and insect life histories. These intimate associations (symbioses) of biocontrol organisms are the subject of the proposed COST Action. The Action will bring bacteriologists, nematologists, entomologists, molluscologists, biochemists and a number of kinds of molecular biologists together with industrialists with the aim of integrating their efforts to understand and exploit the peculiar attributes of symbioses.

Greater understanding of symbiotic complexes has become important because of the need to:

- understand and control the host specificity of natural biocidal symbioses,

- recognise novel bioactive molecules such as those that mediate symbioses,

- manage symbiotic complexes better in industrial production and quality control,

- create symbiotic complexes that can occupy new niches for biological control and environmental tolerance and

- exploit the broader potential of symbiotic parasitic nematodes as delivery systems for highly targeted pesticides.

Biocontrol nematodes have been produced and marketed in Europe for the past ten years. Currently there are seven European biocontrol companies marketing 21 nematode-based biocontrol products (Blum, 2000). These products are used with success to control vine weevil, a major pest in nursery stock and soft fruit production throughout northern and middle Europe. Another substantial biocontol nematode market is for controlling larvae of mushroom gnats (Sciaridae) in mushroom cultivation (nine products from seven European producers). Other developing biocontrol nematode markets are for controlling beetle grubs (Phylloperta sp.) in amenity turfgrass and the use of Phasmarrhabditis hermaphrotida to control slugs in domestic gardens.

Bacterial symbionts: Bacteria function as internal and external symbionts in a broad spectrum of plant and animal hosts, ranging from protozoa to legumes to vertebrates. The molecular interactions between bacterial symbionts and their hosts are under study by several of the experts consulted. So far, only a small number of economically or biotechnologically important internal symbionts are well understood. Those (e.g. nitrogen-fixing bacteria and the plant transformation vector Agrobacterium tumefaciens) have, however, yielded quite extraordinary biotechnical advances.

Nematode symbionts: The slug and insect killing symbiotic nematode/bacterium symbioses already mentioned are the second most important industrially produced and internationally traded natural enemy, after Bacillus thuringiensis. They are the only industrially produced agents that are capable of actively seeking out target pests in soil. The nematodes deliver their natural bacterial symbionts to the insect's blood system and the insects die of bacterial septicemia. These nematodes could also be used to deliver other biocidal agents to the target pests.

Nematode/external bacterium symbioses: These three genera (Heterorhabditis and Steinernema, and Phasmarhabditis) of biocontrol nematodes are obligatory and lethal parasites of their respective hosts. The bacterial symbiont is carried outside of the nematode cells. It is required to kill the host and to digest the host tissues, thereby providing suitable food for multiplication of the nematodes. Both bacteria and nematodes play complex parts in suppression and overwhelming the immune responses of the host. All three nematode genera can be mass produced in culture media containing their symbiotic bacteria.

The symbiotic relationship between species of Heterorhabditis and their unique symbiotic bacteria is very specific. Heterorhabditis grows best on its own symbiont, and only cells of that symbiont are packaged and carried between insect hosts, in the intestine of the infective migratory stage nematode. In the case of Steinernema the symbiotic relationship is a little less specific, and in Phasmarhabditis, the strength of the nematode bacterial symbiosis and its role in the pathogenic process is less clear. Unlike the other two species, Phasmarhabditis can retain many different species of bacteria in its intestine and it can grow and reproduce on a wide range of bacteria. However, of nine strains of bacteria isolated from the intestine of Phasmarhabditis, only two were pathogenic when injected alone into the blood system of slug hosts. Further, Moraxella osloensis, the bacterium which produced the most pathogenic nematode/bacterium combination, was not toxic when injected alone into the slug haemocoel (Wilson et al., 1995).

Although the importance of the symbiosis between biocontrol nematodes and their symbionts has long been recognised, it is only in the past 2-3 years that a concentrated effort has been made to isolate and characterise the entomotoxins produced by the symbiotic bacteria (reviewed by ffrench-Constant and Bowen, 1999). These entomotoxins are proteins encoded by several genes. They are secreted by the bacterium into the insect haemocoel, but some of these toxin complexes also show oral activity against insects. The insecticidal effects of the symbiotic bacteria also involve secreted proteases, lipases, chitinases and lipopolysaccharides. These bacteria also produce several broad spectrum antibacterial and antifungal antibiotics (reviewed by Forst & Nealson, 1996).

Internal Bacterial Symbionts: The (-proteobacterium Wolbachia pipientis is a very common cytoplasmic symbiont of insects, mites and terrestrial isopod crustaceans. W. pipientis has evolved a variety of mechanisms which enhance its transmission. Its most common effect is to cause crossing incompatibility between infected males and uninfected females. This phenomenon has been extensively studied in Drosophila and in the parasitoid wasp Nasonia. Other mechanisms include the induction of parthenogenesis in which infected virgin females produce daughters, feminisation in which infected genetic males reproduce as females, and male killing in which infected male embryos die while female embryos develop into infected females. Recently a virulent strain of Wolbachia has been identified in Drosophila melanogaster. It is quiescent during the fly's development, but starts to multiply rapidly in adult tissue, causing degeneration of a variety of tissues, resulting in premature death. Wolbachia bacteria have been detected in the majority of filarial nematodes tested so far (reviewed by Taylor and Hoerauf, 1999) - filarial nematodes are important pathogens of humans and animals. This finding is directing attention to a new kind of nematode/bacterial symbiosis that may have major possibilities for both insect and nematode pest management, and for novel molecules.

So these symbiotic partnerships produce immunity suppressants, toxins, novel signalling chemicals, and exploit highly specialised niches. They need to be studied in a wide collaboration, because other biologically active molecules (e.g. Cry of Bacillus thuringiensis) may find application along with nematode toxins in pest control. This COST Action will encompass diverse disciplines and focus on how symbiotic complexes can be manipulated and managed for the effective biocontrol of insect and slug pests.

B. OBJECTIVES AND BENEFITS

The main objective of the proposed Action is to develop a better understanding of the interactions taking place in those biocontrol symbioses that may be used against insect and slug pests in Europe.

This new Action is necessary

- to gain a better understanding of symbiosis biology;

- to extend the usage of biocontrol nematodes and identify new target pests and markets for them;

- to gain a better understanding of biotic interactions in the soil, because successful biocontrol relies on and is affected by a range of biotic interactions in the environment where the biocontrol agents are used and

- to maintain European competitiveness. At present Europe is a world leader in the research, development and application of biocontrol nematodes. The biocontrol industry is a knowledge-based industry. The European biocontrol companies (which are all SMEs) rely on European researchers to elucidate the biology and genetics of biocontrol nematodes in order to exploit the biology of these agents more fully and efficiently in biocontrol. This research can be best coordinated and disseminated by means of a COST Action (see also section D).

Several benefits, outlined below, will result from this proposed COST Action.

Crossing research boundaries: To date a great deal of research on symbioses with potential in biological control has been fragmented in different specialisms. This Action will draw together people working on insects and molluscs, on endocellular symbionts and on extracellular symbionts. The interests of this diverse group meet on the mechanisms by means of which symbiotic organisation is maintained - how the symbiotic partnership gains fitness; what new molecules may be involved; how symbioses may be exploited for pest control and how results may be exploited by sustainable agriculture and biotechnology.

New biotechnological potential: Despite being recognised as a major mechanism through which radical and rapid evolution has taken place in the past, combining organisms as symbionts with new characteristics is only now being examined for biotechnological potential. Further discussion of this is provided under C1 (below). Research at participants' laboratories has improved knowledge of how close symbioses of diverse organisms function and maintain their relationships. The broader context provided by this proposed COST Action will improve the likelihood of these advances being made and carried into biotechnology.

Novel bioactive molecules: Symbioses are situations in which the very different genomes of the organisms concerned are in signal mediated interaction with each other. The cybernetic chemicals may cover a very wide range of activity: growth promoters and retardants, sexual modifiers, elicitors and camouflage chemicals. Discovery of novel molecules and processes is to be expected as more symbioses are studied (and some aspects of this are already receiving commercial attention).

Benefits to sustainable trade, agriculture, food and the environment: Pest control by means of industrially produced biological enemies is in its infancy but has already provided several benefits: trade in the agents is world-wide; some otherwise difficult pests such as Otiorhynchus sulcatus (black vine weevil) can be controlled, and chemical pesticide residues in food and the environment can be reduced.

Cross-border training: Experience of COST Actions has clearly demonstrated that the improved international contact between laboratories results not only in short-term visits, but also encourages postdoctoral exchange; provides training for young scientists in specialisms not available in their home laboratories; encourages scientific collaboration between laboratories and countries; encourages frequent consultation and exchange of information between students and staff of distant laboratories and countries. This is an extremely valuable feature of COST for both scientific purposes and social cohesion.

C. SCIENTIFIC PROGRAMME

C.1. Working Group 1: Symbiosis Biology

Evolutionary scientists consider that symbiosis is one of the important macro-mechanisms by means of which evolution has taken place. This is because, in symbiosis, two different genomes cooperate in creating a "super-organism" which possesses new and different capabilities. These capabilities enable the symbiotic complex to occupy and exploit a new, and sometimes surprising, ecological niche. One of the most striking models is deep-sea hydrothermal vent worms. These animals now live autotrophically like plants due to their exploitation of metabolic processes operated by their symbiotic bacteria (Childress et al., 1991, Biological Bulletin 180: 135-153). A more classical example is provided by lichens which combine fungal and algal genomes to give composite organisms that can occupy extremely poor and harsh environments (Honegger, 1998, Lichenologist 30: 193-212) These examples illustrate the possibilities that symbiosis offer for the conquest of extreme environments and new conditions.

In this COST Action WG1 will concentrate effort on symbioses that present applications relevant to agriculture and biotechnology. In addition to the extracellular bacterial symbionts of slug and insect parasitic nematodes, endocellular symbionts (symbionts that live inside the cells of the host) of these nematodes and their insect and slug hosts will also be investigated. Today we estimate that half of all insect species live with endocellular symbionts such as Wolbachia (Werren et al., Proc. R. Soc. Lond. B 1995, 261: 55-71). Study of these organisms has expanded recently and will almost certainly open up very important biotechnological possibilities. Aside from the high likelihood that they will provide novel bioactive molecules (below), endocellular symbionts interacting with their host nematodes should offer tools for pest control in sustainable agriculture, when the mechanisms of the interactions between the genomes are understood. Genome sequencing projects have been initiated in Europe for the symbiont bacteria Wolbachia and Photorhabdus. The data resulting from these sequencing efforts will allow members of WG1 to carry out functional genomics (e.g. using knock-out mutants) to identify key symbiotic genes in these bacterial symbionts.

C.2. Working Group 2: Bio-active Molecules

This Working Group is proposed because new insights have been reported on the occurrence of toxins produced by the bacterial symbionts Photorhabdus. This emphasises the possibilities offered by exploitation of functional diversity from the bacteriological side of the bacterium/helminthic symbiosis. These toxins are proteins encoded by several genes, which may be cloned and used in transformation. On the one hand this is a window to genetic engineering of insecticidal toxins into plants, and on the other hand it provides the possibility of enhancing the pathogenicity of the symbionts themselves in order to provide new symbiotic entomopathogenic combinations. Other toxins have been described from a strain of the bacterial symbiont Xenorhabdus and more are anticipated.

Several antimicrobial organic molecules are believed to arise from secondary metabolism of the bacterial/nematode complexes. The processes need to be better defined, but in the meantime, some antimicrobial proteinic molecules are already being characterised. The genes for these can then be identified and will again provide new possibilities for industry, medicine and agriculture.

Nematode partners of these complexes are also providing evidence of new molecules, which are involved in the pathogenic process, particularly in the early stages of the parasitism (proteases, immune depressive factors, proteases acting on the proPO system of insects). These should be cloned, sequenced and examined for medical or agricultural value. Molecules involved in the interactions between, on the one hand, the nematode and its symbiont, and on the other hand the symbiotic complex and insect defences, require refined investigation. This will be mainly the characterisation of semiomolecules where signal molecules, hormonal compounds and their corresponding receptors in the target organisms are involved. This new area of investigation is a promising way to examine symbiotic diversity for valuable molecules and ways of modifying the complexes themselves for biological control of pests.

C.3. Working Group 3: Biotechnology

The objective of this Working Group is to promote the commercial exploitation of biocontrol nematodes. The Working Group will deal with production biotechnology and related activities in downstream processing. This includes the harvest and cleaning of nematode material coming out of the liquid cultures and the storage and formulation technologies aimed at prolonging the shelf-life of nematode products. This will help to ease marketing logistics and thus contribute to widening the use of these biocontrol agents. Commercial producers will cooperate with academics in order to improve the product quality and methods of monitoring quality from production down to the end user. This Working Group will also deal with testing new targets for biocontrol nematodes in semi-field and field experiments.

Intensifying the cooperation between industry, extension services and entomologists will result in the development of appropriate technical instructions for nematode use and thus help to transfer innovative nematode technology to farmers and growers. The delivery of the biocontrol nematodes to the pest will be improved through the development of novel application technology. All these tasks will be approached in close contact with the seven European companies currently producing biocontrol nematodes, with distributors and extension personnel, and with the Industrialists' Committee of this COST Action (Section D).

C.4. Working Group 4: Interactions with Field Biota

Successful biocontrol in practice relies on, and is affected by, a multitude of biotic interactions in the environment where the biocontrol agents are used. The agents should be synergistic, or at least compatible, with other applied or naturally occurring enemies of the pest. Such enemies include: entomopathogenic fungi, other biocontrol nematodes, bacteria, parasitoids and predators. Furthermore, the application of bio-pesticides must not have deleterious long-term effects on non-target organisms such as soil detritivores, pollinators, or rare and endangered organisms.

This Working Group will focus on synergistic and antagonistic biotic interactions of biocontrol nematode/bacterial complexes in the target environments, including knock-on effects in multitrophic interactions. The Working Group will consider interactions with both micro-organisms and macro-organisms. The impact of crop management practices that involve biological control agents will be addressed, since they are a very influential part of integrated use of the biological agents. This Working Group will find common interests with several other COST Actions, as listed in the Annex.

The possibility of using biocontrol nematodes for inoculative release will be examined. There is evidence that these nematodes can disappear from the fields during cultivation, or perhaps are naturally extinct in some cases. Re-colonisation of agricultural fields by such nematodes may be feasible. Inoculation and management strategies need to be devised and tested to determine the circumstances under which sustainable biocontrol of pests may be obtained through inoculative release, and which management practices suit persistence of the nematode/bacterial complexes.

C.5. Working Group 5: Socio-economics

Socio-economic factors play a key role in the practical use of nematode/bacterial complexes as insect biocontrol agents. Farmers, extension officers and dealers in crop protection products are familiar with the efficacy, technology, speed and reliability of chemical pesticides, but they know little about modern, living, biocontrol products. One task of WG5 is to provide these user groups with easy, understandable and accessible information.

Direct costs of nematode use are higher than those of competing chemicals. But these higher costs can be compensated for by less frequent application and higher market prices following chemical free production. A good economic analysis of the costs and benefits of the use of nematodes in pest control is required for advice purposes.

Symbiotic nematodes are only one tool for the farmer in crop protection. They must be compatible with other crop protection methods and agricultural practices. The Working Group will work through links with other COST Actions (see Annex) and with the IOBC Working Groups on integrated control of pests and diseases, to identify where compatibilities should occur.

Nematode/bacterial symbioses are exempted from registration in most COST countries, but a form of registration for macro-organisms used in biocontrol is under discussion. An inappropriate set of requirements could hinder commercial development of nematodes greatly. This Working Group will provide members of the public, politicians and registration offices with adequate information on nematodes and the risks and benefits involved in their use as biocontrol agents.

The Working Group will bring together technology generation and technology transfer aspects in cooperation with WG4, in particular. This is a two-way process whereby the issues and questions that need to be investigated and the puzzles to be solved are fed into the laboratory in one direction, and the outcome and findings of the laboratory research are sent back to the field in the opposite direction. In other words, a combined "pull and push" situation has to be created, where the push from the field regarding specific research questions for biocontrol researchers has to be coupled with the pull arising from the research sector to bring the "user community" more into the research programme.

In order to provide special expertise in socio-economic affairs, WG5 will incorporate researchers dealing with participatory research methodology, in addition to biological research.

D. ORGANISATION, TIMETABLE AND DISSEMINATION

Organisation:

Why COST?:

COST is the ideal framework in which the work proposed here can be coordinated because it has convenient provision for countries at all stages of accession to the EU and for many countries not in accession. This allows the inclusion of researchers in Norway with researchers from acceding East European countries, to which biological control and industrial production of the agents is becoming extremely important. It further permits institutions in non-COST countries to participate. This proposal needs the flexibility and scope of the COST umbrella in order to draw together research that is growing rapidly, but at scattered locations, and to give it focus.

Management:

There will be a Management Committee to which Working Groups will report through management meetings. The management meetings will, where possible, be held in conjunction with the scientific workshops or Working Group meetings and at most twice each year.

The Management Committee will assign Working Group Coordinators. These Group Coordinators will organise the cooperation within and between the Working Groups. The Group Coordinators will organise sessions on topics within the objectives of their Working Groups during COST workshops. The objectives and organisation of the workshops will be proposed by the coordinators and discussed at meetings of the Management Committee. The Group Coordinators will edit reports and joint publications of the results of the cooperative research carried out. Short-term visits between laboratories and other facilities, for research purposes, will be approved by the Management Committee which will also receive reports on those visits and incorporate them in its own reports.

The research described in Section C above will be carried out by five Working Groups (WGs) operating in parallel. These five WGs correspond to the five research areas of Section C:

Working Group 1: Symbiosis Biology

Working Group 2: Bioactive Molecules

Working Group 3: Biotechnology

Working Group 4: Interactions with field biota

Working Group 5: Socio-economics

Timetable:

Five-year operating period:

The work of each WORKING GROUP requires input over a long period. This is because the focus is on understanding major biological processes. It will yield short-term results, but it will also be expected to take us towards generic technologies and concepts. For example, progress towards understanding how symbiotic relationships may be created or modified will be expected to help towards constructing composite organisms for novel biotechnical purposes and environmental tolerance. That is why the maximum operating period of five years is sought.
This COST Action is planned to run for five years in total.

Dissemination:

(a) Within the Action:

The joint exploitation of the research potential of the national scientific groups will be realised through exchange of information at the COST workshops (which will be held at least twice a year), an annual symposium, and exchange of experts, postdoctoral fellows and research students between laboratories. The workshops will be a forum for the distribution of results, improved protocols and techniques. They may incorporate scientific meetings but will not be replaced by them. The COST Action will form an Industrialists' Committee (IC) in which producers and marketers of biocontrol agents will be invited to participate and to attend and contribute to the workshops. The IC is intended to add an important problem-orienting element to the research of the national scientific groups and to carry research results directly into the business community. WG3 will devise appropriate technical instructions for nematode use in consultation with this Committee.

(b) Outside the Action:

Website: an Action website will be operated through the COST website.

Open Sessions: Open sessions to which interested public and administrators will be invited will be held by WG5, in particular.

Pamphlets: WG3 will devise, collate and distribute appropriate technical instructions for nematode use, in pamphlet form.

Publications: The Management Committee will try to channel the presentation of results obtained within the collaboration to scientific symposia or meetings of existing scientific organisations, and publication in scientific journals.

E. ECONOMIC DIMENSION

The following COST countries have actively participated in the preparation of the Action or have otherwise indicated their interest:

Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Hungary, Ireland, Israel, Italy, Netherlands, Norway, Poland, Portugal, Spain, Sweden, Switzerland, United Kingdom.

On the basis of national estimates provided by the representatives of most of these countries and taking into account the coordination costs to be covered over the COST budget of the European Commission, the overall cost of the activities to be carried out under the Action has been estimated, at 2000 prices, at roughly EUR 7,4 million per year and 274 person-years per year.

Five-year national costs are therefore: EUR 37 million and 684 person years.

This estimate is valid under the assumption that all the countries mentioned above, but no other countries, will participate in the Action. Any departure from this will change the total cost accordingly.

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