Keywords

1 Introduction

Fungi secrete an array of natural metabolites (extrolites), mostly products of secondary metabolism, which serve different functions depending on the ecological habitat of the fungus (Butt 2002; Calvo et al. 2002; Vey et al. 2001). Secondary metabolism is commonly associated with fungal development (i.e. sporulation, cell differentiation). Calvo et al. (2002) classified the function of these products into three groups: (i) metabolites that activate sporulation; (ii) pigments required for spore structures; and (iii) toxic metabolites secreted by growing fungi (mycotoxins). The latter are of great interest to scientists and legislators, not only because of their powerful and varied biological effects, which can be used for medical or industrial purpose (Baker et al. 2007; Hoffmeister and Keller 2007; Nielson and Smedsgaard, 2003), but also to overcome obstacles in the registration and subsequent commercialization of fungal biological control agents (FBCAs).

The authorisation of BCAs is strictly regulated but there is still much debate in defining the criteria for registration of FBCAs, especially with respect to the potential of the micro-organism to produce metabolites, including toxins (points IIM 2.4 and IIM 3.5.2. OECD 2004; Strasser et al. 2000; Goettel et al. 2001; Strasser et al. 2008).

A global harmonization of registration procedures is still not realised, just because of the fact, that all “old” active BCAs in the EU will be put on the “green track (1097/2007/EC; EU 2007a)” and therefore will be listed in Annex I after examination by the rapporteur member state and the commission (Directive 91/414/EC, EU 1991; see 2008/113/EC; EU 2008a). The peer review will be organised by EFSA and will allow member states and EFSA to comment further on the scientific conclusions of the Draft assessment report. This “fast-track” authorisation has been given because unacceptable negative effects to humans and the environment were excluded (see also Table 9.1: Remarks and specific provisions). There is still no official “lesson learned document” available, which was expected to be published based on the experience of the judgement of 4th list substances (2229/2004/EC, EU 2004). Nevertheless, for the first time the Commission gave a clear indication that all already notified old active biocontrol agents do not have any harmful effects on human or animal health, or on groundwater or any unacceptable influence on the environment.

Table 9.1 Secondary metabolites from fungal biopesticide active products mentioned in the “Handbook of secondary fungal metabolites” (Cole and Schweikert 2003a, 2003b; Cole et al. 2003). All fungal biocontrol agents from the online forum of US EPA (2007a) and/or in Annex I (91/414/EC; EU 1991) or currently suggested for inclusion in Annex I (EU 2008a) were considered
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The focus of this chapter is to provide an overview about information available on fungal secondary metabolites (i.e. toxicants) and their effects, and to suggest how to deal with the registration of fungal BCAs for which no information about metabolites is available. A decision scheme for the assessment of potential relevant metabolites of fungal BCAs is presented, which will help to identify and to characterise low risk FBCAs.

2 Mycotoxins of Fungal Biocontrol Agents

In recent years, significant progress has been made in the development of fungal biocontrol agents (BCAs) for the suppression of pests (insects, nematodes), weeds and diseases of a wide range of forest, horticultural and agricultural crops (Butt et al. 2001). Nevertheless, relatively few of these products have reached the market: for example, at the time of writing this article only 24 FBCAs have been registered or will be registered in the near future in the European Union under the harmonized registration procedure of Council Directive 91/414/EEC (EU 1991). Likewise, only 33 FBCAs have been approved under the Pest Control Products Act in Canada and U.S. Pesticide Data Requirements, respectively (Kabaluk and Gazdik 2005). Today, only 39 FBCAs (i.e. insecticides, fungicides and herbicides), comprising 21 fungal genera are registered in the EU, USA and Canada (Table 9.1).

The existence of fungal secondary metabolites and their toxicological significance, apart from accessible information in the literature, often becomes known only by chance, when acute toxicity studies with products based on FBCAs had led to negative effects (Rochon and Belliveau 2006) and applicants in the registration process were committed to isolate and characterise the toxicants.

Based on two examples, the difficulties related to the registration of potential mycotoxin-producing FBCAs are presented. The type of data that need to be produced and will later be available to the target audience will be described. The circumstances under which authorities will ask for data on relevant metabolites (mycotoxins) will be detailed.

In 2003 Cole and co-authors published the comprehensive standard work “Handbook of fungal secondary metabolites” (Cole and Schweikert 2003a, 2003b; Cole et al. 2003). The authors’ aim was to offer data on all major groups of secondary fungal metabolites assigned to fungal species. Although economically important groups (e.g. the aflatoxins, trichothecenes, fumonisins) have been included in the series, most of the relevant secondary metabolites of commercialised FBCAs (e.g. beauvericine, beauveriolide, destruxins, gliovirin, glioprennins, heptelidic acids, oosporein and viridian) are missing (Table 9.1). This comprehensive handbook is an example for all other relevant publications and demonstrates how difficult it is to get a precise overview on secondary metabolites produced by specific, potential fungal biocontrol agents.

A literature search in the database ISI Web of Knowledge (Thomson Reuters 2008) for all reported secondary fungal metabolites and for all FBACs listed in the online forum of US EPA (2007a) and/or in Annex I (91/414/EC, EU 1991) or currently suggested for inclusion in Annex I (2008/113/EC, EU 2008a) resulted in one new identified secondary metabolite per FBCA and per year on average (Table 9.2). The timeframe for this research was defined for the last 5 years, from January 2003 until August 2008.

Table 9.2 New reported secondary metabolites from fungal biopesticide active products published in peer reviewed Journals. All fungal biocontrol agents from the online forum of US EPA 12/2007 and/or in Annex I (91/414/EC; EU 1991) or currently suggested for inclusion in Annex I (EU 2008a) were considered. The following search terms were always checked in the database “ISI web of knowledge” (Thomson Reuters 2008) for the last 5 years: Species name combined with the general search terms (toxin* OR mycotoxin* OR metabolite* OR bioactive; the asterisk stands for the plural, e.g. toxins)
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Since fungi are the subject of numerous high-throughput screening programmes (Nielsen and Smedsgaard 2003), and metabolic profiling techniques are used to build up and complete comprehensive metabolite data bases (i.e. using NMR spectroscopy and chemometric tools; Seger and Sturm 2007; Holmes et al. 2006), new fungal metabolites are found permanently. One example is the recently re-opened discussion for the safety of the genus Metarhizium. Krasnoff et al. (2006, 2008) presented data on two compounds (i.e. fusarin C analogues) which exhibited mutagenic activity in the Ames assay. The authors concluded that “the impact of these findings on the use of M. anisopliae as biocontrol agents is currently unknown and need further investigation”. The subjects of this study were knockout mutant strains of M. anisopliae, which were characterised as metabolite overproducing isolates. Fromthe scientific point of view the authors raised the right questions, and based on this case study it is understandable why strain-specific data on relevant metabolites are required for a risk assessment of FBCAs. Simply because Metarhizium anisopliae var. anisopliae relevant data could be presented to regulation authority in spring 2008 (i.e. PMRA Canada), the ongoing safety evaluation for this potential BCA in Canada was not suspended. Otherwise, new labour- and cost-intensive studies would have to be presented by the applicant to demonstrate, that their specific production strain is safe and does not produce these fusarin-like mycotoxins.

It is understandable that regulatory authorities want to have a stringent procedure for the assessment of potentially toxic metabolic byproducts by candidate fungi (Rochon and Belliveau 2006). In order not to hinder the marketing of potentially low risk products (see Laengle and Strasser 2010), REBECA experts proposed a tiered scheme to be able to assess the risks of potential metabolites of FBCAs.

3 Standard Procedure for Toxic Metabolite Assessment

REBECA proposed a decision scheme (see Chapter 13), which has been tested initially in case studies on fungal toxic metabolites [i.e. alamethicin, antiamoebins, destruxins (type A, B, E), elsionchrome A, gliotoxin, paracelsin, oosporein (Boss et al. 2007; Favilla et al. 2006; Ganassi et al. 2007; Skrobek and Butt 2005; Skrobek et al. 2006) and selected crude extracts from Beauveria brongniartii, Metarhizium anisopliae, Paecilomyces lilacinus, Stagonospora convolvuli, Trichoderma harzianum and Verticillum lecanii (Boss et al. 2007; Butt et al. 2004; Skrobek and Butt 2005; Skrobek et al. 2006)].

Based on RAFBCA results (EU funded project QLK1-CT2001-01391; Strasser et al. 2007; 2008) and the outcome of REBECA workshops and conferences held in Innsbruck (April 2006), Salzau (September 2006), Alès (June 2007) and Brussels (September 2007), REBECA experts emphasized as a final recommendation that potential effects of relevant metabolites produced by micro-organisms should be handled according to the pre-submission data request (see Chapter 15) and the tiered scheme presented in Chapter 15.

4 Conclusion

While microbials are often reported to pose low risks to the environment (OECD 2007), it is critical for the credibility of microbial pest control products to underline such generic statements with solid data. The “decision tree”, presented in Chapter 15, permits the unbiased generation of a risk assessment of metabolites produced by FBCAs, which have a high toxicological relevance on the basis of scientific data. Unfortunately, only few standardised bioassays are available (such as effect-based ones) to evaluate metabolites and their specific toxicity. To provide a sound basis for a verification of the proposed decision scheme for future risk assessment of FBCAs, crude extracts from a number of well-known mycoparasitic, entomopathogenic or phytopathogenic fungi as well as from new isolates (bacteria included) should be tested (Strasser et al. 2008). Crude extracts, produced from fractions of polar and/or non-polar solvents from different production batches, and selected metabolites displaying different structural nature and mode of actions, should be made available from isolates where analyses showed activity of suspected toxic metabolites. Cultivation methods and extraction protocols have to be developed to meet the conditions for high-level production of toxins. Furthermore, bioassays have to be established and/or standardised to be able to define tolerance levels of metabolite toxicity (i.e. genotoxicity, cytotoxicity and ecotoxicity), because crude extracts are not expected to show zero toxicity.

The benefits and pitfalls in the use of crude extract analysis were extensively discussed by RAFBCA and REBECA experts (Strasser et al. 2008, Chapter 15). Rationales to increase the budget for more publicly funded projects (possibly with matching funds from the industry) for metabolite analysis and their risk assessment have been proposed for many years because consumers, animals and the environment require more protection. These new long-term projects will result in a generic safety registration of each particular agent and will also help to define the basis for new alternative regulation concepts.