Practicalities of developing and registering microbial biological control agents

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Review

Practicalities of developing and registering microbial biological

control agents

Sebastian Kiewnick

Address:Agroscope Changins-Wa¨denswil, Research Station ACW, Plant Protection, Ecotoxicology and Soil Zoology, 8820 Wa¨denswil, Switzerland.

Correspondence:Email: [email protected]

Received: 29 November 2006

Accepted: 30 January 2007

doi: 10.1079/PAVSNNR20072013

The electronic version of this article is the deﬁnitive one. It is located here: http://www.cababstractsplus.org/cabreviews

gCABI Publishing 2007 (Online ISSN 1749-8848) Abstract

There is considerable interest in the exploitation of microbial biological control agents (MBCAs) for the control of crop pests, weeds and diseases. MBCAs can be used where chemical pesticides are banned or being phased out or where pests have developed resistance to standard chemicals. The use of MBCAs can play an important role in crop protection, as a key element in integrated pest management (IPM) programmes. However, despite considerable research efforts on the development of new biological control agents the number of such products on the market in the European Union (EU) is still extremely low compared with the United States or Canada. In areas that previously constrained the commercialization of MBCAs, discovery, fermentation, formul-ation and applicformul-ation, significant progress has been made. The low number of products is mainly due to the slow registration process. In the EU, MBCAs are regulated by and follow Directive 91/414/EEC for placing plant protection products in the market. Once an active ingredient is listed in Annex I, national registrations for the formulated product have to follow. This time consuming and expensive process has forced most companies to suspend their efforts in research and development. Initiatives by stakeholders from industry, science, regulatory authorities, policy and environment are underway to accelerate market introduction of MBCAs.

Keywords: Biological control, Production, Fermentation, Formulation, Registration

Review Methodology:The following databases were searched: CAB Abstracts and Pubmed (Keyword search terms used: registration, biological, control, agent, registration, microbial, biopesticide). References from the articles obtained by this method were used for additional relevant material. In addition, information from a symposium at the APS, CPS, MSA joint meeting July 29–August 2, 2006 in Que´bec City, Que´bec, Canada entitled ‘Research, Development, and Adoption of Biopesticides in the 21st Century’ and a workshop on ‘Current Risk Assessment and Regulation Practice’ 18–22 September 2006 in Salzau, Germany with more than 100 experts (scientists from public research and industry, and government regulators) were included.

Introduction

Biopesticides, by the general definition, are pest limiting agents of biological origin which include microbial living systems (bacteria, fungi, viruses), entomopathogenic nematodes, insect predators and natural parasites, plant derived products (botanicals) and insect pheromones (natural and semiochemicals) [1, 2]. Under United States Environmental Protection Agency (US EPA) regulation, the use of plant incorporated protectants (such asBacillus thuringiensis (Bt)-toxins in transgenic plants), genetically engineered micro-organisms, biochemical compounds

with a non-toxic mode of action and biochemical like compounds appropriate for reduced data are also con-sidered biopesticides [3]. Given the large number of bio-pesticide products [4], this review can cover only a fraction of this large group and refers to microbial pesti-cides or microbial biological control agents (MBCAs). Products containing bacteria, fungi or viruses are cur-rently receiving a lot of attention from researchers, industry and authorities [2, 5, 6].

In 1996, the predicted market size for biocontrol products for the year 2000 was 10% of all pesticides sold around the world, with a value of$3 billion [7]. Today, the

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world pesticide market is estimated at $25 billion, but only 1% ($300 million) is spent on biopesticides. In the European Union (EU), the total pesticide market is esti-mated at $5 billion with 2% of this total volume being biopesticides ($100 million). Of this$100 million only 25% comes from sale of MBCAs. The largest proportion of these MBCAs are Bt products with 80–90% of the market and only $2.5–5 million comes from non Bt-sales [2]. Despite the fact that scientists have been working for over 50 years on biological control and integrated pest management (IPM) systems, the biocontrol business is growing a rate of only 10% per year [8]. Consequently, only a few decent medium-sized companies make a profit selling biological products. Most biopesticide companies are unprofitable or marginally profitable [9].

Perceptions of MBCAs

There are three major unique features of MBCAs which make them of interest to a farmer: (i) resistance man-agement (ii) restricted entry intervals and (iii) residues. Because most MBCAs have multiple modes of action [10, 11] there is less chance of resistance developing in a particular pathogen, insect or weed. Therefore, MBCAs are an excellent tool in IPM programmes, where syner-gistic effects can be utilized to reduce the input of chemicals or to re-establish efficacy [6, 12]. The majority of MBCAs if not all have low restricted entry intervals, normally in the range of 0–4 h [9]. In addition, they gen-erally have no pre-harvest interval, which essentially allows the farmer to harvest a crop directly after an MBCA has been applied. Finally, MBCAs are generally considered exempt from tolerances (maximum residue limits) under US EPA regulation. The US EPA considers these products safe and therefore that residues on leaves and fruit do not pose an unacceptable risk [9]. Under EU regulation, residue data are not relevant only if no adverse effects are identified from the proposed use. Residues are here considered as toxins produced by micro-organisms that may be of biological significance. If produced by a micro-organism, maximum residue levels (MRLs) have to be established for these toxins.

MCBAs in the EU, US and Canada

Despite progress made in research and development associated with the use of MBCAs, the use of these products in the EU is still very limited. As demonstrated in Tables 1a, b, c and 2, the number of products registered in the EU in comparison with other countries (e.g. the US) is very small. Tables 1a, b and c list 68 MCBAs used as bactericides, fungicides, herbicides, insecticides and nematicides that fulfill at least one of the following requirements: (1) registration as an active substance in the US; (2) registration as an active substance in Canada; (3)

registration as an active substance in an Organization for Economic Co-operation and Development (OECD) member state outside the EU; (4) registration in an EU member state as an ‘old active’ substance or with a pro-visional registration; (5) inclusion of the active substance in Annex I of Directive 91/414/EEC; (6) under evaluation as a ‘list 4’ substance as either a ‘new existing’ or an ‘old active’ substance. In order to limit the list to a reasonable length Bt and insect virus products are not listed in detail. The Bt group alone represents 13 active ingredients in 106 products on the US market only [17]. A detailed list of products containing Bt or viruses as active substances can be found elsewhere [18, 19]. A total of 53 MBCAs is currently registered in the US. In Canada and other OECD countries 15 and 20 MBCAs are registered, respectively. For the EU, 21 MBCAs are listed that have a registration in a least one member state, either pro-visional or as ‘old active’ substances. Only five MBCAs are currently listed on Annex I of Directive 91/414/EEC, with four more active substances under evaluation. Concerning the last stage of the EU-evaluation process, the fourth list, 18 MBCAs have been notified with an additional three considered as ‘new existing’ active substances. Besides the groups of Bt and virus products, only three other organisms, namelyB. subtilis(Strain MBI 600),Streptomyces griseovirides (Strain K61) andTrichoderma harzianumhave registrations in the US, Canada, other OECD and EU countries (Table 1a and b). The main reason for the dif-ference in the number of products registered lies in the regulatory system that applies to microbial plant protec-tion products in the EU.

Regulation of MBCAs in the US, Canada and the EU

The US regulatory requirements for microbial pest con-trol products are outlined in Microbial Pesticide Data Tables 40 CFR 158.740 established in 1988. However, US EPA recently proposed a change in data requirements for biochemical and microbial pesticides, taking into account that the current regulation was established 24 years ago and therefore data requirements needed adjustment [20]. In Canada, the Pest Management Reg-ulatory Agency (PMRA) establishes the data requirements for microbial pest control products which are outlined in Regulatory Directive DIR2001-02. In the EU, the placing of plant protection products on the market is regulated by Council Directive 91/414/EEC. Plant protection products containing micro-organisms as active substances are sub-mitted to this directive, which has been modified by Commission Directive 2001/36/EEC to specify require-ments for micro-organisms.

The main differences between the regulatory systems in the US, Canada and EU are not within the overall data requirements. They are quite similar, but in the US and Canada, the description of the data requirements is more

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Table 1a Comparison of the registrations of MBCAs – bactericides and fungicides based on bacteria – in the US, Canada, OECD countries and the EU MBCA Target Registration1 US Canada OECD2 EU (Directive 91/414 EEC) National3 Annex I inclusion Notiﬁcation List 4 Bactericides

Agrobacterium radiobacterStrain K84 Crown gall + + + 7 7 New existing

A. radiobacterStrain K1026 Crown gall + 7 + 7 7 –

Pantoea agglomeransStrain C9-1 Fireblight + 7 7 7 7 –

Pseudomonas ﬂuorescensA506 Fireblight + 7 7 7 7 –

Fungicides (Bacteria)

Bacillus cereusStrain UW85 Fungal soil-borne diseases + 7 7 7 7 –

B. licheniformisStrain SB3086 Fungal soil-borne and leaf diseases + 7 7 7 7 –

B. pumilusStrain GB34 Damping-off + 7 7 7 7 –

B. pumilusStrain 2808 Root and leaf fungal diseases + 7 7 7 7 –

B. subtilisStrain GBO3 Damping-off and soil-borne fungal diseases + 7 7 7 7 –

B. subtilisStrain IBE 711 Damping-off and soil-borne fungal diseases 7 7 7 + 7 New existing

B. subtilisStrain MBI 600 Soil and seedborne fungal diseases + + + + 7 –

B. subtilisStrain QST713 Powdery mildew + 7 + + Pending N/A

B. subtilis Damping-off + 7 + 7 7 –

B. subtilissubsp.amyloliquefaciens Strain FZB24

Soil-borne fungal diseases + 7 + 7 7 –

Paenibacillus polymyxaStrain AC-1 Damping-off, powdery mildew 7 7 + 7 7 –

Pseudomonas aureofaciensStrain TX-1 Turf leaf diseases + 7 7 7 7 –

Pseudomonas chlororaphisStrain MA342 Cereal leaf diseases 7 7 7 + + –

P. chlororaphisStrain 63-28 Soil-borne diseases + 7 7 7 7 –

P. syringaeStrains ESC-10, ESC-11 Fruit and dry rot + 7 7 7 7 –

Streptomyces griseoviridesStrain K61 Damping-off and soil-borne fungal diseases + + + + 7 Notiﬁed

S. lydicusStrain WYCD108 Soil-borne fungal diseases + 7 7 7 7 –

Modiﬁed after Hynes and Boyetchko [13]. 1

Organisms are included that were listed in one of the following sources: [14–16].

2_{OECD countries other than US, Canada, and Europe.}

3

Provisional registration or registration as old active substance in EU-member states (before 23 July, 1993).

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Table 1b Comparison between the registrations for MBCAs – fungicides and herbicides based on fungi – in the US, Canada, OECD countries and the EU MBCA Target Registration1 US Canada OECD2 EU (Directive 91/414 EEC) National3 Annex I inclusion Notiﬁcation List 4 Fungicides (Fungi)

Ampelomyces quisqualisStrain M10 Powdery mildew + 7 7 + + –

A. quisqualisStrain 94013 Powdery mildew 7 7 + 7 7 –

Aspergillus ﬂavusStrains AF36 and NRRL 21882 Aspergillus ﬂavus + 7 7 7 7 –

Coniothyrium minitansStrain CON/M/91-08 Sclerotiniaspp. + 7 + + + N/A

Fusarium oxysporumStrain Fo47 Fusarium wilt diseases 7 7 7 + 7 –

Gliocladium catenulatumStrain J1446 Damping-off and soil-borne

fungal diseases

+ 7 7 + + N/A

G. virensStrain GL-21 Damping-off + 7 7 7 7 –

Muscodor albusStrain QST20799 Pre- and post-harvest diseases + 7 7 7 7 –

Ophiostoma piliferum Bluestain fungi 7 + 7 7 7 –

Phlebiopsis gigantea Pine root rot 7 7 7 + 7 Notiﬁed

Pseudozyma ﬂocculosaStrain PF-A22 Powdery mildew + + 7 7 Pending N/A

Pythium oligandrumStrain DV74 Soil-borne fungal diseases + 7 + + 7 New existing

Trichoderma harzianum, several strains Soil-borne and foliar fungal diseases

+ + + + 7 Notiﬁed

T. polysporum Pruning wound infection + 7 7 + 7 Notiﬁed

T. viride Soil-borne fungal diseases + 7 7 + 7 Notiﬁed

Verticillium dahliaeKleb. Dutch elm disease 7 7 7 + 7 Notiﬁed

Verticilliumisolate WCS850 Dutch elm disease + 7 7 7 7 Notiﬁed

Herbicides

Alternaria destruansStrain 059 Cuscutaspp. + 7 7 7 7 –

Chondrostereum purpureumStrain HQ1 Hardwood tree spp. 7 + 7 7 7 –

C. purpureumStrain PFC2139 Hardwood tree spp. + + 7 7 7 –

Colletotrichum gloeosporioidesf. sp.aeschynomene Northern joint vetch + 7 7 7 7 –

C. gloeosporioidesf. sp.malvae Round-leafed mallow 7 + 7 7 7 –

Phytophthora palmivoraStrain MWW Milkweed vine + 7 7 7 7 –

Puccinia thlaspeos Dyer’s wood + 7 7 7 7 –

For footnotes (1–3) see Table 1a.

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Table 1c Comparison between the registrations for MBCAs – insecticides and nematicides – in the US, Canada, OECD countries and the EU MBCA Target Registration1 US Canada OECD2 EU (Directive 91/414 EEC) National3 Annex I inclusion Notiﬁcation List 4 Insecticides (Bacteria)

Bacillus thuringiensis(Berliner) Several insect species + 7 7 + 7 Notiﬁed

B. thuringiensissubsp.aizawai Several insect species + 7 + + 7 Notiﬁed

B. thuringiensissubsp.israelensis Several insect species + + + + 7 Notiﬁed

B. thuringiensissubsp.kurstaki Several insect species + + + + 7 Notiﬁed

B. thuringiensissubsp.tenebrionis Several insect species + + + + 7 Notiﬁed

B. thuringiensis(engineered strain) Several insect species + 7 7 7 7 –

B. sphaericusStrain ATCC 1170 Mosquito larvae + + 7 7 Notiﬁed

Paenibacillus popilliae Japanese beetle + 7 7 7 7 –

Insecticides (Fungi)

Beauveria bassiana, several strains Several insect species + 7 + + 7 Notiﬁed

B. bassianaStrain 447 Fire ants + 7 7 7 7 –

B. bassianaStrain HF23 House ﬂies + 7 7 7 7 –

B. brongniartii Sugar cane beetle 7 7 7 + 7 Notiﬁed

Lagenidium giganteum Mosquito larvae + 7 7 7 7 Notiﬁed

Metarhizium anisopliaevar.acridiumStrain IMI330189

Locust, Wingless grasshopper

7 7 + 7 7 –

M. anisopliaevar.anisopliaeStrain EFS-1 Termites + 7 7 7 7 –

M. anisopliae, several strains Several insect species + 7 + + 7 Notiﬁed

Paecilomyces fumosoroseusApopka strain 97

White ﬂy + 7 7 + + N/A

P. fumosoroseusStrain FE9901 White ﬂy 7 7 7 7 Pending N/A

Verticillum lecanii4_{(2 Strains)} _{White ﬂy and Aphids} ₇ ₇ ₊ ₊ ₇ _Notiﬁed

Insecticides (Viruses)

Granuloviruses, several strains Several arthropod species + + + + 7 Notiﬁed

Nucleopolyhedroviruses, several strains Several arthropod species + + + + 7 Notiﬁed

Nematicides

Bacillus ﬁrmus Plant parasitic nematodes 7 7 + 7 7 –

Paecilomyces lilacinusStrain 251 Plant parasitic nematodes + 7 + + Under evaluation N/A

For footnotes (1–3) see Table 1a. 4

Taxonomic revision means the correct name for this genus is now:Lecanicilium.

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detailed and waivers (scientifically based arguments for non-submission of studies) are generally accepted. In ad-dition, there is a guaranteed time line for the registration process [21]. Table 2 shows the time line for the regis-tration of MBCAs by the US EPA compared with that under EU Directive 91/414/EEC for nine different bio-control agents. On an average, the time needed for registration of a product in the US was 26 months (range 12–60), whereas more than 80 months (63–104) were needed for inclusion of an active substance in Annex I of Directive 91/414/EEC. During the years 1994 to 2003 more than 50 MBCAs were registered in the US, but in the EU only five active substances were added to Annex I. Furthermore, once the active substance has been included in Annex I, national registrations in EU member states of the formulated product still have to follow. As of August 2006, 78 microbial products were registered with the US EPA [3]. In the EU, eight new active substances are still under evaluation for inclusion in Annex I. Currently, stage four, which is the final stage of the review programme for the active substances already on the market in EU member states before 23 July 1993 is underway [22]. Dossiers for 140 of these ‘old active’ substances have been submitted by notifiers, of which 19 dossiers are for microbial active substances (Tables 1a, b and c).

With respect to the risk assessment, the evaluation in the US and Canada is based on a maximum hazard testing and not provided by the applicant. Conversely, in the EU, risk is evaluated on a hazardexposure basis and the applicant has to provide the risk assessment for the MBCA in the dossier submitted to the authorities.

One of the major differences between the EU regula-tions and those of other countries lies in the requirement by the EU to provide efficacy data for the MBCA for all intended specific uses in specific crops [10]. Therefore, a product has to be evaluated in several experiments, in different geographic zones and for two consecutive years. As a consequence, products that have registrations for use on multiple crops against several diseases in the US

will have a registration in the EU for a specific use only [10]. In the EU, this approach is used to prevent the application of products with no efficacy but potential unwanted side effects. Conversely, US EPA does not require efficacy data, because the attitude is that the market should decide whether a product is accepted by farmers or not. As a consequence, several products are currently registered with US EPA, but are not sold in significant quantities.

Additional Reasons for the Low Number of Products Registered in the EU versus US and Canada

As described above, the registration process is the main hurdle to overcome in the successful commercialization of an MBCA. There are some major problems within the regulatory process that are responsible for the delays in registration (Table 2). The guidelines currently used to evaluate MBCAs were originally developed for chemical pesticides and are mostly not appropriate for micro-organisms [23]. In some cases (e.g. sensitization proper-ties), authorities have recognized that methods for testing dermal sensitization are not suitable for testing micro-organisms. Sensitization by inhalation is considered to be most probably a greater problem than dermal exposure, but no validated test methods are available [24]. As a con-sequence, all micro-organisms are regarded as potential sensitizers. This presumptive safety approach also takes into consideration immuno-compromised or other sen-sitive individuals in the population [24]. However, as a consequence, the formulated product carries an Xn-label which indicates that the content is classified as a sensitizer (R42/R43). From the standpoint of commercialization, this safety precaution has a tremendous impact on the hand-ling, shipping and storage of a product that supposedly is a safe alternative to chemical pesticides. This definitely contradicts the general perception of biological control Table 2 Comparison of the time needed (in months) for registering nine microbial biological control agents in the EU under Directive 91/414/EEC (i.e. inclusion in Annex I) compared with the US EPA

Organism (strain) Product

Time needed for registration (months)

Europe (Annex I) US EPA

Ampelomyces quisqualis(M10) AQ101

104 ?

Bacillus subtilis(QST713) Serenade1

>741 14

Coniothyrium minitans(CON/M/91-08) Contans1

63 23

Gliocladium catenulatum(J1446) Prestop1

67 13

Paecilomyces fumosoroseus(Apopka 97) Preferal1

85 60

Paecilomyces lilacinus(251) Bioact WG1

>531 21

Pseudomonas chlororaphis(MA342) Cedomon1

99 –

Pseudomyza ﬂocculosa(PF-22) Sporodex1

>661 39

Spodoptera NPV Spodex1

>1101 12

Average >80 26

1_{Dossiers currently under evaluation; ?}₌_{no data available.}

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products. In addition, products with an Xn-labelling can-not be stored longer with food in cold storage and the shipping and handling costs are significantly increased because a dangerous good is shipped. Consequently, the end user price increases, which makes an MBCA less attractive to the farmer and the product is no longer considered as safe by the end user [21].

The question whether toxins produced by micro-organisms pose a risk to workers and consumers has fuelled the discussions on the safety of biocontrol agents in past years. Two EU funded Research, Technological Development (RTD)-projects, BIPESCO (Biological Con-trol of Soil Dwelling Pests, FAIR6-CT989-4105) and RAFBCA (Risk Assessment of Fungal Biocontrol Agents, QLK1-2001-01391), were initiated to enhance the pro-duction, formulation, and efficacy of fungal biocontrol agents and evaluate the risks involved with the application of these MBCAs [25]. One of the major concerns during the registration process of an MBCA, the production of metabolites, or toxins, was intensively investigated in the RAFBCA project. The objectives of the project were to identify and characterize metabolites produced by fungal BCAs, and to establish whether they entered the food chain and posed a risk to human and animal health based on new risk assessment tools. The major outcome of this research was that the potential for exposure to

metabolites produced by certain fungal MPCAs was con-sidered to be low [26–29] (for more details see www. rafbca.com).

Changes in the Requirements for the Development of an MBCA

The development of an MBCA requires several steps. However, the importance of each step and the impact of a single step on the whole process of discovering, developing and commercializing an MBCA has certainly changed over the last decade. This is especially true for EU member states and in part also for the US and Canada. Ten years ago, the strategies for selecting and developing microbial biopesticides for the control of insect weeds and plant pathogens focussed on technological constraints [30].These technological constraints included the lack of low-cost production methods, stable formulations with reasonable shelf-lives and efficacy under field conditions. Strategies for selecting potential biocontrol agents and experimental approaches to overcome the constraints were of key importance at that time.

Figure 1 demonstrates how the general scheme of the steps important in the development of an MBCA has changed. This scheme, presented by Montesinos

(a)

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c c

National registrations of formulated

product

Figure 1 Basic procedures for the development and commercialization of a microbial biocontrol agent (a) general scheme modiﬁed after Montesinos [31] and (b) scheme demonstrating the requirements under current EU regulation

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[31], placed emphasis on the initial steps of isolation, identification and characterization (Figure 1a). Until a few years ago, these first steps were rather laborious and resource intensive with a maximum success rate of 1% or less. Due to the advances in understanding the mech-anisms involved and responsible for biocontrol activity, nowadays selection of potential biocontrol agents can be based on certain traits that are responsible for biocontrol efficacy [32]. Therefore, potential organisms can be selected at an early stage for further testing with an increased success rate [32].

Initial trials with the candidate strains are done under a range of conditions to fully evaluate their potential for disease control. In the new scheme (Figure 1b) this phase is of great importance for commercial and regulatory reasons [33]. At this point, the researcher should identify potential crops and cropping systems and determine effects such as physical, chemical or cultural factors that limit the effectiveness of the MBCA. Furthermore, in the context of regulatory issues, application timing and rates as well as pathogen thresholds have to be established early on in the development process.

Once a candidate strain has been selected for further development, a decision on patenting has to be made. Patenting is particularly important for researchers who want to later licence their invention to a company. However, costs for obtaining and maintaining patents are high and although large number of patents on the use of MCBAs have been filed, only a fraction has materialized into registrations of the active substance [31]. This indi-cates that factors other than patents are more important for success in developing and commercializing MBCAs.

Major improvements have been made in recent years in the process of mass production and formulation of micro-organisms. The development and improvement of solid state fermentation technologies for filamentous fungi [34, 35] has led to the development of several new MBCAs. The advantages of solid state fermentation such as low capital investments and energy requirements combined with cheap and simple media have reduced the production costs to a level competitive with chemical pesticides [36, 37]. Furthermore, the development of new formulations of MBCAs have led to better storage stability, compat-ibility with standard application equipment and increased efficacy [13, 37]. The advances in the fermentation and formulation process have also led to an increase in the quality of biocontrol products. The possibility of con-taminants (unwanted pathogens, toxins or toxic meta-bolites) in a biocontrol product needs to be considered at an early stage of development [38].

Field studies to evaluate the efficacy of the candidate organism must be conducted before an MBCA can be considered seriously for further development. Evaluation of product efficacy under diverse practical conditions can help to identify the limits of the MBCA. Furthermore, a compatibility profile of the MBCA with other crop pro-tection inputs is essential for successful commercialization

[39]. Lack of consistency in efficacy of biological products is still a problem [10]. However, a recent metadata ana-lysis of efficacy data for MBCAs by Oijambo and Scherm [40] revealed that some of the previous assumptions concerning the efficacy of biocontrol products are not correct. Their study revealed a moderate effectiveness on average, but no differences between the effects of bio-control agents in greenhouse versus field studies, between the effects on soil-borne versus aerial diseases, or under conditions of low, medium and high disease pressure. However, effects were greater on annual than on perennial crops, but were not different for fungal versus bacterial biocontrol agents or for those targeting fungal or bacterial pathogens. Interestingly, on average the efficacy of Bacillus spp. was lower than that of other antagonists. Finally, the effect of one or two sprays for control of aerial diseases was significantly greater than the application of eight sprays or more; this indicates that in an attempt to compensate for anticipated poor per-formance of biocontrol agents, more applications than necessary are made [40].

Recent developments in application technologies for MBCAs were reviewed by Gan-Mor and Matthews [41] who found several improvements in application tech-nologies for biopesticides but concluded that yet more research and development is needed to make sure that promising products can be applied by farmers. Further-more, advances in downstream processing and formul-ations of MBCAs, such as Bt and viruses, which have been in use for decades, allow further significant improvements in economy, shelf-life, ease of application and field efficacy [19, 42]. By improving production and formulation of these products even wider use can be expected in the future.

In Figure 1a, the registration process is considered of equal importance to all other steps in the process of developing and commercializing an MBCA. Conversely, under the current EU regulatory system, several major hurdles have to be negotiated before the successful regis-tration of a biocontrol agent (Figure 1b). Toxicological and environmental impacts are amongst the key issues in the current discussions concerning the registration process for MBCAs in the EU [25, 43, 44]. Better knowledge of the fate and behaviour of an introduced micro-organism in the environment is also crucial for appropriate assessment of potential side effects. It is desirable that the biocontrol agent is established in the area to which it is applied only for the period necessary for the control of the pathogen or pest in order to minimize risks that may ensue from its application. Longer persistence would increase exposure and the possibility of unwanted side effects [44].

MBCAs represent a complex array of approaches towards pest control. Their use for pest control presents challenges in understanding target selectivity, and the occurrence and fate of MBCAs in the environment [45]. Therefore, careful testing of human health risks

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posed by the biocontrol agents is necessary [46]. However, experiences so far do not suggest an unrea-sonable risk of adverse health effects associated with MBCAs although the possibility of infectious and immu-nological responses needs consideration in the context of human exposure [45].

Guidelines that can be used to appropriately address the risks involved in using an MBCA are still lacking. More research is needed on the development of test systems and guidelines to adequately measure the risk a micro-organism poses to the environment [47]. The method-ology developed by Van Lenterenet al. [48] to assess the risks of import and release of exotic enemies used in inundative forms of biological control cannot be extra-polated for MBCAs [47, 49]. La¨ngle [50] further devel-oped the model that integrates information on the potential of an organism to establish and disperse, its host range and possible direct and indirect effects on non-target organisms, to better fit the needs for MBCA risk assessment.

All the above mentioned requirements for MBCAs have significantly contributed to the increase in the costs for obtaining a registration. A decade ago, the costs for registration of an MBCA were estimated at $200 000– 500 000 in the US and Canada with a total of$1–2 million for its development, registration and commercialization [11, 51]. However, more recent estimates were in the range of $500 000 to 1 million for the EPA registration process [9]. Moreover, the costs for obtaining a regis-tration in a single EU member state for on a specific crop can reach $300 000 [37]. Krause et al. [33] calculated that based on the current EU registration requirements an investment of $7–9 million over the course of 6–10 years is needed for registering and commercializing a new MBCA in the EU. Therefore, before a decision is taken on the commercialization of biocontrol product a thorough market analysis is necessary. The development should be market- and not product-driven to avoid failure and ensure that a return on investment is possible within the first three years after sales have begun [7].

Outlook

Although almost all the companies that undertook regis-trations of the products listed in (Table 2) have currently suspended their research and development efforts for new biocontrol agents, there are still many reasons for being optimistic. There are several initiatives underway that will help to promote the use of biocontrol products in the future. Concerning efficacy, a certification scheme was developed that provides a farmer with data on the efficacy of an MBCA to counteract the impression of ‘snake oil’: products that are sold as biopesticides but without proof of efficacy [6]. From the regulators side there have been new initiatives to promote the use of alternative products. The Pesticide Safety Directorate

(PSD) in the UK has launched a new biopesticide scheme to facilitate more alternative products entering the mar-ket. Key elements are the appointment of a ‘Biopesticide Champion’, an expert providing the initial contact to help applicants through the application process. Secondly, speci-fic guidance to applicants is provided via pre-submission meetings. Thirdly, the cost of evaluations, which has been one of the main concerns for applicants, has been reduced [21] (for more details see www.pesticides.gov.uk). Cur-rently, the EU funded specific support action, acronym ‘REBECA’ (Regulation of Biocontrol Agents), is underway with the objective of bringing together stakeholders from industry, science, regulatory authorities, policy and en-vironment to form a network of expertise within the EU (for more details see www.rebeca-net.de). This expertise should help to improve regulatory procedures for MBCAs [52]. With this in mind, the use of MBCAs can play an important role in future crop protection, as a key element in IPM programmes.

References

1. Copping LG, Menn JJ. Biopesticides: a review of their action, applications and efﬁcacy. Pest Management Science 2000; 56:651–76.

2. Warrior P. Opportunities and challenges for microbial pesticides in the global market place. Phytopathology 2006;96 Suppl.:155.

3. Anderson J. EPA’s role in biopesticide development. Phytopathology 2006;96 Suppl.:156.

4. Copping LG (editor). The Manual of Biocontrol Agents. 3rd ed. British Crop Protection Council. Hampshire, UK; 2004. 5. Belanger R. From lab bench to marketplace: how far should

University researchers go? Phytopathology 2006;96 Suppl.:156.

6. Marrone P. Faciliating commercialization: helping both start-ups and established industry. Phytopathology 2006;96 Suppl.:156.

7. Cross JV, Polonenko DR. An industry perspective on registration and commercialization of biocontrol agents in Canada. Canadian Journal of Plant Pathology 1996;18:446–54.

8. Redbond M. Biocontrol 2003. Pesticide Outlook 2003;14:168–70.

9. Evans J. Shifting perceptions on biopesticides. Agrow 2004;455:18–21.

10. Alabouvette C, Olivian C, Steinberg C. Biological control of plant diseases: the European situation. European Journal of Plant Pathology 2006;114:329–41.

11. McSpadden Gardener B, Fravel DR. Biological Control of Plant Pathogens: Research, Commercialization and Application in the USA. Online. Plant Health Progress 2002; doi:10.1049/PHP-2002-0510-01-RV.

12. Jacobsen BJ, Zidack NK, Larson BJ. The role of

Bacillus-based biological control agents in integrated pest management systems: Plant diseases. Phytopathology 2004;94:1272–5.

(10)

13. Hynes RK, Boyetchko SM. Research initiatives in the art and science of biopesticides formulations. Soil Biology and Biochemistry 2006;38:845–9.

14. Kabaluk T, Gazdik K. Directory of Microbial Pesticides for Agricultural Crops in OECD countries. Available from: URL: www.agr.gc.ca/env/pdf/cat_e.pdf (last accessed October 12, 2006).

15. Environmental Protection Agency. Biopesticide Active Ingredients and Products containing them. Available from: URL: http://www.epa.gov/pesticides/biopesticides/ product_lists/bppd_products_by_AI.pdf (last accessed October 12, 2006).

16. European Union. Status of Active Substances under EU Review (doc. 3010) [updated 17 October 2006] Available from: URL: http://ec.europa.eu/food/plant/protection/evaluation/ stat_active_subs_3010_en.xls.

17. Kiewnick S. Potential of Microbial Control Agents: Fungi. Available from: URL: http://www.rebeca-net.de/downloads/ REBECASalzauDocuments/Salzau%20Minutes.pdf (last accessed November 17, 2006).

18. Lacey LA, Frutos R, Kaya HK, Vail P. Insect pathogens as biological control agents: do they have a future? Biological Control 2001;21:230–48.

19. Brar SK, Verma M, Tyagi RD, Vale´ro JR. Recent advances in downstream processing and formulations of

Bacillus thuringiensisbased biopesticides. Process Biochemistry 2006;41:323–42.

20. Environmental Protection Agency. 40 CFR 158 and 172, Pesticides; Data requirements for biochemical and microbial pesticides; Proposed rule. Federal Register

2006;71(45):12072–116.

21. Kiewnick S. European regulatory hurdles and prospects for new biocontrol product development. Phytopathology 2006;96 Suppl.:156.

22. Smeets L. Revision of the Council Directive 91/414/EEC and current progress with the EU review of existing active substances. European and Mediterranean Plant Protection Organization. 2006; p. 3. Available from: URL: http// www.eppo.org/PPPRODUCTS/EUreport.pdf (last accessed November 7, 2006).

23. Sixth Framework Programme. Contract for Speciﬁc Support Action: Regulation of Biological Control Agents (REBECA): Available from: URL: http://www.rebeca-net.de/downloads/ REBECAPartBFinal.pdf (last accessed May 31, 2006). 24. KEMI. Microbiological plant protection products-workshop

on the scientiﬁc basis for risk assessment. National Chemicals Inspectorate of Sweden, Stockholm. 1998; p. 69.

25. Strasser H, Butt TM. The EU BIPESCO project – latest results on safety of fungal biocontrol products. In: Papierok B, editor. Insect Pathogens and Parasitic Nematodes. IOBC/wprs Bulletin 2005;28(3):207–10.

26. Dudley E, Wang CS, Skrobek A, Newton RP, Butt TM. Mass spectrometric studies on the intrinsic stability of destruxin E fromMetarhizium anisopliae. Rapid Communications in Mass Spectroscopy 2004;18(21):2577–86.

27. Michelitsch A, Ru¨ckert U, Rittmannsberger A, Seger C, Strasser H, Likussar W. Accurate determination of oosporein in fungal culture broth by differential pulse polarography. Journal of Agricultural Food Chemistry 2004;52:1423–6.

28. Skrobek A, Butt TM. Toxicity testing of destruxins and crude extracs from the insect-pathogenic fungusMetarhizium anisopliae. FEMS Microbiology Letters 2005;251(1):23–8. 29. Wang C, Skrobek A, Butt TM. Investigations on the destruxin

production of the entomopathogenic fungusMetarhizium anisopliaein liquid and solid media. Journal of Invertebrate Pathology 2004;85:168–74.

30. Wilson CL, Jackson MA. Commercializing of

biologically-based technology for the control of plant pest. Journal of Industrial Microbiology and Biotechnology 1997;19:156.

31. Montesinos E. Development, registration and

commercialization of microbial pesticides for plant protection. International Microbiology 2003;6:245–52.

32. McSpadden Gardener B, Driks A. Overview of the nature and application of biocontrol microbes:Bacillusspp.

Phytopathology 2004;94(11):1244.

33. Krause MS, Vanachter ACRC, De Ceuster TJJ. Commercial research and development of disease-suppressive

microorganisms. In: Raaijmakers JM, Sikora RA, editors. Multitrophic Interactions in Soil and Integrated Control. IOBC/ wprs Bulletin 2006;29(2):63–6.

34. Lu¨th P, Eiben U. Solid-state fermenter and method for solid-state fermentation. United States Patent 2003; US 6,620,614 B1.

35. Pandey A, Soccol CR, Mitchell D. New developments in solid state fermentation: I-bioprocesses and products. Process Biochemistry 2000;35:1153–69.

36. Cherry AJ, Jenkins, NE, Heviefo G, Bateman R, Lomer CJ. Operational and Economic analysis of a West African pilot-scale production plant for aerial conidia of

Metarhiziumsp. for use as a mycoinsecticide against locusts and grasshoppers. Biocontrol Science and Technology 1999;9:35–51.

37. Kiewnick S. Advanced fermentation and formulation technologies for fungal antagonists. In: Sikora RA, editor. Integrated Control of Soil Pests. IOBC/wprs Bulletin 2001;24(1):77–9.

38. Kiewnick, S. Production of low risk fungal biopesticides using proprietary solid state fermentation and spore separation technology. Int. Symp. Bioactive Fungal Metabolites – Impact and Exploitation. April 22–27, 2001; University of Wales, Swansea. British Mycological Society; 2001.

39. Brannen PM, Kenney DS. Kodiak1_{-a successful}

biological-control product for suppression of soil-borne plant pathogens of cotton. Journal of Industrial Microbiology and Biotechnology 1997;19:169–71.

40. Ojiambo PS, Scherm H. Biological and application-oriented factors affecting plant disease suppression by biological control: A meta-analytical Review. Phytopathology 2006;96:1168–74.

41. Gan-Mor S, Matthews GA. Recent developments in sprayers for application of biopesticides – an overview. Biosystems Engineering 2003;84(2):119–25.

42. Szewcyk B, Hoyos-Carvajal L, Paluszek M, Skrzecz I, Souza ML. Baculoviruses-re-emerging biopesticides. Biotechnology Advances 2006;24:143–60.

43. La¨ngle T, Bauer T, Pernfuss B, Seger C, Raffalt J, Strasser H. A GLP/GEP based ﬁeld study onBeauveria brongniartiiwith respect to Commission Directive 2001/36/EC. In: Papierok B,

(11)

editor. Insect Pathogens and Parasitic Nematodes. IOBC/wprs Bulletin 2005;28(3):109–12.

44. Rumbos C, Kiewnick S. Effect of plant species on persistence ofPaecilomyces lilacinusstrain 251 in soil and on root colonisation by the fungus. Plant and Soil 2006;283:25–31. 45. Sudakin DL. Biopesticides. Toxicology Reviews

2003;22(2):83–90.

46. Kiewnick S. Effect of temperature on growth, germination, germ-tube extension and survival ofPaecilomyces lilacinus

strain 251. Biocontrol Science and Technology 2006;16(5):535–46.

47. Kiewnick S. Risk assessment of fungal biocontrol agents. In: Sikora RA, Gowen S, Hauschild R, Kiewnick S, editors. Multitrophic Interactions in Soil. IOBC/wprs Bulletin 2004;27(1):137–43.

48. Van Lenteren JC, Babendreier D, Bigler F, Burgio G, Hokkanen HMT, Kuske S,et al. Environmental risk assessment of exotic natural enemies used in inundative biological control. BioControl 2003;48:3–38.

49. Biological control agents: Safety and regulatory policy [Letter to the Editor]. Biocontrol 2003;48:477–84.

50. La¨ngle T. Developing a risk index for microbial control agents. Available from: URL: http://www.rebeca-net.de/downloads/ REBECASalzauDocuments/Salzau%20Minutes.pdf (last accessed November 17, 2006).

51. Utkhede RS. Potential problems of developing bacterial biocontrol agents. Canadian Journal of Plant Pathology 1996;18:455–62.

52. Ehlers R-U, Strauch O. REBECA – a project to review regulation of BCAs. DIAS Report 2006;119:81–4.