SID 5 Research Project Final Report -...

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SID 5 Research Project Final Report

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Project identification

1. Defra Project code PS2135

2. Project title

A desk study of current knowledge on the combined use of microbial biopesticides and chemical pesticides in Integrated Pest Management

3. Contractor organisation(s)

University of Warwick School of Life Sciences, Gibbet Hill Road, Coventry CV4 8UW UK ADAS UK Ltd ADAS Boxworth, Battlegate Road, Boxworth, Cambridgeshire, CB23 4NN

54. Total Defra project costs £ 30,000

(agreed fixed price)

5. Project: start date ................ 01/12/2010

end date ................. 31/05/2011

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6. It is Defra‟s intention to publish this form.

Please confirm your agreement to do so. YES X (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.

Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.

In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary

7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

Arthropod pests are a major constraint on food security. For half a century, pest management in the industrialised economies has been based on the intensive use of synthetic chemical pesticides. However this strategy is now under considerable strain because the availability of effective chemical active substances is declining as a result of resistance in pest populations plus product withdrawals precipitated by new health and safety legislation. Most experts believe that the way to make pest management more sustainable is through Integrated Pest Management (IPM). IPM is a systems approach based on minimal use of synthetic pesticides combined with alternative control methods and pest monitoring. Alternative pest management methods include resistant crop varieties, physical controls, cultural techniques and biologically based control agents such as natural enemies and natural products.

This report centres on microbial biopesticides, which are mass-produced, commercial plant protection agents based on entomopathogenic microorganisms and used for the biological control of arthropod pests. The main groups of entomopathogenic microbes used in biopesticides are the bacterium Bacillus thuringiensis (Bt), fungi, baculoviruses and nematodes. Bt, fungi and baculoviruses all have to be approved under EU/UK Plant Protection Products (PPP) legislation in order to be used legally for crop protection, In contrast entomopathogenic nematodes are exempt from PPP authorisation (although approval for use of non native nematode species is covered by the ACRE committee).

For PPP microbial biopesticides, there are currently 17 different active substances approved in the EU (9 Bt, 4 fungi, 4 baculoviruses) while 7 different nematode species are also being sold. These products are all used on horticultural crops and they are applied using an “inundation” strategy, in which the control agent is applied at high dose rates in the close vicinity of the target pest. The attractions of using microbial biopesticides include operator and bystander safety, lack of toxic residue, narrow pest activity spectrum, a short interval between application and harvest, low cost of product development and potential for self-sustaining control. However, microbial biopesticides can be less efficacious than fully effective conventional pesticides, they are slower acting, they tend to be more expensive to purchase and they can be adversely affected by environmental conditions.

We reviewed the scientific literature to examine whether pest management can be improved in an IPM approach by combining together different types of microbial biopesticide or by using microbial biopesticides with chemical pesticides. We set out some fundamental principles

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relating to combined treatments before critically evaluating the current “state of the art” from the scientific literature and by talking with IPM practitioners.

We identified 143 papers from Web of Science that reported on combination treatments involving different microbial biopesticides or microbial biopesticides plus chemical agents. The chemical agents included both chemical pesticides and other materials such as diatomaceous earth. Unfortunately, a large number of the studies did not provide sufficient data to enable rigorous statistical analysis for identification of the nature of the interaction (synergism, antagonism or additive effect). Many of the studies employed methods that can give misleading or unreliable results. One exception is a study showing that Bt and the entomopathogenic fungus Beauveria bassiana can give improved control of Colorado potato beetle. We established that UK growers are using combinations of microbial biopesticides and chemical pesticides on the basis solely of anecdotal evidence. The theoretical basis of understanding the molecular biology, biochemistry, genetics, physiology and ecology of combination treatments in IPM is currently poorly developed. However this could change rapidly. For example, recent empirical work has been done using entomopathogenic nematodes and fungi that tests ecological theories of mixed pathogen infections. Research has also been done showing that inhibition of the insect immune system by a chemical pesticide can enhance the action of an entomopathogenic fungus. In pharmacological research, algorithms have been developed based on the dose-effect relationship (derived from mass action principles in chemistry) which enable the type of interaction in 2-drug or 3-drug combinations to be identified with a high degree of confidence. This approach has been shown to be applicable to Bt and there is no reason why it could not be used for other microbial biopesticides.

A small number of high quality studies have been published indicating that combination treatments have potential in three areas: (i) Microbial biopesticides can slow down the development of resistance to chemical

pesticides in arthropod pest populations; (ii) Microbials can reduce the expression of chemical pesticide resistance once it has

evolved; (iii) “Potentiator” chemicals can significantly improve the effectiveness of microbial

biopesticides. A potentiator is a compound that is not pesticidal but which causes in increase in pest mortality when used with a pesticidal agent. As an example, researchers in the USA have developed a simple sugar that inhibits a pathogen detection molecule that forms part of the immune system of termites. When used in combination with an entomopathogenic fungus, the sugar makes the termites far more susceptible to fungal infection.

We make the following recommendations for future studies:

A priority is to establish whether combinations of entomopathogens, or entomopathogens plus chemical pesticides, interact synergistically, antagonistically or give additive effects for selected pests of importance to UK agriculture and horticulture.

The potential for microbial biopesticides to delay the onset of resistance evolution to chemical pesticides, or to reduce the expression of pesticide resistance in pest genotypes, could be a very significant way of extending the availability of chemical pesticides.

There are some exciting opportunities to develop potentiators for microbial biopesticides by exploiting new knowledge on arthropod pest molecular biology, in particular by better understanding of arthropod defences against pathogens using –omics technologies and molecular engineering.

Most of the research work to date has been done at the laboratory scale, with less research at the glasshouse or field scale. There is a need for more glasshouse and field-based research. In particular, we need to investigate the combinations of treatments currently being used by growers against UK pests exhibiting pesticide resistance, and for which there is anecdotal evidence of a beneficial effect of using a combination treatment.

Not enough information is available about the ecological, physiological, biochemical and molecular mechanisms of interaction between microbial biopesticides and between microbials and chemical pesticides / other agents. This is a major impediment to the development of combination treatments.

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Project Report to Defra

8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include:

the scientific objectives as set out in the contract;

the extent to which the objectives set out in the contract have been met;

details of methods used and the results obtained, including statistical analysis (if appropriate);

a discussion of the results and their reliability;

the main implications of the findings;

possible future work; and

any action resulting from the research (e.g. IP, Knowledge Transfer).

A desk study of current knowledge on the combined use of microbial biopesticides and chemical pesticides in Integrated Pest Management Introduction The development of sustainable systems for managing arthropod pests is a significant challenge for the agricultural industry. Since the 1950s, pest management in the UK has been based mainly around the intensive use of synthetic chemical pesticides. While these have helped increase crop yields substantially, their use is becoming increasingly problematic:

Pesticide products based on „old‟ chemistry are being withdrawn from sale because of health and safety legislation.

The rate at which safer replacement chemicals are being made available is very low owing to a fall in the discovery rate of new active molecules and the increasing costs of registration.

Pesticide spray programmes based on a small number of active ingredients can result in management failure through the development of heritable resistance. Worldwide, over 500 species of arthropod pests have resistance to one or more insecticides or acaricides (Hajek, 2004).

Given these problems, alternative crop protection tactics are required that can alleviate the overreliance on chemical pesticides and reduce the problems of pesticide resistance. A range of alternatives is already available to farmers and growers including plant varieties with total or partial pest resistance, physical and cultural controls (e.g. crop covers), natural compounds and biological control agents. However, these alternatives tend to be less efficacious than conventional pesticides. To make best use of alternative controls, it is generally agreed that they should be used as part of an Integrated Pest Management (IPM) programme. IPM is a systems approach that combines different crop protection practices with careful monitoring of pests and their natural enemies. The idea behind IPM is that combining different practices together overcomes the shortcomings of individual practices. The EU passed a package of legislative measures in 2009 based around IPM, including the Framework Directive on the Sustainable Use of Pesticides. Adoption of IPM principles will become mandatory in 2014, and member states have been encouraged to use incentives to get farmers implementing IPM before this date. An important consequence of the EU reforms is that a reduction in use of broad-spectrum pesticides should make the adoption of IPM more likely (van Lenteren, 2000; Raymond et al., 2006).

This literature review concerns one group of alternative crop protection agents, microbial biopesticides (referred to as “microbials” for brevity later in the report), These are mass-produced, commercial plant protection agents based on entomopathogenic microorganisms and are used for the biological control of arthropod pests. We focus on whether pest management can be improved in an IPM approach by combining different types of microbial biopesticide together or by using microbial biopesticides with chemical pesticides. We describe some fundamental principles relating to combined treatments before critically evaluating the scientific literature. We finish the report with a set of recommendations.

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Project Objectives The project had the following Objectives: 1. Summarise the different types of microorganisms currently being used as commercial microbial

biopesticides. Summarise the current availability of microbial biopesticides in the UK and the EU. 2. Review the scientific literature on the effects of combined applications of microbial biopesticides

and chemical pesticides on the control of invertebrate pests. 3. Review the scientific literature on the effects of combined applications of different microbial

biopesticides (e.g. pathogenic fungi combined with pathogenic viruses) on the control of invertebrate pests.

4. Liaise with other scientists and IPM practitioners who are investigating / using combined applications of microbial biopesticides and chemical pesticides or combined applications of different microbial biopesticides.

5. Summarise the different types of interactions documented between microbial biopesticides and pesticides (e.g. synergism, additive effects, or inhibition) and between different microbial biopesticides. Identify the general principles (in terms of mechanisms of interaction) that help determine the particular outcome.

6. Review what is known about whether microbial biopesticides could be used to increase the susceptibility to chemical pesticides of resistant genotypes of pest species.

The Objectives are not written up in numerical order in this report. The results that we obtained in the research meant that it was logical to have them in a different order. Microbial biopesticides (Objective 1, Task 1.1) A range of microbial biopesticides based on entomopathogenic fungi, bacteria, viruses and nematodes are now available worldwide, although the number of registered products varies from country to country. A summary of the modes of action of the different entomopathogen groups is given in Table 1. Approved microbial biopesticides are considered by regulatory authorities to be low risk products and their use is being encouraged on the grounds of human and environmental safety (Chandler et al., 2011). New EU legislation, for example, gives a specific status to non-chemical and “natural” alternatives to conventional synthetic chemical pesticides and requires them to be given priority. Microbial biopesticides based on fungi, baculoviruses and bacteria are regulated according to EU and UK Plant Protection Products (PPP) legislation and require authorisation at EU and / or national level. The UK regulator is the Chemicals Regulation Directorate. Entomopathogenic nematodes – which are classified as “higher” organisms – are exempt from PPP legislation. In the UK, permission to introduce non-indigenous nematode species falls within the remit of ACRE (Advisory Committee on Releases in the Environment). At present only indigenous nematode species are used in the UK. Insect pathogenic bacteria. Bacillus thuringiensis (Bt) is a pathogen of a range of insect orders and is the most widely used microbial biopesticide. It consists of a range of different subspecies and strains, each of which is pathogenic to a small number of taxonomically-related insect species. Bt synthesizes a plasmid encoded parasporal protein crystal, known as the Bt δ-endotoxin, which is toxic to insects on ingestion. Wild-type strains of Bt usually have more than one crystal (cry) gene so that the parasporal crystal consists of a combination of different proteins. More than 130 genes encoding δ-endotoxins have been identified to date (Siegel, 2001). Following ingestion, the δ-endotoxin is cleaved by insect gut proteases to an activated, N terminal form. The activated toxin binds to midgut epithelial cell membranes resulting in the formation of pores that lead to rapid cell death (Gill et al., 1992). In some highly susceptible hosts, ingestion is followed by a general paralysis leading to death within an hour. In the majority of insects, only the gut is paralysed and death occurs in about 48 h depending on dose. Because its activity is based on the activity of an insect specific toxin, microbial Bt is comparable in many ways to a chemical pesticide (although the overall activity of Bt can be co-dependent on bioactive compounds other than the Cry toxins, such as the cytolytic protein produced by Bt subsp. israelensis). This may well explain its relative popularity as a microbial biopesticide. The currently available microbial Bt products are used against Lepidoptera (moth pests), Coleoptera (beetles and weevils) and haematophagous Diptera (blackflies, mosquitoes) but new toxins have been discovered against a wider array of targets including mites, cockroaches, grasshoppers and others (van Frankenhuyzen, 2009). Microbial Bt is safe to workers, beneficial organisms and the environment (Lacey & Siegel, 2000). At present, microbial Bt is used mainly on fruit, vegetable and ornamental crops where its selectivity and safety are considered desirable and where there are problems with resistance to conventional insecticides. In the USA, for example, microbial Bt foliar sprays are applied to about 10% of the total

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apple production area (NASS, 2008). Prior to the mid 1990s, microbial Bt sprayable products were also sold for use on arable crops, however these have now been superseded by Bt transgenic plants. The crops that are currently available synthesize δ-endotoxins active against lepidopteran pests, particularly those that are major pests of maize, such as European corn borer Ostrinia nubilalis, and pests of cotton such as the bollworm Helicoverpa zea. Bt maize with resistance to western corn root worm Diabtrotica virgifera (Coleoptera) is also available. In the USA, 63% of the area of maize planted, and 73% of the area of cotton, now consists of GM varieties expressing Bt δ-endotoxin genes. Resistance management is a legitimate concern, as resistance has developed to microbial Bt (Li et al., 2007). In N America a successful resistance management strategy is in place based on the use of non-GM crop refugia (Shelton et al., 2002). Pest resistance to Bt cotton has recently occurred in India where the refugia strategy is not used (http://www.sciencemag.org/content/327/5972/1439.full). Entomopathogenic viruses. Baculoviruses are entomopathogenic DNA viruses that are genetically distinct from viruses recorded from vertebrates, are infective only for insects, and thus they are considered inherently safe to humans (Tanada and Kaya, 1993). There are two baculovirus genera: Nucleopolyhedroviruses (NPVs) infect over 400 insect species, mainly Lepidoptera (34 families), Hymenoptera and some other orders (Diptera, Coleoptera, Neuroptera). Granuloviruses (GVs) infect lepidopteran hosts, and each GV species is usually specific for just one lepidopteran species. Baculoviruses infect larval hosts per os. The virus initially infects the cells of the midgut before spreading to the fat body. During infection the host becomes debilitated, resulting in reduction of development, feeding and mobility and increasing exposure to predation. Death occurs in 5 – 8 days depending on the dose, but it can be longer. Because they have such a narrow host range, baculoviruses are considered to pose minimal environmental risk (Groner; 1990; Huber, 1986).

Baculoviruses require living insect cells in order to reproduce, and hence mass production can only be done by growing the virus in host insects. In the USA, baculovirus products are available as inoculative biopesticides for the control of forest pests such as douglas fir tussock moth and gypsy moth, and as inundative biopesticides against pests of cotton such as Helicoverpa and Spodoptera (see below for a description of inoculative versus inundative application strategies). In the US and Europe the Cydia pomonella granulovirus (CpGV) is used as an inundative biopesticide against codling moth on apples. In Brazil, the NPV of Anticarsia gemmatalis, the soybean caterpillar, was used in the 1990s on 4 million ha (approx. 35%) of the soybean crop (Moscardi, 1999). Entomopathogenic fungi. Most species of entomopathogenic fungi used for biological control occur in two phyla, the asexual Ascomycetes and the Zygomycetes (Entomophthorales). Entomopathogenic fungi cause infection in all the major taxonomic groups of insects and mites. They infect their hosts using spores that grow through the host cuticle using a combination of enzymatic action and mechanical pressure. Usually the fungus grows into the host‟s haemocoel. If it is able to overcome the host immune system it then goes on to proliferate and spread throughout the rest of the body, often in the form of yeast-like cells that do not naturally survive outside of the host. The host is killed by a combination of mechanical damage, nutrient exhaustion and cell death caused by fungal inhibitors. The relative importance of these mechanisms varies with the fungal species, isolate or host species. Death occurs within 4 – 7 days of infection, followed by the production of large numbers of spores on the cadaver. Thousands or millions of spores may be produced on large insects. The spores of many species of Entomophthorales are actively discharged from the cadaver in order to transmit the fungus to new hosts. They also show a range of other adaptations to increase transmission including timing the release of spores to periods of the day that are most favourable to infection, and manipulating host behaviour so that diseased insects die in exposed positions (Roy et al., 2006). The Entomophthorales contain species that cause natural outbreaks in a number of agricultural pests and some systems have been developed to exploit them for crop protection, for example Neozygites fresenii which causes predictable outbreaks in cotton aphids, Aphis gossypii, in the southern USA (Hollingsworth et al., 1995; Pell et al., 2010). However many entomophthoralean fungi are difficult to mass produce and so they are not yet used widely for commercial biological control.

Entomopathogenic fungi that occur in the asexual Ascomycetes are associated less commonly with natural epizootics, but they are popular choices for use as biopesticides because they can be mass-produced easily. Worldwide at least 130 different fungal products are currently commercially available, registered, or undergoing registration (Faria & Wraight, 2007). About half of these have been developed in South and Central America, around one fifth in N America, about one tenth each in Europe and Asia, and 3% in Africa (Faria & Wraight, 2007). They are used against a wide range of target pests including Hemiptera, Coleoptera, Lepidoptera, Diptera, Orthoptera and Acari. The majority

http://www.sciencemag.org/content/327/5972/1439.full

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of products are based on Beauveria bassiana or Metarhizium anisopliae. They have been used successfully as control agents of insect and mite pests in glasshouse crops, fruit and field vegetables. The largest single area of use is in Brazil, where commercial biopesticides based on M. anisopliae are used on over a million ha against spittlebug pests of sugarcane and grassland (Li et al., 2010). Part of the reason for the success of Metarhizium biopesticides is attributed to government support for research and development (Li et al., 2010). Different strains of M. anisopliae have also been developed commercially for the control of locust and grasshoppers in Africa and Australia (Lomer et al., 2001; FAO, 2007). These pests are usually treated with conventional insecticides sprayed over very large areas, particularly organophosphates, and hence there are genuine concerns about environmental and human safety. When mass produced Metarhizium spores are sprayed in an oil-based formulation they cause up to 90% locust and grasshopper control in 14 – 20 days. Reasons for effectiveness includes high virulence, persistence of fungal spores on the soil and vegetation and the production of new spores on infected hosts, which then spread to uninfected insects. In Europe, Lecanicillum muscarium (“Mycotal”) and Isaria fumosorosea (“PreFeRal”) are approved for the control of whiteflies on protected crops, M. anisopliae (“Met52”) is registered for use against black vine weevil on soft fruit and ornamentals, while two different strains of B. bassiana are available as the products “Naturalis” and“BotaniGard” for the control of a range of pests of greenhouse vegetable and ornamental crops. Entomopathogenic nematodes. Two nematode families - the Steinernematidae and the Heterorhabditidae – are virulent parasites of insects and are the most important nematodes for biological control. Infection is done by 3rd stage juveniles (known as „dauer‟ juveniles). Host finding can be an active process in response to physical and chemical cues. Dauer juveniles of steinernematid species infect their hosts by being ingested or enter through the spiracles and penetrate the tracheae, whereas the heterorhabditids are able to enter a host by actively burrowing through the cuticle. Infective juveniles introduce symbiotic, mutualistic bacteria of the genus Xenorhabdus (Steinernematidae) or Photorhabdus (Heterorhabditidae) into the haemocoel of their hosts following penetration. Subsequent multiplication of the bacteria contributes to host death due to the action of bacterial toxins, which can occur within as little as 48 h of infection. The bacterium also acts as a source of food: the nematode can kill its host without its associated bacterium but is unable to reproduce without it. After the host has died, the dauer juvenile nematodes mature into adults and the infection cycle terminates with the production of large numbers of progeny juveniles. If adequate moisture is present, the next generation dauer juveniles leave their hosts through the cuticle. The dauer juveniles can remain active for a long period of time and, under laboratory conditions, some will live for 1 year (Koppenhofer, 2006). Nematodes are adapted to the soil environment and hence they tend to be used for biocontrol of soil-dwelling pests. They are usually applied as a drench or through drip irrigation lines. A number of products are sold by different companies based on Steinernema feltiae for the control of larvae of sciarid flies, Bradysia spp. on protected crops, and products based on Steinernema kraussei or Heterorhabditis spp. are used for control of larvae of the black vine weevil, Otiorynchus sulcatus on soft fruit and ornamental crops. Strategies for using microbial biopesticides The majority of microbial biopesticides are used according to an augmentation strategy, in which biological control is done using a species of control agent that lives naturally in the country or region of use. This strategy is aimed at increasing the effectiveness of control by releasing an indigenous species of control agent in crop environments where it is absent or at low levels. Normally, the microbial biopesticide is applied using an “inundation” method, in which it is applied at high dose rates in the close vicinity of the target pest. The intention is to achieve rapid pest control without the need for the control agent to reproduce and persist in the environment. As a consequence, applications have to be repeated if the pest population increases again. Microbial biopesticides used according to the inundation method are usually formulated as liquids, emulsions, powders or granules so that they can be applied using the same kind of equipment used for the application of chemical pesticides. The inundation method is based on a hypothesis that natural factors limiting the persistence and spread of the biological control agent (such as abiotic conditions, poor dispersal of the control agent, or a patchy distribution of the target pest) can be overcome by repeated applications of relatively large amounts of the control agent (Hallett, 2005).

An alternative is to use an “inoculation” method in which a relatively small initial amount of the microbial agent is applied with the expectation that it will reproduce and persist in the environment and henceforth respond to changes in the size of the pest population. In this case the environment must be

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suitable to support the reproduction and localised spread of the microbial agent. At the present time, the inoculation method is not used with microbial biopesticides applied to field or glasshouse crops. However it is used on long lived crops in stable environments with pests that have high economic action thresholds, for example the use of baculoviruses for the control of pests in forestry in N America. In practice all entomopathogens have potential to reproduce within their invertebrate hosts, hence there is not always a straightforward distinction between inundation and inoculation. Thus, control agents applied according to the inundation method will exhibit a certain amount of localised reproduction and spread, resulting in a small amount of self-sustaining control. In inoculation, most of the control may be given by the released organisms with the effects of the progeny declining over time (Hajek, 2004). Microbial biopesticides – pros and cons Microbial biopesticides have a range of attractive properties for IPM, including operator and bystander safety, lack of toxic residue, narrow pest activity spectrum, a short interval between application and harvest, low cost of product development and potential for self sustaining control. They can be used against pests for which conventional chemical pesticides are not available or no longer effective. Entomopathogenic nematodes can actively seek out their hosts and hence are useful against pests occupying “cryptic” habitats such as the root zone. The negative sides of microbial biopesticides are generally based on a comparison with conventional chemical pesticides. They can have a lower efficacy than fully effective conventional pesticides, are slower acting, they tend to be more expensive to purchase and they can be adversely affected by environmental conditions (temperature, humidity, u.v. radiation). The narrow host range of many microbial biopesticides limits them to niche markets, which can deter companies from developing new products (Gelernter, 2005).

These issues raise two important questions: can the performance of microbial biopesticides be improved, and how can microbial biopesticides be used with other crop protection methods in IPM in order to make best use of their beneficial properties? To date, more research effort has been put into developing microbial products and looking for ways to improve their efficacy than on integrating them in IPM. The development of microbials has generally been done according to a chemical pesticides model that has concentrated on the technical challenges facing new products, such as mass production, improving product shelf life etc. These are important areas of biopesticide research, but unfortunately the “pesticides model” has also created unrealistic expectations of chemical-like efficacy for microbial products, while at the same time the attractive biological characteristics of microbial agents have not received enough attention (Waage, 1997). Current availability of microbial biopesticides (Objective 1, Task 1.2) Worldwide there are c. 1400 biopesticide products being sold (Marrone, 2007); this includes all entities categorised as a “biopesticide” (microbial agents, semiochemicals and other natural products). As far as microbial biopesticides are concerned, in the EU there are 9 different microbial Bt active substances approved for use, 4 different entomopathogenic fungi and 4 different baculoviruses (total = 17) (see Table 1). These products are all sold for use on horticultural crops. There are another 17 microbial control agents listed as active substances on Annex I, mainly for use as microbial antagonists of plant pathogens. As of 2006, there were 6 producers of entomopathogenic nematodes in Europe, of which 3 were classed as large-scale producers (Kaya et al., 2006), selling products based on seven species of nematode: S. feltiae, S. kraussei, Steinernema carpocapsae, Steinernema scapterisci, Steinernema glaseri, Heterorhabditis megidis, Heterorhabditis bacteriophora. These are mainly targeted against Coleoptera, Lepidoptera and Diptera on horticultural crops. Microbial biopesticides represent a small fraction of the total pesticides market, but there is rapid growth in the sector with a five-year compound annual growth rate of 16% compared to 3% for synthetic pesticides and an expected global market worth up to $10 billion by 2017 (Marrone, 2007). Microbial biopesticides make up about 85% of the value of the market for all biologically based pest control agents (Gelernter, 2005).

As described previously, microbial Bt, baculoviruses and entomopathogenic fungi all have to be approved for use according to EU / UK plant protection products legislation. The biopesticide registration data portfolio that is submitted for approval is normally a modified form of the one in place for conventional chemical pesticides and is used by the regulator to make a risk assessment. It includes information about mode of action, toxicological and eco-toxicological evaluations, host range testing and so forth. This information is expensive for companies to produce and it can deter them from commercialising biopesticides which are usually niche market products. Therefore, the challenge for the regulator is to have an appropriate system in place that ensures product safety and consistency but which does not inhibit commercialisation.

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Microbial biopesticides and IPM Many farmers and growers are experienced at using combinations of pest control methods in IPM. In general, the adoption of a wider range of IPM tactics is correlated with farmer education, experience and crop typewith IPM being adopted more on horticultural crops (Lohr and Park, 2002). IPM is often perceived by growers who currently rely on pesticide programmes to be more expensive in terms of direct costs and labour time. The relative costs on outdoor crops have not yet been established as comprehensive IPM programmes have not yet been developed. However, IPM on protected crops is cost-effective and widely adopted by growers (see below). On arable and field vegetable crops, IPM is based around targeted pesticide use, choice of crop variety and crop rotations but use of biocontrol agents including microbial biopesticides is still relatively undeveloped (Bailey et al., 2009). In N America, China, India and elsewhere, there has been widespread adoption of GM Bt maize, cotton and soya for control of lepidopteran pests. On soft fruit and top fruit, pesticide resistance has been a significant issue for some key pests for over 30 years, and IPM programmes are based on targeted sprays of selective pesticides designed to conserve natural enemies such as the predatory phytoseiid mite Typhlodromus pyri which is important for the control of two-spotted spider mite Tetranychus urticae (Blommers, 1994). As described previously, microbial biopesticide products based on Bt and baculoviruses are also available, and the codling moth sex pheromone is used for mating disruption on about 160,000 ha of apple orchards worldwide (Witzgall et al., 2008). The most sophisticated, biologically based IPM systems are currently used on edible and ornamental greenhouse crops which in Europe include crops grown in glasshouses and polythene tunnels. These IPM programmes were first initiated over 40 years ago in response to widespread pesticide resistance. The pressure to reduce insecticide usage was reinforced by the adoption of bumblebees within greenhouses for pollination on some crops such as tomato. Some highly effective IPM programmes are in place based on the use of multiple natural enemies and supplementary applications of selective chemical pesticides against a range of pest species occurring on greenhouse crops. IPM adoption was also helped by the fact that greenhouse crop production is labour intensive, technically complex, and thus growers already had a high level of knowledge and were used to innovation prior to IPM (Pilkington et al., 2010). The advantages of the biologically based IPM system used in greenhouse crops include effective pest control in the face of pesticide resistance, lack of phytotoxic effects, a short or zero pre-harvest interval, and operator safety. These advantages are quickly appreciated by growers of protected crops when they first adopt IPM. Even growers who are initially sceptical about the perceived increased costs and labour time involved compared with using pesticide programmes, soon realise that IPM can be cost-effective and even labour-saving. New IPM converts soon appreciate the relative benefits and quickly gain confidence in IPM as long as they are supported by well-informed technical advice and guidance (Bennison 2004). While most of the biological control in greenhouses uses predators and parasitoids, pathogens are also valuable treatments, for example using B. bassiana as a knock down spray in combination with predators against spider mites and western flower thrips (Chandler et al., 2005; Jacobson et al., 2001).

Combination treatments involving microbial biopesticides and chemical pesticides (Objective 2, Task 2.1; Objective 3, Task 3.1) At present, it is not standard practice to systematically combine different microbial biopesticides in IPM, or to combine a microbial biopesticide with a conventional chemical pesticide. Instead, microbials tend to be used as “stand alone” products in situations where chemical pesticides are undesirable or not effective, for example if there is pesticide resistance in the target pest population or if there are concerns expressed from consumers or retailers about pesticide residues. However, there are sound reasons why combination treatments should be considered:

A combination of microbials and chemical pesticides with different modes of action may have synergistic effects.

A combination treatment may allow the control of multiple pests or enable pest control under a broader range of environmental conditions than for a single treatment.

Microbials could be used to reduce the chances of resistance developing to a chemical pesticide or as a way of reducing the severity of resistance after it has evolved. It should be possible to alternate between chemical pesticides and microbials in order to reduce the selection pressure for chemical resistance or it may be possible to tank mix a microbial and a chemical pesticide for use against pests that have evolved pesticide resistance. On the face of it, microbials are attractive products for combining with chemical pesticides as they can usually be applied using conventional spray equipment.

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Most microbials have a significantly shorter interval between application and crop harvest compared to conventional chemical pesticides. Thus a farmer or grower could use chemical pesticides for most of the time but switch to a microbial prior to harvest.

Using combinations of drugs to treat infectious and non-infectious diseases is widespread in medicine. We believe there is potential for pest management scientists and practitioners to learn from this, since the principles of managing agricultural pests (treatment thresholds, tackling resistance, impact on non targets) have parallels in medicine and pharmacology.

Individual species / strains of entomopathogenic microorganisms synthesize a number of different bioactive compounds, including enzymes, inhibitors and others, that interact to cause disease in a susceptible host insect or mite. In this regard, therefore, an entomopathogen already consists of a “combination treatment” and it is not such a big leap of the imagination to think about developing the “combination” further using other microbials or chemical pesticides. As we have already mentioned, strains of Bt each synthesize a cocktail of δ-endotoxins, each toxin having a slightly different potency or host range. Entomopathogenic fungi synthesize a wide range of virulence compounds including enzymes and inhibitors that overcome the host immune system and contribute to successful infection. The mutualistic bacteria carried by insect pathogenic nematodes synthesize different insect-active toxins. Entomopathogenic virus strains show variation in coat proteins and mixed strain infections are common in nature: it is thought that there may be mutualistic interactions during infection between the different virus strains (Tanada and Kaya, 1992).

Quantifying combination treatments involving microbial biopesticides and chemical pesticides (Objective 5, Task 5.1) In this section we discuss different strategies for combining microbial and chemical pesticides and how their effects can be quantified. When one considers the numbers of entomopathogens and chemical pesticides that could be used together, the wide range of crops that are grown, and the fact that integrated treatments combining microbial biopesticides and chemical pesticides can be done over a wide range of spatial and temporal scales, then the total number of possible interactions is very large indeed. Moreover, farmers, growers and IPM practitioners usually have to control multiple pest species, and so there are issues concerning the compatibility of the different microbial and chemical pesticide treatments used for different pest species. We are dealing with a highly complex situation, therefore. However, we can still identify some general principles. Firstly, we consider four different types of interaction that can occur when microbials and / or chemical pesticides are used together:

When two pesticidal agents are combined, then there are three types of interaction. An additive effect is where the effect of the combination is equal to the sum of the effects of the individual agents. Synergism is where the effect of the combination is greater than the sum of the individual agents. Antagonism is where the effect of the combination is less than the sum of the individual agents. However, as we explain below, identifying when synergism, antagonism or an additive effect has taken place is not a straightforward task.

Potentiation occurs where two agents, A and B, are combined for pest management: Agent A is pesticidal when used on its own while agent B has a non-lethal effect on the pest, however when used in combination their joint lethal effect is greater than that of agent A. For this report we are excluding formulation chemicals, which improve the efficacy of microbial and chemical pesticides by increasing their shelf life, survival time on foliage, spray characteristics etc. but which do not have a direct effect on the target pest. However some agents act as both potentiators and formulants. For example, optical brighteners increase the efficacy of baculoviruses by affecting host cell membranes and thereby enhancing virus infection (a potentiating effect) but they also protect virus particles from damage by u.v. radiation (i.e. a formulation effect) (Thorpe et al., 1999; Shapiro & Farrar, 2003; Ibargutxi et al., 2008).

Secondly, we make a distinction between sequential and simultaneous applications of control agents:

Sequential treatments, i.e. two or more agents applied one after the other. This has two subcategories: (a) an application of a microbial biopesticide is followed by an application of a different microbial biopesticide against the same pest target; (b) an application of a chemical pesticide is followed by an application of a microbial biopesticide (or vice versa) against the same pest target. Note that “pest target” includes a population of one pest species or a community of different pest species feeding on the same crop. The time interval between applications and the total number of applications may vary. For example, a microbial may be used one season and a chemical pesticide used the next season. In such a case, the two agents are unlikely to be in direct

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contact with each other, although they could interact indirectly via their effects on the pest population. Another option could be to alternate between using a microbial biopesticide and a chemical pesticide over a shorter time interval. In this case the two agents may come into direct contact with each other in the same pest organism depending on how long they persist.

Simultaneous treatment, i.e. two or more agents applied at the same time. Again, this has two subcategories: (a) two or more microbial biopesticides are applied together; (b) a combination of microbial biopesticide(s) and chemical pesticide(s) are applied together. The aim of simultaneous treatments is to achieve a synergistic interaction between the agents in the mixture. The different pest control agents may well be tank mixed. They will be in direct contact with each other in the tank, on the surface of the crop and inside individuals of the target pest.

Thirdly, we note that combination treatments can be thought about from a physicochemical,

biochemical, physiological, ecological or evolutionary perspective. Algorithms are now available from pharmacological research, based on a physicochemical description of dose-effect relationships that enable synergism and antagonism to be identified with a good level of confidence (Chou, 2006). A major shortcoming of research on combination treatments involving microbial biopesticides and chemical pesticides has been the lack of a reliable method of identifying synergism and antagonism. Without proper mathematical quantification of the interaction, the biochemical / physiological mechanisms behind the interaction cannot be determined with certainty. Finally, considering the interaction problem from an ecological / evolutionary perspective may help identify biological traits that determine the outcome of interaction. Combined treatments: a physicochemical perspective When two therapeutic agents have a dose effect curve that can be normalised using the same procedure (e.g. using a logistic transformation) then their joint effect can be analysed as an isobologram based on a physicochemical equation. The theoretical basis and analysis of drug combination studies has been reviewed by Chou (2006). The author‟s findings are highly relevant to combination treatments for IPM. He points out that researchers rarely provide a mathematically rigorous method for identifying synergism or antagonism in drug combination studies, and as such faulty or unsubstantiated claims of synergy have ended up being put into clinical practice. He notes two key pitfalls in drug combination studies: Firstly, the failure to differentiate between potentiation and synergism. Secondly, a failure to identify what is meant by synergy, antagonism and additive effect. An additive effect is not simply the arithmetic sum of two drugs, for example if two antibiotics each kill 70% of a population of bacteria, then the combined efficacy is clearly not 140%. Drug combination effects are sometimes calculated using a fractional product method. In the case of a two drug combination, for example, the effect of the combination is given as (1 – X), where X = (1 – A)(1 - B) and where A = the proportional inhibition caused by drug A on its own, and B is the proportional inhibition given by drug B on its own. Thus if A = 0.7 and B = 0.7, then (1- X) = 0.91. However, this calculation does not take into account the shape of the dose-effect curve and is not valid when two drugs have similar mechanisms, i.e. they do not operate independently. The dose-effect relationship of a combination treatment can be analysed using an “isobologram” method, which is derived from basic principles of enzyme kinetics (e.g. the Michaelis-Menten equation). The approach can be summarised as follows: The effect of a drug is proportional to its dose. When two drugs are mixed, the mixture behaves as a third drug in terms of the dose-effect relationship. An isobologram is a plot of the dose of drug 1 (D)1 versus the dose of drug 2 (D)2 in a mixture over the effective dose range for each drug on its own (usually from zero to the median effective dose, ED50). If there is no interaction between the two drugs in the mixture (i.e. an additive effect) then the relationship is a straight line with a negative gradient that is plotted simply by connecting the ED50 for drug 1 on axis D1 to the ED50 for drug 2 on axis D2 (see figure 1). Deviation from the straight line in actual combination data indicates synergism or antagonism. In synergism, the effective dose of each drug is lowered in the mixture, and hence combination data points are below the straight line. For antagonism, the effective dose of each drug is increased in the mixture, and combination data points are found above the straight line. Software has been developed to analyse drug combinations based on using mixtures at different proportions of the ED50 for each drug. The software is able to analyse the effects of two or three drug combinations and gives predictions for the optimal combination ratio to maximise synergy (Chou, 2006). The experiments are done by recording (a) the effect of each drug on its own at a range of concentrations and (b) the effect of the two drugs as a mixture at a range of concentrations. Chou (2006) recommends that the concentration of each drug singly and in combination ranges from 0.25xED50 to 4xED50.

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Combined treatments: an ecological / evolutionary perspective Current ecological theory suggests that pathogens evolve to optimise their transmission. Host death is a product of pathogen growth and reproduction within the host because the pathogen takes resources away from its host and overcomes the host immune system as it grows. For a horizontally transmitted pathogen (i.e. transmission between individual hosts in a cohort), mutations that enhance transmission will be favoured by natural selection. High virulence will be selected against if it reduces transmission, i.e. if a pathogen kills its host very quickly then it impairs its ability to be successfully transmitted. Thus there is a trade off between pathogen reproduction and virulence that is mediated by transmission. Strains of entomopathogen selected in screening programmes for use as inundative microbial biopesticides are usually chosen on the basis of high virulence.

There is currently a poor understanding of the ecological interactions that occur between co-infecting pathogens. This is despite the fact that mixed infections are thought to be a normal occurrence in nature (Staves & Knell, 2010). Co-infecting pathogens are likely to compete for host resources – nutrients, manipulation of host behaviour, space, water etc. - unless there is niche complementarity between them (for example if they naturally occupy different parts of the host). The interaction between two or more entomopathogens will also be mediated by the host‟s immune response, which can be thought of as apparent competition (Mideo et al., 2008; Mideo, 2009). These interactions are normally considered in terms of the outcome for the competing pathogens; in theory, one pathogen may competitively exclude the other, or they may co-exist. The outcome will depend upon each pathogen‟s virulence, its life history strategy (r or K selected), the starting dose, and the immune status and physiological condition of the host. In general, competition between two pathogens will result in a reduction in the net reproductive rate of both pathogens. However for this study we are more concerned with understanding the outcome of a co-infection in terms of the survival of the host, i.e. whether there is synergism, antagonism or an additive effect in a combination treatment and how this affects pest control. Antagonism would occur if competition between two pathogens results in reduced pathogen growth inside the host. However increased host death could occur if two pathogens produce inhibitors or other bioactive chemicals that interact synergistically, for example by affecting different target sites. For IPM, the desired outcome of a combined treatment is synergism.

Alternatively, a pest may be infected by one pathogen and then be challenged by a different pathogen (i.e. a secondary infection attempt). This could occur if a pest population is sprayed with two different microbial biopesticides, one after the other. If the first pathogen is established within the host then it may be well placed to prevent a secondary infection. Some entomopathogens, such as the fungus B. bassiana, synthesize antibiotics that are thought to prevent secondary infections (Zimmerman, 2007). However the outcome will depend on pathogen virulence, dose, host immunity and the time interval between the two infection events.

A third scenario is where a pest is infected by a pathogen, successfully overcomes the infection but is later exposed to the same or a different species / strain of pathogen. Insects and mites have evolved multiple defences against pathogens including physical barriers, behavioural responses and an innate immune system comprising humoral and cellular reactions (macrophages, Toll, Imd and JAK / Stat pathways, prophenoloxidase activity). Until recently it was thought that the innate immune system was not adaptive. However there is evidence now that successfully fighting an infection can “prime” an arthropod‟s immune system to make it less susceptible to subsequent infection (Roth et al., 2009). Primed immunity can also be passed to the subsequent generation (Sadd et al., 2005).

Host-pathogen interactions are major drivers of evolutionary change. Hence it is important to consider the evolutionary context when thinking about combined treatments of biopesticides or biopesticides + chemical pesticides. Entomopathogenic microorganisms are widespread in the environment and hence it is very likely that insects and mites are subject to co-infection attempts by different pathogens in nature. In addition, phytophagous insects and mites are subject to the effects of host plant defence chemicals (which we can think of as natural pesticides). Therefore, we can hypothesize that herbivorous arthropods will have evolved mechanisms to fight co-infections and to counteract the combined effects of plant defence compounds and entomopathogens. On the other hand, there will also have been opportunities for plants and entomopathogens to have evolved mutualistic relationships. Unfortunately, we know little about the ecological and evolutionary interactions of arthropods, pathogens and plants. Uncovering some basic principles could be very useful in informing the development of combined treatments of entomopathogens and chemical pesticides. Wild maize plants, for example, synthesize a sesquiterpene volatile, (E)-β caryophylene, in response to root feeding by western corn rootworm Diabrotica virgifera and which recruits entomopathogenic nematodes to aid plant defence against the pest. This trait has been lost in commercially grown maize varieties. However, transforming a nonemmitting maize line with a (E)-β

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caryophylene synthase gene resulted in constitutive expression of the sesquiterpene that cause a 60% improvement in nematode-mediated protection against corn rootworm in pest infested field plots in which nematodes were applied (Degenhardt et al., 2009). Similarly, research on the effects of entomopathogens on pest fitness is starting to be exploited as a chemical pesticide resistance management strategy (see below).

Combination treatments involving microbial biopesticides and chemical pesticides: a summary of different types of interactions based on an analysis of the literature (Objective 2, Task 2.1; Objective 3, Task 3.1; Objective 5, Task 5.1) We ran a literature search in Web of Science to identify peer reviewed papers concerning the effects on pests of combining either different microbial biopesticides or microbial biopesticides and chemical pesticides (including conventional chemical pesticides and alternative treatments such as neem or diatomaceous earth). The search terms used are given in Appendix 1. These terms produced 143 papers from Web of Science that report on combination treatments involving different microbial biopesticides or microbial biopesticides plus chemical agents. The chemical agents included both chemical pesticides and other materials such as diatomaceous earth. There were 76 examples reported in 72 papers of interactions between microbial biopesticides and chemical pesticides. Of these, the majority (54) concerned entomopathogenic fungi. There were 57 examples reported in 50 papers of combinations of different microbial biopesticides; these included combining different taxonomic groups of entomopathogen (e.g. a fungus with a nematode; total of 39 different examples) or combining entomopathogens from the same taxonomic group (e.g. a fungus with a fungus; 17 different examples). A list of the papers is given in Appendix 1. We have grouped them according to the type of pest control agent used in the combination (microbial biopesticide, chemical pesticide or other material) and the taxonomic group of entomopathogen (bacterium, virus, fungus, nematode) (see Table 2). Each paper has been evaluated for the following criteria: (1) scale of experiment (laboratory, glasshouse or field); (2) strategy (were the agents applied simultaneously or sequentially?); (3) result (did the combination of agents result in an increase in the level of pest control, a decrease or no change?); (4) mechanism (was the mechanism of interaction identified?). It is not our intention to provide a detailed analysis of each article. Rather, we flag up the key points to come out from the literature and we discuss selected research areas that we believe are particularly relevant. Interactions between microbial biopesticides and chemical pesticides The effect on pest control of simultaneous applications of microbial biopesticides and chemical pesticides has been investigated in a range of studies motivated by a desire for higher pest mortality and improved speed of kill. Research has also been done to investigate the role of microbial biopesticides in preventing or delaying the development of chemical pesticide resistance, and also to look at the effect of sublethal quantities of chemical pesticide on the performance of microbial biopesticides. These aspects are discussed in separate sections below.

The majority of studies have concerned entomopathogenic fungi. For example, Hornbostel et al. (2005) investigated a combination of M. anisopliae and the pyrethroid permethrin for the control of the deer tick (Ixodes scapularis). While M. anisopliae was moderately pathogenic to tick larvae the addition of permethrin did not significantly affect larval mortality. Results from a second study combining M. anisopliae with a pyrethroid were more positive. Kpindou et al. (2001) combined the entomopathogen with lambda-cyhalothrin in order to improve speed of kill of Sahelian grasshoppers. Here the chemical pesticide gave rapid knockdown with mortality due to the M. anisopliae beginning two days after application.

Delgado et al. (1999) evaluated the effect of combined applications of B. bassiana with a small amount of the chitin synthesis inhibitor diflubenzuron against savannah grasshopper populations in large scale field experiments in Mali. Insects can effectively remove fungal pathogens that are invading the cuticle by moulting; hence intuitively it would appear reasonable to assume that chemical pesticides that inhibit moulting may enhance fungal infection. In this case there was a simple additive effect of the combined treatment which nonetheless could be useful within an IPM context. Taking a similar approach, Irigaray et al. (2003) combined B. bassiana with benzoylphenyl urea, another chitin inhibitor, for the control of two-spotted spider mite (Tetranychus urticae). Here, the combination actually gave reduced mortality compared with the entomopathogen used on its own. This may have been because the pesticide was inhibitory to the fungus, as it was observed to reduce mycelial growth in laboratory tests, although conidial germination of B. bassiana was unaffected.

Koppenhöfer et al. (2003) investigated the potential of combining entomopathogenic nematodes with imidacloprid for control of white grubs (Scarabaeidae). Here, imidacloprid had little effect on

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survival or pathogenicity of Heterorhabditis bacteriophora. Similarly, other neonicotinoids (thiamethoxam and acetamiprid) were found to be compatible with entomopathogenic nematode species, although high rates of acetamiprid did affect Steinernema feltiae behaviour. Where combinations of a nematode and a neonicotinoid led to higher levels of insect mortality the nematode populations in the soil were also found to be higher. It was suggested that these higher nematode populations could provide extended control of the pest, however, this will depend in part of the remaining pest population and the ability of the nematode to survive in the soil.

A number of studies have looked at the potential to combine biopesticides with the botanical insecticide neem, which is straightforward to produce and hence is attractive as an alternative “cottage industry” pesticide. Shah et al. (2008) found both M. anisopliae and neem cake (a by-product of neem oil production) to be effective against early instars of the black vine weevil when incorporated into compost and that the addition of neem cake enhanced the efficacy of M. anisopliae. It was suggested that the neem cake caused greater movement of the larvae by acting as a repellent or antifeedant leading to increased acquisition of fungal spores. The apparent antifeedant properties of the neem cake also resulted in reduced larval growth. Reduced feeding may in turn have weakened the larvae making them more susceptible to the entomopathogen. Similarly, Mohan et al. (2007) found most isolates of B. bassiana tested to be compatible with neem oil and that a combination was more effective against tobacco budworm. This improved efficacy was seen both by increased mortality and faster speed of kill. Barčić et al. (2006) investigated the efficacy of Bt, neem and pyrethrins for the control of the Colorado potato beetle (Leptinotarsa decemlineata). Here the combinations were found to have greater efficacy and persistence compared to the individual components. Neem has also been used to overcome limitations on the use of baculoviruses in pest management. Although viruses are of great interest as a pest management tool, their use is limited by slow speed of action, instability to UV light, high production costs and short shelf life. Work by Nathan and Kalaivani (2006) investigated the potential of combining a neem extract with Spodoptera nucleopolyhedrovirus (SpltNPV). In this case the combination of the virus with the neem caused tobacco budworm (Spodoptera litura) larvae to die significantly faster than when either component was used on its own. It was also found that the concentration of the virus could be reduced in the combination, helping to reduce the cost of these applications. As for the work with Bt, these benefits are thought to be due to the neem disrupting the insect gut epithelium, facilitating the penetration of the virus. In contrast, a combination of spinosad with Spodoptera frugiperda multiple nucleopolyhedrovirus appears to work with the pesticide providing an initial kill and the virus killing surviving individuals (Mendez et al. 2002). It was found that the dose of spinosad used in the combination could be reduced while maintaining efficacy, helping to reduce the effect of the chemical pesticide on beneficial insects.

Relatively few studies have investigated the potential of combining microbial biopesticides with chemical pesticides sequentially. Sequential applications of biopesticides and chemical pesticides may be used to enhance the efficacy and speed of kill of the biopesticide in a similar way to simultaneous applications. Alternatively, sequential applications may be used in Integrated Pest Management (IPM) programmes that seek to benefit from the different modes of action of biopesticides and chemical pesticides. For the work published in this area, sequential applications of microbial biopesticides and chemical pesticides to improve the efficacy of the biopesticide are usually applied close together. For example, Santos et al. (2007) investigated the potential of using imidacloprid to improve the performance of an entomopathogenic fungus to control the leaf cutting ant (Atta sexdens rubropilosa). Here a sub-lethal dose of imidaclorpid was applied first. Previous work has shown that imidacloprid disrupts the movement and social grooming of termites, helping to increase conidial spore attachment (Boucias et al. 1996; Quintela & McCoy, 1998). Results indicated improved efficacy of moderately virulent isolates when imidacloprid was also applied but no improvement was recorded for highly virulent isolates, which were effective with or without the addition of imidacloprid. Cuthbertson et al. (2008, 2010) have investigated the potential of combining the entomopathogenic fungus Lecanicillium muscarium with chemical pesticides in an IPM programme for the tobacco whitefly. The fungus was applied to plants 24 hours after the chemical pesticide. Although spore germination was affected by chemical pesticides such as spiromesifen or physically acting products such as fatty acids, mortality of second instar whitefly larvae was higher in combinations than when the fungus or the pesticides were applied on their own. Similarly, James (2003) combined azadirachtin (from neem) with the entomopathogenic fungus Paecilomyces fumosoroseus for control of the same pest. Again higher levels of mortality were recorded when the azardirachtin and the entomopathogenic fungus were combined in sequential sprays separated either by two hours or three days.

Currently we have a poor understanding of the causal mechanisms of interaction between microbial biopesticides and chemical pesticides. Most of the studies of combined biopesticide-chemical

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pesticide treatments are based on measuring the effect of the combination treatment on pest survivorship. Only a handful of studies have been done on mechanisms. For example, infection of scarab grubs, Anomala cuprea, by M. anisopliae was enhanced in the presence of fenitrothion because of inhibition of the insect immune response by the insecticide (Hiromori and Nishigaki, 2001). Research on the interaction between entomopathogenic fungi and sub-lethal doses of imidacloprid suggests that fungal infection can be enhanced as a result of insecticide-induced starvation or behavioural modification (see below). New research in this area is clearly warranted to strengthen the theoretical basis for the development of combination treatments.

Combining treatments of microbial and chemical pesticides as a resistance management strategy (Objective 6, Task 6.1) The insecticide and acaricide products that are currently available are based on a relatively small group of active chemicals and modes of action (MoA). There are currently 25 known MoA (primary sites of action) that are exploited in pest control against insects and mites as defined by the Insecticide Resistance Action Committee (IRAC). Of these, 16 MoA groups are currently registered in the UK. However for each crop-pest combination the range of MoA available is greatly reduced. Development of resistance to insecticides and acaricides by pests of arable and horticultural crops is one of the main threats to pest management programmes that rely on chemical controls. Where resistance to a synthetic pesticide develops this has the effect of reducing the range of MoA available to control a pest and thereby increasing the selection pressure for resistance to the remaining pesticides. Resistance may develop to a single group of pesticides with a shared MoA or there can be cross resistance, i.e. resistance to more than one MoA. In addition, pests may develop different forms of resistance (multiple resistance) to pesticides with different MoA.

Key pests in the UK that have evolved resistance to chemical pesticides are listed in Table 4. Resistance in these pests has developed either through enhanced metabolic activity (e.g. elevated levels of esterases, cytochrome-P450 monoxygenases and glutathione S- transferases) or though target-site resistance (e.g. resistance to carbamates and pyrethroids in peach-potato aphid). In order to delay or prevent development of resistance, management strategies have been developed in which products with different MoA are alternated or the number of applications of a single MoA restricted.

Recent studies have investigated the potential to combine microbial biopesticides with synthetic pesticides in order to improve the control of some pest species that show pesticide-resistance in the UK. Cuthbertson et al. (2005; 2008a & 2010) have investigated the compatibility of synthetic insecticides with the entomopathogenic fungus L. muscarium for the control of tobacco whitefly (Bemisia tabaci). This work highlighted the potential to apply the biopesticide sequentially with a range of synthetic insecticides, where the fungus would only come into contact with dry residues of the chemicals on the leaf surface (see above). Cuthbertson et al. (2008b) have also investigated the compatibility of the entomopathogenic nematode S. carpocapsae with a range of synthetic insecticides. Here the use of nematodes in combination with the neonicotinoid thiacloprid resulted in higher levels of mortality in B. tabaci than when the chemical was used on its own. Er & Gökçe (2004) investigated the compatibility of synthetic insecticides with the entomopathogenic fungus Isaria fumosorosea (= Paecilomyces fumosoroseus) for control the glasshouse whitefly Trialeurodes vaporariorum in laboratory experiments. The results suggested potential for combining the fungus with a range of different insecticides. Positive results have also been recorded when M. anisopliae or B. bassiana were combined with oils (Malsam et al. 2002) or neem extract (Islam et al. 2010) against whitefly. In contrast, work by James and Elzen (2001) recorded a decrease in the expected level of pest control of B. tabaci when B. bassiana was combined with imidacloprid. There is no clear explanation for this result other than a possible behavioural effect caused either by the imidacloprid or the B. bassiana.

Populations of the western flower thrips (Frankliniella occidentalis) are currently resistant to most synthetic insecticides approved for use in the UK. The development of effective biopesticides and IPM programmes is therefore currently of great interest. Ansari et al. (2007 & 2008) found M. anisopliae to be effective against soil dwelling stages of this pest. However, reduced rates of imidacloprid or fipronil applied with the fungus did not significantly improve control. This was at least in part due to the high level of efficacy of M. anisopliae on its own. Shah et al. (2007) did though suggest that the insecticide may still be important by providing immediate crop protection and thereby giving the fungus more time to be effective. Indeed, in a separate study investigating the use of imdacloprid combined with B. bassiana to control the brown plant hopper (Nilaparvata lugens), Feng and Pu (2005) suggested that the dose of imidaclorpid should be selected depending on the speed of kill required.

Gatarayiha et al. (2010) found that potassium silicate may enhance the efficacy of Beauveria bassiana in controlling two-spotted spider mite (T. urticae). Here the potassium silicate was applied as

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a plant nutrient and the B. bassiana applied to leaves conventionally. It was hypothesised by the authors that the potassium silicate increased plant resistance, which resulted by reduced feeding by the mites and increased their vulnerability to the fungus.

These studies point to the potential of biopesticides to contribute effectively to the management of some UK pests that have developed pesticide resistance but they have not investigated how microbial biopesticides affect the evolution or expression of pesticide resistance. Research by Farenhorst et al. (2009) with African malaria mosquitoes showed that infection by B. bassiana or M. anisopliae significantly reduced the expression of resistance to the public health insecticides permethrin and dichlorodiphenyltrichloroethane when resistant mosquito genotypes were treated with these insecticides three days after fungal infection. In subsequent experiments (Farenhorst et al., 2010) the order of treatment was varied (fungal infection followed by pesticide exposure three days later and vice versa). This indicated a reciprocal relationship between fungi and insecticides: fungal infection increased insecticide mortality in resistant mosquito genotypes, while conversely insecticide exposure increased subsequent mortality caused by fungal infection. However the highest levels of mortality were observed when insecticide and fungal biopesticide were applied at the same time. If these findings were applicable to other insecticide-resistant pests (and there is no reason to think that they would not) then this could be an important new method of pesticide resistance management.

Arthropods can also evolve greater levels of defences against entomopathogens. This appears to be primarily an issue for entomopathogens that do not have multiple modes of action including Bt. However, entomopathogens in which virulence is determined by many genes appear to be far less likely to select for resistance. For example, the response to selection in Drosophila melanogaster for resistance B. bassiana was much weaker compared to other species of natural enemy, particularly parasitoids, which if extrapolated to other insect species may indicate that resistance to fungal biopesticides is unlikely to evolve (Kraaijeveld & Godfray, 2008). The development of resistance to Bt is a very real concern and a number of authors have reported on studies that use combined applications of Bt and other microbials against resistant pest genotypes. For example, Jung & Kim (2007) investigated using X. nematophila (the bacterial symbiotic mutualist of the entomopathogenic nematode Steinernema carpocapsae) in combination with microbial Bt against Bt-resistant diamondback moth larvae, Plutella xylostella. A co-application of X. nematophila and microbial Bt resulted in greater mortality than when X. nematophila was fed to larvae on its own. In addition, X. nematophila cells were only recovered from larval haemocoel when used in combination with microbial Bt, suggesting that Bt facilitated entry for X. nematophila by causing damage to gut epithelial cells. Raymond et al. (2006) used the isobologram method to analyse the interaction between an NPV and Bt Cry1Ac toxin co-applied against Cry1Ac-resistant and susceptible populations of P. xylostella. There was no evidence for an interaction of the virus and Cry1Ac used against the resistant insects, but in contrast there was an antagonistic interaction against susceptible insects at the Cry1Ac LC25. The authors hypothesised that low doses of Cry1Ac causing limited mid-gut cell death may have inhibited infection by the virus. Artificial selection experiments with different P. xylostella populations showed that selection for resistance to Cry1Ac had no effect on the susceptibility of larvae to the NPV (i.e. there was no cross resistance). Subsequent research based on further laboratory selection experiments and simulation modelling showed that NPV increased the fitness cost of Bt resistance in P. xylostella and confirmed that spray mixtures of Bt and NPV had potential to slow the evolution of Bt resistance, although the effectiveness of the strategy depended on precisely controlling dose applications and / or the presence of a spray-free refuge (Raymond et al., 2007). Rotating applications of Bt and NPV was found to be a better option for slowing the evolution of resistance as simulations indicated it worked over a wider range of environmental conditions and was more cost effective (Raymond et al., ibid.). Elsewhere, entomopathogenic nematodes were found to increase the fitness cost of resistance to Cry1Ac in pink bollworm Pectinophora gossypiella, with simulation modelling indicating that application of nematodes would slow the evolution of resistance to Bt Cry1Ac GM crops (Gassmann et al., 2006; Gassmann et al., 2008).

Singh et al. (2007) have proposed an alternative approach to resistance management by combining Bt with the main active ingredient of neem, azadirachtin. They identified and then combined sub-lethal concentrations of both components in bioassays using third instar larvae of the cotton bollworm (Helicoverpa armigera). These combinations were found to increase the speed of kill and to result in 100% mortality. The efficacy of these combinations may in part be explained by azadirachtin disrupting the insect gut wall, which aids the binding of Bt toxins to the midgut. It is suggested that this approach has the benefit of reducing the cost of pest control while at the same time reducing the probability of resistance development.

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Interaction between microbial biopesticides and potentiating chemicals A potentiator is a compound that is not pesticidal but which causes in increase in pest mortality when used with a pesticidal agent. In theory, there are many different stages in the life cycle of an entomopathogen that could be made more effective using a potentiator: attachment or ingestion of the pathogen, invasion, infection establishment, growth and proliferation, reproduction, and manipulation of host behaviour to enhance pathogen spread. The literature suggest that there are good prospects for developing potentiators but a more systematic research approach needs to be adopted.

Some compounds are known to enhance the effect of entomopathogens by altering insect behaviour. For example, application of the alarm pheromone E β farnesene increased the movement of aphids on leaf discs of pepper in a laboratory bioassay, which caused them to pick up more spores of the entomopathogenic fungus Lecanicillium longisporum (= Verticillium lecanii) leading to an increase in fungus induced mortality (Roditakis et al., 2000). Elsewhere, a phagostimulant based on flour and ground maize cob applied with multiple nucleopolyhedrovirus (SfMNPV) increased the virus induced mortality of fall armyworm Spodoptera frugiperda in field experiments by causing the insect to ingest more virus particles from foliage (Castillejos et al., 2002).

Stilbene optical brighteners are known to potentiate the pathogenicity of NPVs to lepidopteran larvae (Thorpe et al., 1999; Ibargutxi et al., 2008). It is hypothesised that the optical brightener causes degradation of the insect peritrophic membrane and thereby facilitates entry of virus into midgut cells. However the effect may vary with insect species (Shapiro & Farrar, 2003). Optical brighteners can also act as protectants for virus particles from u.v. radiation damage (Shapiro & Farrar, 2003) so this needs to be taken into account when studying their role as potentiators in the field.

Other studies have used sublethal doses of an insecticide as a potentiator. For example Cisneros et al., (2000) reported that a sub lethal dose of boric acid combined with SfMNPV in a phagostimulant of maize flour granules reduced the median lethal concentration of the virus in a laboratory bioassay against S. frugiperda larvae. This effect was also found in the field. The largest number of studies in this area has been done using sublethal doses of imidacloprid combined with entomopathogenic fungi (e.g. Broderick et al., 2000; Jaramillo et al., 2005; Brito et al., 2008). Ye et al. (2005) modelled the mortality of chrysanthemum aphid, Macrosiphoniella sanborni, in response to time and dose of B. bassiana applied alone or with sublethal concentrations of imidacloprid in laboratory experiments. These experiments provided very strong evidence of a potentiating effect of imidacloprid on fungal virulence. The first evidence of an effect of imidacloprid on fungal pathogenicity was published eight years previously when Quintela and McCoy (1997) demonstrated that 100 ppm of imidacloprid resulted in an increase in the mortality of the root weevil, Diaprepes abbreviates treated with B. bassiana and M. anisopliae. However fungus mortality was reduced when imidacloprid was applied at 1000 ppm. In later experiments it was observed that imidacloprid increased the number of spores adhering to D. abbreviates cuticle, however at higher insecticide doses the attachment of spores was reduced, which was attributed to effects of the inert insecticide carrier (Quintela and McCoy, 1998a). It was also observed that B. bassiana and M. anisopliae on their own had no effect on larval movement but that imidacloprid impaired larval movement and this effect was increased when the insecticide was applied in combination with the fungi (Quintela and McCoy, 1998b). Similarly, a combined application of a sublethal dose of imidacloprid and B. bassiana caused an increase in the mortality of leaf-cutting ants Atta sexdens compared to the fungus on its own, and it was found that the insecticide reduced the movement of ants at this dose (Santos et al., 2007) (see above). In contrast, Roditakis et al. (2000) observed that imidacloprid applied at 1% of the recommended dose caused an increase in the movement of aphids leading to enhanced secondary pick up of spores of L. longisporum (see above). Furlong & Groden (2001) reported that applying sublethal concentrations of imidacloprid together with spores of B. bassiana resulted in increased mortality of larvae of Colorado potato beetle Leptinotarsa decemlineata in laboratory tests. This occurred when imidacloprid was applied at the same time as the fungus or when it was applied 24h before the fungus, but not when the insecticide was applied 24h after the fungus. It was found that the imidacloprid inhibited larval feeding and it was proposed therefore that starvation-induced stress factors made the larvae more susceptible to the fungus. In this case, the possibility that fungal infection made the insect susceptible to normally sublethal concentrations of imidacloprid can be ruled out as there was no increase in mortality when the insecticide was applied after the fungus. This would need to be investigated in any future studies of using sublethal doses of insecticide combined with a microbial. There is also the issue of whether routinely using a sublethal dose of insecticide in a combined treatment would increase the chances of the pest developing insecticide resistance. As far as we know this has not been investigated in detail. Finally, Shah et al. (2007) recovered more M. anisopliae conidia from black vine weevil (Otiorhynchus sulcatus) larvae when the entomopathogen was applied in combination with sub-lethal doses of either

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imidacloprid or fipronil. However, here the efficacy of M. anisopliae on its own was so high that the addition of either insecticide did not significantly improve efficacy.

Looking ahead, there is considerable scope to develop new potentiators based on improved understanding of insect–pathogen molecular interactions. An indication as to how this can work comes from a groundbreaking study of termites by Bulmer et al. (2009) at Northeastern University Boston and MIT. These authors investigated gram-negative binding proteins (GNBPs) which are receptor molecules that signal the presence of pathogen infection and initiate a cascade reaction resulting in the synthesis of antimicrobial peptides. One particular termite receptor, tGNBP-2, was found to have ß (1,3) glucanase activity (ß (1,3) glucan is a component of fungal cell walls). Termites secrete this receptor and “paint” it in their nests, where it is involved in the degradation of the cell walls of invading fungal pathogens and primes termites to turn on antimicrobial defences in their immune system when a pathogen is sensed in the termite colony. Using a molecular engineering approach, a simple, inexpensive and nontoxic sugar molecule was designed that blocked the active site of tGNBP-2. When termites were treated with the sugar they became significantly more susceptible to M. anisopliae as a result of their antimicrobial immunity being suppressed. While the immune systems of insects remain poorly understood, they are relatively simple and hence it would be possible to characterise the pattern recognition receptors of a range of pest species and investigate the possibility of designing inhibitors that would potentiate entomopathogens. Interactions between microbial biopesticides One of the criticisms of microbial biopesticides is that their speed of kill and overall efficacy are usually less than that of many chemical pesticides. Hence it is no surprise that researchers have looked to improve pest management with microbial biopesticides by combining different microbial products in an IPM approach. Generally the main intention is to identify synergistic interactions between microbials that give greater pest mortality, faster speed of kill or which enable a reduction in application rates in order to save money. Most of the work has been done in the form of laboratory bioassays although greenhouse and field scale experiments have also been done. The majority of experiments consisted of applying two different entomopathogens to the test insect as a co-infection (i.e. a simultaneous treatment). Only a small number of investigations have examined the effects of applying microbial biopesticides sequentially. Unfortunately, many of the papers in this area provide insufficient data for rigorous statistical analysis of interactions. Common failings include inadequate controls and too few pathogen doses. Many of the papers compare the observed mortality from the co-infection against an expected mortality calculated from the fractional product method or a simple sum of mortalities of the individual pathogens. This can result in inappropriate conclusions because it does not take account of the dose effect. Moreover, very few of the papers identified in our literature search based their experiments on hypotheses derived from theoretical knowledge of pathogen ecology. An exception is Staves and Knell (2010), who investigated the relationship between pathogen virulence and reproduction during intraspecific competition between different strains of M. anisopliae within larvae of the waxmoth, Galleria mellonella and interspecific competition between M. anisopliae and S. feltiae. While most theoretical models predict that virulent pathogens will be more competitive in mixed infections, Staves and Knell‟s (2010) data suggest that competitive ability varies depending on whether competition is intra- or interspecific: Thus in mixed infections involving different strains of M. anisopliae, virulent strains were better competitors, whereas less virulent M. anisopliae strains were more competitive in mixed infection with S. feltiae. It was suggested that the different outcomes may depend on the type of competition, either direct competition (via antimicrobial metabolites) or indirect competition (i.e. scramble competition for resources).

Ansari et al. (2004) observed that a co-infection of M. anisopliae and Heterorhabditis megidis in the scarab Hoplia philanthus resulted in a reduction in the production of progeny nematodes. Reducing the amount of within-host reproduction will also reduce the degree of self-perpetuating control by a biopesticide, so from this perspective it is not a good strategy unless the combination results in a very strong synergistic effect. As we outlined previously, the outcome of a co-infection in terms of the survivorship of the host insect population is difficult to pre-judge and is not well understood. Intuitively we would expect a reduction in the rate of mortality in a mixed infection if competition results in a reduction in pathogen growth, simply because host death is a by-product of the development of the pathogen in the host. However, the true result is likely to depend on the nature of the competition: if co-infecting pathogens produce bioactive metabolites that have synergistic effects then the result could be faster kill of the host. Tabashnik (1992) and Fernandez-Luna et al. (2010) have both demonstrated that the cytolytic protein (Cyt toxin) produced by Bt subsp. israelensis interacts synergistically with Bti Cry toxins. Intuitively, we would also expect that co-infecting pathogens with similar modes of action are

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likely to compete more strongly than pathogens that have different modes of action. For example, Liu et al. (2006) observed that co-infection of H. armigera with HaNPV and Bt Cry1Ac resulted in a lower than expected amount of insect mortality. The authors speculated that this was because NPV and Cry1Ac toxin both act on midgut epithelial cells and hence are likely to be competing for the same resource. Alternatively, the authors noted that Bt can rapidly inhibit insect feeding and this would prevent host insects from consuming sufficient virus to initiate an infection. Mnyone et al. (2009) showed that combining M. anisopliae and B. bassiana in different proportions had no effect on the survival of the malaria mosquito Anopheles gambiae, i.e. there was an additive effect. Wraight and Ramos (2005) investigated the effect of combining B. bassiana and microbial Bt against field populations of Colorado potato beetle Leptinotarsa decemlineata (CPB). This study used a well thought out rationale that should serve as a model for other research. There is a requirement for alternative control agents for CPB, which has a high level of resistance to most chemical pesticides. It was known before the study that B. bassiana was very infectious to Colorado beetle larvae in laboratory experiments but gave slow and inadequate control in the field. Microbial Bt could give some control of CPB in the field but it was considered to be more expensive to use than chemical pesticides. Hence strategies were sought that could bring down the cost of using Bt. Research by other investigators had shown previously that CPB exhibited retarded development when treated with sublethal doses of Bt. It was reasoned therefore that Bt would prolong the interval between moults for CPB larvae and thereby enhance the activity of B. bassiana (i.e. more time would be given for the fungus to penetrate the insect cuticle before being lost though moulting). Alternatively, starvation induced by Bt could affect the susceptibility of CPB to B. bassiana, as demonstrated by Furlong and Groden (2001) with CPB treated with sublethal doses of imidacloprid prior to exposure to B. bassiana. Microbial Bt and B. bassiana were used as commercial biopesticide products and applied as a tank mix to field plots, with two spray applications made in total, one week apart. The B. bassiana product gave zero or very low-level control of CPB in the field while microbial Bt gave between 40 – 50% control depending on dose. However when B. bassiana was combined with microbial Bt the level for CPB control increased to between 80 – 85%. Combination treatments currently being used by farmers, growers and IPM practitioners in the UK (Objective 4) Discussions with IPM practitioners (consultants and growers) who have experience of using combinations of biopesticides or biopesticides with pesticides on commercial crops confirmed that only anecdotal evidence is available to them on the efficacy of these combinations. Observations made on commercial nurseries include:

A tank mix of L. muscarium (Mycotal) and teflubenzuron (Nemolt) has been considered to give improved control of glasshouse whitefly, Trialeurodes vaporariorum. The suggested mechanism is that as teflubenzuron is a chitin inhibitor, preventing the immature insect developing a new exoskeleton during the moulting process, this might benefit fungal infection by allowing easier fungal access to the insect‟s body.

A tank mix of B. bassiana (Naturalis-L) and abamectin (Dynamec) has been considered to give improved control of two-spotted spider mite, T. urticae. The suggested mechanism is that the biopesticide and pesticide are effective against different life stages of the pest, thus using a tank mix gives improved control. Abamectin is effective against the adult and immature mites, whereas B. bassiana is ovicidal to spider mite eggs, both reducing percentage hatch and reducing female mite fecundity.

A tank mix of B. bassiana (Naturalis-L) and spinosad (Conserve) has been considered to give good control of sciarid fly (Bradysia spp.) and shore fly (Scatella spp.) in glasshouses used for ornamental bedding plant propagation. Although neither product is recommended for control of these pests, B. bassiana is known to have activity against flies including shore fly (Stanghellini & El-Hanalawi 2005; Castrillo et al., 2008) and spinosad (Tracer) is recommended for control of cabbage root fly in horticultural brassicas. The suggested mechanism is that the two products have an additive effect when tank-mixed.

A tank mix of pyrethrum (Pyrethrum 5 EC) and B. bassiana (Naturalis-L) has been considered to give improved control of sciarid and shore flies. The suggested mechanism is that the two products have different modes of action with different persistences and are active against different life stages of the pests. The short-persistence pyrethrum is reported to give quick „knock-down‟ of adult flies whereas the B. bassiana, although applied as a foliar spray, is considered to persist on the surface of the compost for a longer period, giving longer-term control of the ground-dwelling larvae. A

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similar effect against the same pests is claimed to have been achieved by using a tank mix of pyrethrum with entomopathogenic nematodes.

Using a tank mix of two biopesticides, B. bassiana (Naturalis-L) and entomopathogenic nematodes has been considered to give improved control of the ground-dwelling life stages of thrips, sciarid and shore flies in protected herb production. The suggested mechanism is that the two products have different modes of action and thus the effect may be additive.

Using biopesticides in combination with chemical pesticides in a sequential programme, rather than as a tank mix, is used by many growers. This could be to target the same or different pests infesting the crop, using products with different modes of action. One reason for using such a programme is that the number of applications per crop of some pesticides is restricted, to meet resistance management guidelines. For example, neonicotinoid applications are restricted to two per crop. Thus for example, in poinsettia crops, B. bassiana (Naturalis-L) might be used early in the production stages for control of whitefly, when it is easier to make contact with the pest underneath the leaves, and one or two neonicotinoid applications might be used in later production stages, when a product with systemic action gives better control due to the denser crop canopy. Thus, using a biopesticide in a control programme can help to control pests on long-term crops and can prolong the effective life of a pesticide by offering an alternative mode of action and by reducing the resistance pressure.

Tank mixes of fungicides with entomopathogenic fungi need to take account of any potential side effects on the beneficial fungi.

It is striking that growers are using these combinations based on anecdotal evidence alone. While this “learning by doing” approach can be successful, it is clear that an evidence based approach is needed, using rigorously designed experiments. Conclusions and recommendations It is certainly the case that progress to develop combination treatments of microbial biopesticides and chemical pesticides has been impeded by some inadequate experimental practices. However, there are also enough high quality publications in the literature to indicate that combination treatments have potential to make pest management more effective. Three areas stand out:

(i) microbial biopesticides have potential to slow down the development of resistance to chemical pesticides in arthropod pest populations:

(ii) microbials have potential to reduce the expression of pesticide resistance once it has evolved; (iii) potentiators can significantly improve the effectiveness of microbial biopesticides.

Further work in these areas is required. We make the following recommendations for future studies:

A key priority is to establish whether combinations of entomopathogens, or entomopathogens plus chemical pesticides, interact synergistically, antagonistically or give additive effects for selected pests of importance to UK agriculture and horticulture. This needs to be done using well designed and analysed laboratory experiments. As a general point there is a need for a more systematic, standardised approach to studies of potential synergism / antagonism.

The potential for microbial biopesticides to delay the onset of resistance evolution to chemical pesticides, or to reduce the expression of pesticide resistance in pest genotypes, could be a very significant way of extending the availability of chemical pesticides. More research in this area is clearly warranted.

There are some exciting opportunities to develop potentiators for microbial biopesticides by exploiting new knowledge on arthropod pest molecular biology, in particular by better understanding of arthropod defences against pathogens using –omics technologies and molecular engineering.

Most of the work to date has been done at the laboratory scale, with less research at the glasshouse or field scale. There is a need for more glasshouse and field-based research. In particular, we need to investigate – as a starting point – the combinations of treatments currently being used by growers against some “model” UK pests exhibiting pesticide resistance, and for which there is anecdotal evidence of a beneficial effect of using a combination treatment. Rigorously designed experiments could then be done to provide the evidence base for the success or otherwise of these treatments. This would include research on the effects of applying different microbial biopesticides at different times in the growing season, on using combinations of microbials with different tolerances of environmental conditions, and on making better use of the ability of entomopathogenic nematodes and fungi to persist within the environment, particularly in the soil.

However, it is essential that glasshouse and field scale experiments as described above are underpinned by knowledge on mechanisms. Not enough information is available about the

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ecological, physiological, biochemical and molecular mechanisms of interaction between microbial biopesticides and between microbials and chemical pesticides / other agents. This is a major impediment to the development of combination treatments.

References to published material

9. This section should be used to record links (hypertext links where possible) or references to other published material generated by, or relating to this project.

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Ansari, M. A., Shah, F. A., Whittaker, M., Prasad, M. & Butt, T. M. (2007) Control of western flower thrips (Frankliniella occidentalis) pupae with Metarhizium anisopliae in peat alternative growing media. Biological Control 40, 293-297.

Ansari, M.A., Shah, F.A., and Butt, T.M. (2008a). Combined use of entomopathogenic nematodes and Metarhizium anisopliae as a new approach for black vine weevil, Otiorhynchus sulcatus, control. Entomologia Experimentalis et Applicata 129, 340-347.

Ansari, M.A., Shah, F.A., and Butt, T.M. (2008b). Combined use of entomopathogenic nematodes and Metarhizium anisopliae as a new approach for black vine weevil, Otiorhynchus sulcatus, control. Entomologia Experimentalis et Applicata 129, 340-347.

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Castillejos, V., Trujillo, J., Ortega, L.D., Santizo, J.A., Cisneros, J., Penagos, D.I., Valle, J., and Williams, T. (2002). Granular phagostimulant nucleopolyhedrovirus formulations for control of Spodoptera frugiperda in maize. Biological Control 24, 300-310.

Castrillo, L.A., Ugine, T.A., Filotas, M.J., Sanderson, J.P.; Vandenberg, J.D. & Wraight, S.P. (2008). Molecular characterization and comparative virulence of Beauveria bassiana isolates (Ascomycota: Hypocreales) associated with the greenhouse shore fly, Scatella tenuicosta (Diptera: Ephydridae). Biological Control 45, 154-162.

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Chandler et al., 2011 Chandler, D, Bailey, A.S., Tatchell, G. M., Davidson, G., Greaves, J. & Grant, W. P. (2011). The development, regulation and use of biopesticides for Integrated Pest Management. Philosophical Transactions of the Royal Society B 366, 1987 - 1998.

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Cisneros, J., Perez, J.A., Penagos, D.I., Ruiz, J., Goulson, D., Caballero, P., Cave, R.D., and Williams, T. (2002). Formulation of a nucleopolyhedrovirus with boric acid for control of Spodoptera frugiperda (Lepidoptera : Noctuidae) in maize. Biological Control 23, 87-95.

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sweetpotato whitefly, Bemisia tabaci. Mycopathologia 160, 35-41. Cuthbertson, A.G.S., Blackburn, L.F., Northing, P., Luo, W.Q., Cannon, R.J.C., and Walters,

K.F.A. (2008a). Further compatibility tests of the entomopathogenic fungus Lecanicillium muscarium with conventional insecticide products for control of sweetpotato whitefly, Bemisia tabaci on poinsettia plants. Insect Science 15, 355-360.

Cuthbertson, A.G.S., Mathers, J.J., Northing, P., Prickett, A.J., and Walters, K.F.A. (2008b). The integrated use of chemical insecticides and the entomopathogenic nematode, Steinernema carpocapsae (Nematoda : Steinernematidae), for the control of sweetpotato whitefly, Bemisia tabaci (Hemiptera : Aleyrodidae). Insect Science 15, 447-453.

Cuthbertson, A.G.S., Blackburn, L.F., Northing, P., Luo, W., Cannon, R.J.C., and Walters, K.F.A. (2010). Chemical compatibility testing of the entomopathogenic fungus Lecanicillium muscarium to control Bemisia tabaci in glasshouse environment. International Journal of Environmental Science and Technology 7, 405-409.

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Er, M.K., and Gokce, A. (2004). Effects of selected pesticides used against glasshouse tomato pests on colony growth and conidial germination of Paecilomyces fumosoroseus. Biological Control 31, 398-404.

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