Info

+ to +++++: Increasing amount of attention needed during formulation. - : Not relevant.

+ to +++++: Increasing amount of attention needed during formulation. - : Not relevant.

M. anisopliae, B. bassiana and P. farinosus (Hallsworth and Magan 1994) and T. harzianum (Agosin et al. 1997). Virulence can be influenced by the nutrients in the production as was demonstrated in M. anisopliae by Shah et al. (2005) and by repeated sub-culturing in vitro (Nahar et al. 2008). Degeneration of entomogenous fungi on nutrient rich media has been described for many species (Butt et al. 2006) and needs to be monitored in commercial productions. The lipid content in dauer juveniles (DJs) can be influenced by the production medium and eventually this may have an effect on shelf-life and infectivity (Patel et al. 1997). These examples demonstrate that formulation is not merely adding components to the propagules once they have been collected from the production. Ultimately, formulation considerations start from the beginning of the production process and formulation features can be influenced by medium components and culture conditions.

Formulation can also be part of the downstream process. Protectants and other compounds may be added before or during drying. This depends on the method used and on the type of formulation. For instance, when granules are made by spray-drying, protectants may be added in the drying process, as well as formulants with other functions that play a role in spraying characteristics or in enhancing persistence. Standardization must be realized in formulation. Each product batch must contain similar numbers of viable propagules within a narrow range. This often requires counting, germination tests and/or bio-assays whose results are used to set the standard content or potency of the active ingredient. A difficult issue in setting the standard is whether over-formulation can be done to compensate for loss of viable propagules over time. An example: if a shelf-life of 1 year is aimed for, but survival of propagules is only 80%, over-formulation can be done to 125% so that after 1 year 100% is still viable, which is in absolute numbers the required amount. Registration generally demands stability over the shelf-life period within a narrow range, but the possibility of over-formulation is not well laid down in the regulations. Death of 20% of the propagules is likely to be accompanied by a quality decrease in the still viable ones. This is an aspect that has hardly been studied in pathogens. Apart from the quality concerns, costs also play a role. Generally, 10% over-formulation seems acceptable, but there is no information available on what companies actually do in this respect.

The formulation needs to be physically stable, i.e. no clump-forming in wettable powders or irreversible sedimentation of propagules in suspensions should occur, nor separation of carriers in the case of emulsions. Besides protecting the active ingredients from degradation, formulation also serves to inhibit growth of possible contaminants. Other unwanted microorganisms may be present in low numbers and need to be killed or limited in their presence and growth as much as possible. Not only could they harm the active ingredient during storage, they could interfere with the biological activity once applied, or pose a risk to the applicator and consumer. Also registration requirements reply to product stability with regard to physical-chemical stability as well as to biological stability.

3.1.2 Second Function of Formulation: Efficacy and User-Friendliness

The second function of the formulation is related to its use in the field, where the formulation should enhance the desired effect on the target pest. Pests can acquire pathogens by direct contact and by secondary pick-up from the spray deposit via contact or per oral. Another possible route is pick-up after a primary growth cycle on the leaf or secondary growth after cycling through a dead insect. The latter is not influenced by formulation. A primary consideration is the spray characteristics in case of a foliar application that is aimed at an optimal coverage of the target pest and/or the target's host plant. Next, good contact with the target insect should be aimed for, as well as enhancement of the activity of the organism such as germination and growth on the insect and on the foliage. Stimulants could also be added to the formulation to enhance oral uptake or to modify the insect's behaviour through which more propagules may be picked up. In the case of a soil application, an even distribution matters more than spray characteristics. Spray characteristics are a well-studied area in chemical products, but this is an almost neglected area in biopesticides. Droplet size spectrum and droplet behaviour during spraying (evaporation and drift) and when droplets hit the plant surface (bouncing off, spreading and drying) can be manipulated by the formulation and the spraying equipment. Propagules should be easily dissolvable and well dispersed in the spray solution and be evenly distributed over the droplets, and to the plants and targets. These spray characteristics can be influenced by additives such as surfactants, spreaders, stickers, humectants, etc. The list of compounds for these functions is huge. Finding the right additives requires an extensive range of investigations on the target and target crops. Few papers refer to these parameters as part of a biopesticide formulation, but they are a relevant part of successful targeting the pest. Bateman and Alves (2000, Fig. 1) illustrated where spray application effects can be manipulated by the formulation of biopesticides. Co-formulants can influence propagule distribution in the spray droplet spectrum, evaporation of the spray solution during atomization, and effects on the deposit on the leaves. The latter can be retention, spreading, drying, survival and availability of active ingredients for the target insect. Particularly persistence, adherence, protection, propagule growth and feeding stimulants are important for mycoinsecticides (see Fig. 3.2). Evans (1999) reviewed the principles of dose acquisition and transfer of pathogens. He emphasized the connection between the biology of the microbial agent and the biology of the target, as well as the delivery system. Bateman and Alves (2000) studied the effects of equipment type in relation to the numbers of spores per droplet and the impact on delivery to the target. Their work underlines the role of these factors for biopesticides. A formulation should be appropriate for a variety of crops and field conditions.

Other aspects are convenience of use and compatibility with the end-users' equipment and practices. The formulation should be an acceptable quantity with regard to logistics, storage, handling, and to measuring the desired quantity for an application. An unmanageable quantity that needs cold storage is a big disadvantage that will

Spraying

Spraying

Plant surface effects

Environmental effects

Death of propagules over time

Fig. 3.2 Spray application, deposit and pick-up processes of the active ingredient. The shaded box represents effects which can be manipulated by the formulation and which are particularly important for mycoinsecticides. The dark box represents effects only relevant in mycoinsecticides (modified after Bateman and Alves 2000)

Plant surface effects

Environmental effects

Death of propagules over time

Fig. 3.2 Spray application, deposit and pick-up processes of the active ingredient. The shaded box represents effects which can be manipulated by the formulation and which are particularly important for mycoinsecticides. The dark box represents effects only relevant in mycoinsecticides (modified after Bateman and Alves 2000)

cause failure of the product in the market. An example from my own experience in the mid 1980s is R350, a fungal nematicide based on Arthrobotrys irregularis: it needed cold storage and the rate of application was 1,000 kg/ha. Further, the formulation consisted of mycelium-overgrown cereal kernels that formed one big mass that needed to be separated manually into small pieces that could be spread over the soil. Besides efficacy problems and costs, the difficulties with the formulation and with logistics caused this product to fail in the early days of biopesticides.

3.1.3 Third Function of Formulation: Protection and Persistence

The third function of formulation is to protect the propagule from harmful environmental effects and increase its persistence on the target site. Protection is needed against desiccation, high temperatures, de-activation by sunlight, and washing off by irrigation or rain, particularly when used outside the greenhouse. Good persistence increases the chance of secondary pick-up and pick-up after a growth cycle of the pathogen on the leaf. The addition of growth stimulants to the formulation may give rise to the formation of new propagules which increase the chance of secondary pick-up. This is particularly useful with fungi as illustrated by the following example. Additives in the formulation of Mycotal allow the fungus L. muscarium to grow on the leaf surface and form new conidia. These conidia are formed in clusters and covered with a sticky slime that prevents desiccation and improves adherence to insects. This slime dissolves in water and as a result is not present anymore on spores that have been sprayed onto the plant. Newly formed spore clusters are produced in slime heads and enhance secondary pick-up by passing insects such as thrips (Hall 1980).

3.1.4 Fourth Function of Formulation: Registration and Safety

The fourth function of formulation relates to registration demands and concerns possible harmful effects of the formulation on, especially, the applicator. In the toxicological risk assessment of the product, exposure to the microorganisms is a concern during loading, mixing and spraying. Especially dusty formulations may pose a hazard to the person measuring the needed amount and during pouring this into the spray liquid. Therefore, the trend in agrochemicals is to change from powder formulations to liquids and granules. Most biopesticides are formulated as a powder, and small particles as spores can easily can be inhaled and thus pose a possible risk. This may cause sensitization by or allergenicity to the microorganism. Liquid formulations are difficult to make with microorganisms, because water activity has to be low to keep the propagules inactive and to prevent contaminants from developing. However, currently an increasing number of oil suspension formulations is being developed, which minimize inhalation risks during loading and mixing. The aspect of microbial impurities is also a concern in registration and needs attention during formulation.

3.1.5 Formulation in Academic Research

Many authors have recognized the importance of a good formulation (Couch and Ignoffo 1981; Rhodes 1990; Guillon 1993; Hofstein and Fridlender 1994; Jones et al. 1997; Jones and Burges 1997; Couch 2000; Boyetchko et al. 1999; Brar et al. 2006; Jackson et al. 2010) and many aspects of formulation have been studied by scientists and by the biocontrol industry. A comprehensive overview of formulation of microbial pesticides is given by Burges (1998). This book covers formulation of beneficial microorganisms and EPNs, including usages as insecticides, herbicides, fungicides and seed treatments. It discusses the principles of formulation and specific features per group of microorganisms. This review is very helpful for any product developer. Jones and Burges (1998) recognized four basic functions of formulations; Couch (2000) listed six criteria for Bt's that need to be satisfied for commercial formulations. All criteria are similar to the first three described above. These authors, however, do not mention the aspects related to registration, but these are surely relevant and need to be considered seriously. For an overview of the different types of formulations, such as dry products (wettable powder, granules, etc.) and liquid suspensions (water or oil-based, emulsions, etc.), I refer to Jones and Burges (1998). They also included the advantages and disadvantages of equipment that can be used for various specific application methods for each group of ento-mopathogens. A more recent overview is given by Lisansky (CPL 2006) with a specific focus on commercial formulations. He also provided a number of tables with the advantages and disadvantages of wettable powders, flowable concentrates, granules and high-potency formulations.

In academic research, formulation has received much attention. Often, investigations have focused on a specific aspect of the formulation. Rarely are all aspects covered. In Burges (1998) the state of the art for the mid 1990s is presented for the insect pathogens covered in this book. I will review developments since then per type of microorganism and relevant to bio-insecticides for greenhouse applications, particularly for foliar applications. The details of formulated commercial products, however, are rarely published or patented and in most cases details are kept as trade secrets. I will also discuss possibilities to improve efficacy of biopesticides by using adjuvants, by other enhancing agents, and new developments in formulation.

3.1.6 Formulation of Entomopathogenic Bacteria

Standardization, spray characteristics and protection from environmental factors must be considered in formulations of bacteria. The formulation of bacteria seems to be the least demanding when the four pathogen types are compared, at least when it concerns bacterial spores and proteineous substances, as in Bacillus species. Spores formed by these bacteria are long-lived and resistant to heat and desiccation. There is a big difference between these dormant stages versus metabolically active stages. For bacterial cells as in the case of Serratia spp. and Pseudomonas spp., formulation is much more complicated. As only Bt's are used in greenhouses, these will be considered here. The good stability and easy handling of Bt formulations surely are part of their success. Shelf-life is generally 2 years at room temperature. Points of attention here are the concentration of the spores, the drying technology, and the determination of the potency standard (Skovmand et al. 2000). Most formulations are wettable powders or water dispersible granules, some are oil-based or water-based or mixtures thereof; detailed information is given by Burges and Jones (1998a) and Couch (2000). Every conceivable type of formulation has been developed with Bt. Brar et al. (2006) review recent advances in formulations of Bt's. They summarize screening factors for which additives need to be chosen and they give a list of different types of additives and their function that are used in microbial formulations. Formulation requirements and formulation types are different depending on the usage of a Bt product, in agriculture, forestry or in aquatic habitats.

Advances in formulation technology like encapsulation and controlled release formulations offer possibilities to improve efficacy. Brar et al. (2006) also review possibilities to better protect Bt's from harmful factors such as sunlight, rainfall, dew, extremes in temperature, pH and foliage effects. They recommend studying the whole process from fermentation to formulation since many factors are interconnected. The large Bt producing companies have developed successful and convenient formulations, but details of commercial formulations are proprietary. Improvements are always possible and new formulations appear on the market. In most Bt products the potency has been doubled in recent years in order to make the products more cost-effective and to comply with the trend to reduce spray volumes. To increase the effect of Bt's, feeding stimulants have been studied, but satisfactory improvements have not been found and feeding stimulants are generally not included in formulations nor added as tank-mixes (D. Ave, personal communication).

3.1.7 Formulation of Entomopathogenic Fungi

Formulation of fungal propagules is the most challenging among the four types of pathogens discussed in this study. This is related to their mode of action which is primarily by adhesion to the outer parts of an insect and penetration through the cuticle. This infection process is described in detail by Butt (2002). The infection process is complicated compared to bacteria and baculoviruses with which infection takes place after take-up per oral, or compared to the active search and penetration behaviour of EPNs as part of the infection process. Formulation of fungi poses challenges related to targeting, persistence, protection against harmful environmental factors, growth stimulation and enhancing agents. Generally, formulations of fungal products contain spores, either conidiospores or blastospores, sometimes mycelial particles.

The literature on formulation of fungal spores is vast. Burges (1998) reviewed the developments in formulation of mycoinsecticides and provided an excellent overview of the various formulations and additives that stabilize and protect the propagules and enhance the biological activity. Vega compiled a bibliography called "references dealing with formulations of fungal entomopathogens", see the website

http://www.sipweb.org/fungi.htm. There are about 150 references, updated to 1999. Most products are based on aerial conidia, some on conidia and hyphae on a granular substrate, and some on blastospores (De Faria and Wraight 2007). Most entomopathogenic fungi produce large amounts of aerial conidia which are responsible for dispersal and infection. These spores are thick-walled and relatively stable. In solid state production these spores are also formed in high numbers. These characteristics make them the preferred propagule for formulating final products. Some fungi are difficult to produce efficiently on solid media and submerged produced blastospores are used for formulation. Blastospores may also be more infectious as is shown in I. fumosorosea against Bemisia argentifolii (Jackson et al. 1997) and in M. anisopliae against Locusta migratoria (Kleespies and Zimmermann 1994). Wraight et al. (2001) reviewed stabilization and formulation in relation to production of the different kind of propagules of entomopathogenic and herbicidal fungi. Feng et al. (1994) reviewed formulations of B. bassiana based on conidiospores, blastospores and mycelium. Jackson et al. (2010) reviewed the ecological considerations in producing and formulating fungal entomopathogens with emphasis on interactions between the propagule and the insect cuticle, and the role of a surfactant.

Formulation-related aspects can be improved during production and this area has received considerable attention in entomopathogenic fungi. Hallsworth and Magan (1994) demonstrated that inoculum quality (in this case virulence and shelf-life) can be improved by the culture conditions (low water activity) and nutrient composition in conidia of B. bassiana, M. anisopliae and P.farinosus. Jackson et al. (2003) studied blastospore production in I. fumosorosea and showed that blastospore tolerance to desiccation can be positively affected by supplementing certain medium components. Ypsilos and Magan (2004) showed that modifications in physico-chemical culture conditions such as water stress, and washing treatments of blastospores of M. anisopliae have the potential to improve spore quality in terms of drying-resistance and storage stability. Tolerance to desiccation is an integral factor in the process of drying blastospores and for long shelf-life and this should be considered early on in the production. Many features are influenced in the production and this needs to be studied and used in the product development. Formulation additives are another tool to improve the propagules' stability and activity. Tolerance to desiccation can be further improved by formulation additives. Blastospores of I. fumosorosea mixed with starch-oil mixtures improved survival of spores during freeze-drying and improved storage stability (Jackson et al. 2006).

Oil-based formulations have been developed for conidiospores of M. anisopliae var. acridum for locust control. This proved to be a major breakthrough, enabling the usage of this fungus in hot and dry environments with ULV applications (Bateman et al. 1993). This formulation technology has several advantages that made it so successful: shelf-life is quite long for a mycoinsecticide (>18 months at 17°C); good persistence in the field; enhanced biological activity; and usage by conventional equipment at ultra low volumes (Bateman 1997). The precise role of the oil in the infection process is not well understood. Bateman and Alves (2000) suggested that secondary pick-up from plants plays a greater role than direct impaction of spray droplets in the case of locusts. This would mean that persistence of spores was improved by the oil as well as the pick-up and adherence to insects. These aspects relate to efficacy, user-friendliness, and costs, and resemble the use of a chemical, making acceptance of this product easy. This technology was developed in the Lubilosa Programme (www.lubilosa.org) and the product Green Muscle, based on M. anisopliae var. acridum, is formulated in mineral oil.

Oil formulations have been developed with mycoinsecticides (Botanigard ES, Naturalis, Green Muscle) based on mineral and paraffin oils. It is believed that oil formulations increase spore adherence to insects because they improve contact between spores and the hydrophobic cuticle of insects. In oil-based formulations sprayed with water as a carrier, the assumed mode of action is that the mixture once sprayed with a pressure of 10-15 bar forms an emulsion with fine oil droplets. Under high pressure and considerable shear forces an inverted emulsion may be formed with oil droplets that contain water and spores. These oil droplets may enhance adherence to insects once the water has evaporated. In these droplets the spores may germinate and grow out of the droplet and infect an insect. In this way, spore germination and adherence are enhanced, resulting in greater insect mortality and reduced dependency on a high relative humidity and the availability of free water. Where an invert emulsion does not occur, the result might be protection of spores from dehydration under a thin film of oil (Auld 1993) and a better adherence to insects, still resulting in improved efficacy. These effects were seen using a vegetable oil-based adjuvant with Mycotal, which contains hydrophilic conidia of L. muscarium, for control of thrips (Van der Pas et al. 1998). Many other studies indicate that oil formulations improve the efficacy of mycoinsecticides; a review is given by Inglis et al. (2002). On the other hand, Ugine et al. (2004) showed that there was no increased secondary acquisition of conidia of B. bassiana by Frankliniella occidentalis adults and larvae between unformulated conidia and two formulations with conidia. These were the WP and the ES (emulsifiable suspension) formulations of Botanigard. It was tested, however, under optimal conditions for the fungus, at 25°C and a 100% RH. Formulations are developed to enhance product efficacy under sub-optimal conditions, e.g. lower RH conditions. Testing under optimal conditions is not likely to show enhancement of efficacy by a formulation. It also may explain why the oil-based formulation (ES) did not increase conidia acquisition. B. bassiana spores posses a hydrophobic cell wall (Inglis et al. 2002), so any adherence effect of oil is much less relevant, even under optimal conditions for the fungus.

Vegetable oil formulations could also be developed to improve shelf-life of vulnerable fungal material. This has been studied with mycelium of Lagenidium giganteum in water-in-oil emulsions, together with prevention of sedimentation of cells (Vandergheynst et al. 2007). Oil thickeners based on silica nanoparticles proved to be effective in preventing sedimentation to a large extent in the formulation as well as in the mixture with water. The authors assume that shelf-life was improved by protecting propagules from desiccation in the water-in-oil matrix. In biocon-trol of weeds with pathogenic fungi various kind of oil formulations have been tested, including invert emulsions, oil suspension emulsions, and many adjuvants. An overview is given by Green et al. (1998).

Many compounds have been shown to protect spores and to enhance biological activity. This area of research offers interesting ideas that could be useful in developing formulations with entomopathogenic fungi. Other non-agricultural areas where microorganisms are used for pest control should also be considered. Examples are the control of mosquitoes and weeds in aquatic environments, insect control in forestry where specialized formulations, often oil-based, have been developed for low volume aerial applications, and control of termites, ants or cockroaches in domestic usages.

Some formulations consist of the production substrate including the spores. These are usually fungi grown on rice or cereal kernels where the spores are not separated from the production substrate and where fungus-overgrown granules are applied to soil. Examples are Melocont from Kwizda (Austria) and Engerlingspilz form Andermatt Biocontrol AG for control of white grubs in meadows, lawns and orchards. The production matrix is here the final formulation and these products are not formulated with any additives.

A small number of products have been formulated with mycelium. The product Laginex AS (Agraquest, USA) for control of mosquitoes, based on the fungus L. giganteum, is formulated as an aqueous suspension with mycelial parts since production of oospores gives uneconomic yields (Vandergheynst et al. 2007). The product is no longer on the market due to an unstable formulation with a short shelf-life and a limited market (D. Edgecomb, AgraQuest; personal communication). A formulation of M. anisopliae, based on mycelial pellets, was developed by Bayer, but it was never marketed due to economical constraints (Zimmermann 2005). The product Mycar, based on mycelium of H. thompsonii was put on the market, but it was withdrawn because results were insufficient (McCoy et al. 1975). In mycoherbicides mycelial formulations are also used. An example is the fungal herbicide Chontrol Paste, from MycoLogic Inc., Canada, which contains mycelium of Chondrostereum purpureum for inhibition of re-sprouting of tree stumps (www.epa.gov.). A definition of formulation types and an overview of mycoinsecticides and mycoacaricides, including the type of the commercial formulation, are provided by De Faria and Wraight (2007).

3.1.8 Formulation of Baculoviruses

Key aspects in the formulation of baculoviruses are standardization, microbial contamination, protection against harmful environmental factors, improvement of the speed of kill by faster and greater uptake of viruses, as well as enhancement of the pathogenicity of the virus. Shelf-life is of minor importance because products can be stored for many years when kept at approximately -20°C, or 1 year at refrigerated temperatures, and even for a few weeks at room temperature. Products can also be re-stored from one temperature to the other to some extent. Spray characteristics should ensure an even distribution on foliage for optimal oral uptake by caterpillars.

A main issue in virus formulations is microbial contaminants. Purification of the crude technical product is needed for product stability and registration requirements, and various methods can be used (Huber and Miltenburger 1986). On the other hand, it is well known that remainders of the dead insects enhance stability of the virus during storage and that these particles also aid in the protection against UV radiation (Huber 2005). This creates a formulation challenge for producers who will have to find a compromise between these two aspects. At the same time, the formulation needs to be set at certain potency, and counting occlusion bodies (OBs) or polyhedral inclusion bodies (PIBs) is difficult and unreliable. Particles in the formulation can be hard to distinguish from the OBs or PIBs and inactive ones are also counted. Bio-assays are needed to standardize the product.

Many researchers have focused on formulation improvements such as adding feeding stimulants, stickers for rain-fastness, and protectors against UV. It is generally accepted that exposure to sunlight rapidly inactivates baculoviruses. Accordingly, most formulation studies focus on protection from harmful wavelengths. Overviews of formulation methods and research are given by Burges and Jones (1998a) and by Hunter-Fujita et al. (1998); both papers include extensive lists of wetting agents, stickers, phagostimulants and UV protectors. Encouraging results have been obtained in apple using CpGV with various additives against the codling moth (Ballard et al. 2000). The use of these compounds (molasses, sorbitol, a-farnesene) led to less codling moth damage, although the exact mechanism was not clear. Phagostimulants, built into a granular formulation of SfMNPV, increased infection rate in Spodopterafrugiperda in maize and improved persistence on crop foliage compared to an aqueous spray application (Castillejos et al. 2002). Sunscreens to block damaging UV wavelengths have been studied widely (Burges and Jones 1998a). Optical brighteners can give protection against UV radiation and at the same time enhance baculovirus activity (Dougherty et al. 1996). The results, however, are considered insufficiently effective for open field applications. The use of adjuvants based on lignin and particle films were studied as putative solar pro-tectants, but showed no significant improvement in field trials (Arthurs et al. 2008). The situation for feeding stimulants and stickers is similar and the use of these compounds is not recommended in practice (M. Andermatt, personal communication). On the other hand, Lasa et al. (2007) showed that the use of an optical brightener, i.e. a UV blocker, in the formulation increased mortality in greenhouse trials with SeMNPV against larvae of Spodoptera exigua in Spain. Mortality increased in late instars larvae, whereas persistence was not improved by the additive. The effect on mortality was most likely due to an increased rate of virus acquisition, suggesting that the optical brightener leucophor AP increases pathogenicity of this virus and/or acts as a feeding stimulant. The effect as a UV blocker was not seen. Apparently, UV radiation is much less harmful in a plastic-covered greenhouse than in the field.

The formulation ingredients of current baculovirus products are not known as the information is proprietary. Formerly, virus products were often formulated as wettable powders, particularly those used in forestry (Young and Yearian 1986). Oil formulations have been studied, but oils are often repellent or act as an anti-feedant to the target pests (Cherry et al. 1994). Nowadays, most formulations are aqueous suspensions that are developed for spray applications in orchards and greenhouses and are user-friendly.

3.1.9 Formulation of Entomopathogenic Nematodes

Nematodes require formulation for similar reasons as do microorganisms, although they present unique features. Shelf-life and user-friendliness are integral to an optimal nematode formulation. Persistence in the soil cannot be influenced by adding formulation ingredients, and registration is not an issue of great importance. The DJs are relatively large survival stages that are able to move actively. They also present challenges to oxygen and moisture requirements and to temperature conditions during storage, and are more sensitive in this respect than the microorganisms. Generally, nematodes are applied to the soil which poses less formulation considerations then when pathogens are applied onto foliage where spray characteristics and protection from environmental factors are necessary. On the other hand, when nematodes are applied through irrigation systems, blocking of filters and small drippers, either by the DJs itself or formulation lumps, needs to be avoided. DJs can be stored in refrigerated and aerated water to supply sufficient oxygen and to prevent settling. For storage and transport of products, formulation is needed. The main goal is maintenance of quality for as long as possible. In formulation, the stage of DJs of nematodes is used in which they become metabolically inactive and can withstand a relatively high degree of desiccation and low temperatures. Further, the growth of contaminants must be prevented. Species, and even strains, differ in their biology, and storage and formulation needs to be studied case by case (Strauch et al. 2000). Temperature should be kept as low as possible to ensure a long shelf-life. Once temperature allows activity and mobility, DJs use up oxygen and their energy resources, which will negatively influence shelf-life and ultimately quality and efficacy, although it can vary depending on the species (Patel et al. 1997). Once DJs have moved inside the package, the formulation ingredients protect them less efficiently and quality rapidly decreases, even when the temperature is restored to a level where they can not move anymore. In this respect nematodes are much more sensitive than non-moving propagules of pathogens. High temperature can be lethal. The moisture content of the formulation demands a balance between the level that keeps the DJ's from desiccation and the level that prevents them from moving. Moisture content should also be low to prevent contaminants from developing.

Formulations need to be tailored towards the species and the type of formulation, and requires studying the physiological chemistry of a nematode and its ecology and behaviour. A historical overview of formulation with EPNs is given by Georgis and Kaya (1998). The authors also identified significant steps in the process of formulation (Table 9.6, p. 298). A more recent overview of the various types of formulations is presented by Grewal (2002). Many types of commercial formulations have been developed and marketed, including water-dispersible granules. This last formulation was not successful and was taken from the market (Georgis 2002). The dominant and successful formulations are clay powder formulations and gel formulations.

Liquid formulations may have several advantages over solid matrix formulations. The latter have a relatively short shelf-life of about 3 months, are difficult to handle, and the entire package needs to be used up at once because of the non-homogeneous nature of the package contents. High temperature effects are irreversible due to the change of the EPN-matrix mixture when DJs have crawled out of the carrier. On top of that, active nematodes produce heat and gases and contaminants may start to develop, resulting in a negative spiral effect with regard to product quality. Aqueous formulations, however, have the disadvantage of sinking of EPNs and lack of oxygen. Wilson and Ivanova (2004) investigated neutral density liquid formulations based on colloidal silica suspensions which could overcome these problems. Survival and virulence with H. megidis and S. feltiae DJs was improved compared to nematodes stored in aerated water solutions with Ringer's solution at 4°C and at 15°C for S. feltiae. Still they found a decline over time of surviving DJs. This is not acceptable in commercial formulations. The number of DJ/ml was at maximum 10,000 per millilitre which would mean a 5 l volume for a 50 million nematode package which is generally about 200 ml with clay formulations. Both aspects are not acceptable for a commercial formulation and efforts to improve these points were not sufficient (unpublished data of our group). Although a liquid formulation has advantages such as easy measuring off the desired quantity, less or no visible residue on plants, and no blockage of nozzles, this idea has not been developed into a commercial formulation due the disadvantages mentioned above.

3.1.10 Foliar Applications of Entomopathogenic Nematodes

There is an increasing interest in using EPNs for control of foliar insect pests such as leafminers, thrips and caterpillars. To be successful, DJs need sufficient time to find and penetrate the target insect. They are only able to move in a water layer and desiccation within a short period of time after application limits their effect. Formulations are mainly developed to have a long shelf-life. For enhancement of EPNs' effectiveness on foliage through good mobility and survival, formulation requirements are different. This formulation should aim at maintaining the presence of the water layer long enough for the nematodes to find and penetrate the host, usually a matter of a few hours. This can be reached by tank-mixing an adjuvant or by developing a new formulation.

Piggott et al. (2000) studied the formulation of DJs in a polyacrylamide polymer and its effect on leafminers. This formulation improved survival of DJs on leaves because of slower drying of droplets compared to water and gave a significant increase of pest mortality, compared to DJs in water, and water plus a wetting agent. This formulation is marketed by Becker Underwood as Nemasys F for use against thrips and leafminer. The exact formulation is proprietary and not known, however. Enema and Koppert developed similar gel formulations. These formulations have a similar shelf-life as the clay formulations, and can be used for foliar as well as for soil applications. The gel formulations also have the advantage that there is no visible residue on plants, unlike the clay formulations.

The use of formulation additives to enhance EPNs effectiveness on foliage has been studied by Schroer et al. (2005a) against diamondback moth larvae. Many different compounds such as polymers, surfactants, oils, waxes, and mixtures thereof were tested in combination with S. carpocapsae DJs in bio-assays on cabbage leaf discs. The authors found that a mixture of a surfactant and a polymer gave the best results, suggesting that a good deposition and distribution of DJs over the foliage is important as well as a prolonged DJ survival. In leaf bio-assays Schroer and Ehlers (2005) demonstrated that the effect of the surfactant-polymer formulation is mainly due to support of the DJ's performance on the leaf surface rather then to improving DJ survival. Survival was prolonged, but diamondback moth larvae mortality did not increase over time. The results indicate that insect penetration by the nematode on the leaf occurs within the first hour after application. Field trials under tropical conditions did not show any additional effect of the polymer-surfactant or surfactant formulation. This was attributed to humid, rainy conditions during the trial (Schroer et al. 2005b). The authors conclude that the use of anti-desiccants only slightly improves the effectiveness of EPNs on foliage and they recommend not using them due to higher costs and the small additional effect.

Studies in our group confirm that the most significant factor in foliar use of nema-todes is contact between target and nematode, and that conditions in the first hours are determinative. Prolonged survival by means of anti-desiccants does not seem to be very useful. The above-mentioned gel formulations have an anti-desiccant effect and give reasonable control of foliar pests. The use of a spreading agent may improve spray characteristics and contact between DJ's and target insects. Using a separate adjuvant seems to be the most practical. This is because EPN products generally are used for soil applications and do not need this kind of formulation features, so building in features for foliar applications would be unnecessary. The use of adjuvants with EPNs offers a potential improvement in the use of EPNs against foliage-feedings insect pests and needs to be further studied in field trials focusing on initial contact between nematode and host.

The application method for foliar treatments with nematodes needs careful investigation. As DJs are large particles, the suitability of equipment (pressure, nozzle size), droplet size, survival, distribution and canopy structure are determinative. These aspects have been studied by several authors, including the use of adjuvants, but optimal application methods have not yet been developed (Lello et al. 1996; Shapiro-Ilan et al. 2006).

3.1.11 Entomopathogens and Adjuvants

Efficacy of entomopathogens can be enhanced by certain additives in the formulation or by tank-mixing adjuvants. Additives are defined as compounds incorporated into a formulation and their role has been mentioned several times above. Adjuvants are defined as separate products that can be tank-mixed with (bio)pesticides in order to enhance their efficacy. Adjuvants are increasingly used to improve the biological activity of pesticides. An overview of why, where and what kind of adjuvants are used with chemical pesticides is given by Stock (1997). The main reasons are spray characteristics such as atomization, foliar retention, foliar coverage, foliar penetration and enhancement of the mode of action of the active ingredient. Adjuvants should have no biological activity on their own. This is also a requirement related to regulations of adjuvants. Adjuvants are needed because there are limitations to the amount of additives that can be built-in into one pack-formulations (products) and some additives are not compatible with the active ingredient and can only be tank-mixed. Another reason is that an additive may not be compatible with all the foreseen usages of the product; they could be phytotoxic to some crops, but useful in some other crops. There is a limit to a multi-purpose formulation.

The considerations for developing and using adjuvants with biopesticides are given in Table 3.7. The range of adjuvants is increasing; the adjuvant industry is growing, with their own newsletters (e.g. Adjuvant Newsletter) and conferences (the 9th International Symposium on Adjuvants for Agrochemicals was held in 2010) and specific adjuvant market reports. Specialized consultants are active in this field, such as Alan Knowles (Form-AK, UK) and Hans de Ruiter (Surfaplus, the Netherlands). The market of adjuvants is estimated to be about US$1.5 billion in 2005 (Knowles 2006). In biopesticides, similar considerations for the use of adjuvants play a role and adjuvants may enhance the biological activity of pathogens or improve their persistence on the target site.

Adjuvants are sometimes recommended in combination with biopesticides. An example is Nu-Film-17 (Nufarm) that was tank-mixed with Madex as a UV protec-tant and sticker. Results were disappointing and Nufilm is no longer recommended (M. Andermatt, personal communication). Examples with fungi are AddQ, added to AQ10, a mycofungicide based on Ampelomyces quisqualis to control powdery mildew (Kiss et al. 2004), which improved efficacy. And, NuFilm added to Naturalis (B. bassiana) brought the level of control of whiteflies up to the chemical standard level (Mayoral et al. 2006).

Some adjuvants are specifically developed for usage with biopesticides. Our group developed a vegetable oil-based adjuvant Addit to improve the activity and persistence of Mycotal (Van der Pas et al. 1998). Initially, we attempted to develop an oil formulation with the spores of L. muscarium in order to increase the efficacy and persistence on the foliage. Due to technical problems such as sedimentation and clogging of the spores over time, we abandoned this idea. Another reason was the need to register a new formulation, while launching an adjuvant only needs a limited registration or none at all in some countries. The product Addit is recommended to

Table 3.7 Considerations when using tank-mixed adjuvants with biopesticides

Adjuvant aspect

Features that need to be considered

Formulation functions

Efficacy Costs Quantity User-friendliness Undesired effects

Registration

Improve spray characteristics, enhance mode of action, increase persistence, improve secondary pick-up, etc. Should not have direct efficacy on its own Additional end-user's costs should be acceptable Logistics, storage, handling Easy to mix (dilution, emulsion) and apply

Phytotoxicity, growth of sooty moulds, visible residue; harmful effects on natural enemies, etc. Often needed, sometimes exempt; often easier than a new formulation registration

□ mycosed nymphs

□ total of mycosed and not-mycosed nymphs

□ mycosed nymphs

□ total of mycosed and not-mycosed nymphs

MYCOTAL + ADDIT

□ mycosed larvae

□ total of mycosed and not-mycosed larvae

MYCOTAL + ADDIT

MYCOTAL + ADDIT

Fig. 3.3 Mortality of Mycotal and Mycotal + Addit on whitefly nymphs (Trialeurodes vaporar-iorum) (left graph) and thrips larvae (Frankliniella occidentalis) (right graph) 7 days after 2nd application on cucumber (RH 55-80%) (Van der Pas and Ravensberg, 1995, unpublished data)

be tank-mixed with Mycotal at a dose of 2.5 ml/l of the spray liquid. It increases the mortality of whitefly and particularly of thrips (Fig. 3.3). It shows mycosis earlier and more pronounced than in the control, so that mortality is easier to assess by the grower and most importantly, its activity is less dependent on a high relative humidity. The main effect of the oil is the improved secondary pick-up of spores resulting in a quicker death and a higher mortality overall (Van der Pas et al. 1998).

As a result of the development of Addit, the usage of Mycotal has increased, demonstrating the value of an adjuvant. The addition of arachid oil also improved the efficacy of V. lecanii against powdery mildew on cucumber and reduced the humidity dependence (Verhaar et al. 1999). Curtis et al. (2003) studied the effect of nutrient and non-nutrient additives to conidiospores of an Australian V. lecanii strain (FI40) derived from an aphid. They found that nutrient additives in the spray solution, such as whole egg powder and glycerol, increase growth on leaves and infection in aphids. A period of over 15 h of high humidity was still needed to get high mortality. Oil-based additives did not improve mortality rates. Our group found that an oil adjuvant did not improve the efficacy of Vertalec (blastospores), in contrast to Mycotal (conidiospores). Whether this is related to the spore type, the target insect or to strain differences is yet unclear. With Bt, the use of cotton oil as a tank-mix improved the efficacy in sweet corn on three lepidopteran pest species considerably, although the mechanism is not fully understood (Hazzard et al. 2003). The use of adjuvants for foliar applications of EPNs is discussed above.

The quantity and the cost are the issues in using adjuvants. Often, improvements on efficacy can be realized by tank-mixing an adjuvant, but in many studies the quantity required is prohibitive. A typical example is the study of Ballard et al. (2000). They found that adding 10% (v/v) cane molasses or 10% (w/v) sorbitol or 0.08% a-farnesene to CpGV treatments with 250 l/ha in an apple orchard significantly improved levels of codling moth control. Damage reduction was 41% with pure CpGV and 74% with pure CpGV plus molasses. In this case, 10% molasses is 25 l, costing about C25 (purchase price (in 2007)), without any sales margins. Adding sorbitol gave a significant increase of control, but it would mean using 25 kg/ha, costing about €200 (purchase price, in 2007). Efficacy was also improved by adding a-farnesene in a quantity of 200 ml, but the additional cost is about €55 (purchase price in 2007). One treatment with a CpGV product costs a grower about C60/ha in 2007 in the Netherlands. From a practical point of view, this renders the use of these compounds as adjuvants in terms of logistics and costs impossible. If the price of an adjuvant increases the cost of the overall treatment considerably, a grower chooses another product. An adjuvant must be cheap and easy to use.

Undesired effects of adjuvants need also to be taken into account. Molasses may stimulate growth of sooty moulds. Oils may cause phytotoxic effects. Optical brighteners and other compounds may cause visible residues which can be a problem on ornamentals and on shiny fruits like eggplant and sweet pepper. Besides, harmful effects on natural enemies may occur. All these aspects need to be considered and studied.

The use of biopesticides is often criticized for its lack of reliable efficacy. Adjuvants offer a potential improvement, and more research should be dedicated to investigating the combination of biopesticides with both available and new adjuvants. This is a relatively quick and affordable approach by which efficacy of microbial products can be improved. Adjuvants are regulated in most countries, but dossier requirements for registration are much less than those of biopesticides.

3.1.12 New Formulation Technologies

Formulation technology offers a promising field for improvements in terms of efficacy and user-friendliness. This will improve the uptake of biopesticides in the market. New research, new knowledge and new products open ways to develop more sophisticated formulations. The agrochemical industry is constantly trying to improve their formulations and new developments are taking place that could be interesting for the biocontrol industry. The latest are micro-encapsulated formulations, granular formulations and replacement of harmful solvents agents by vegetable oil or water. Some examples: Syngenta and Zeneca are looking at micro-encapsulation techniques (Anonymus 2004; Perrin 2000), Bayer is developing vegetable oil/additives dispersion formulations to optimize coverage and penetration and to improve spray characteristics and rain fastness (Anonymus 2006) and Dow AgroSciences is developing small droplet sized water-based formulations (Anonymus 2007). Incentives are the wish to create more user-friendly products, using less active substances and less to no organic solvents, better efficacy, reduced exposure and safer formulations for human and the environment. Formulation research for biopesticides can learn from these developments as these are going towards safer compounds and formulation concepts that may be useful for biopesticides. There are some examples with biopesticides: granular formulations such as dry flowables and water dispersible granules have been developed for Bt products. Aqueous suspensions have been developed for organic use: Serenade ASO and Madex.

Many new products and new concepts have been developed by the additive and adjuvant industry. Better understanding of the role of surfactants, dispersants, emulsifiers, spreaders, and other additives is resulting in new and safer formulations. These new concepts are used for built-in formulations as well as for tank-mixed adjuvants (Knowles 2006). In the past, adjuvants were mainly studied for use with herbicides, while current studies include usage with insecticides and fungicides. Since many of these developments are focusing on safer additives for humans and the environment, they may also be useful for formulating living propagules for biopesticides. Some examples of new co-formulants that can be used in biopesticides are compounds such as natural and modified alcohols, fatty acids, starches, sugars, polymers, vegetable oils, waxes, etc. Examples of companies involved in the production of these compounds are Akzo Nobel, Clariant, Degussa, Helena, Seppic, Croda and Uniqema.

3.1.13 Enhancement of Entomopathogens Through Formulation

Improvements can be made by manipulating biological characteristics of a pathogen and technical aspects of the formulation. Biological improvements can be made in various ways. A review of potential improvements through biological features is given by Charnley et al. (1997), but few of these have been incorporated in products. A better understanding of production aspects related to virulence and quality of propagules and a longer shelf-life is needed. This requires more fundamental research. Nutrition can influence virulence and quality of propagules (Butt et al. 2006). Fungi produce cuticle-degrading enzymes and bioactive secondary metabolites (Butt 2002) and a better understanding of when and which ones are produced and what their role is in the infection process could help improve fungal products. We also need to know more about these enzymes and metabolites and how to exploit them better in the production process and in the formulation. Once more fundamental knowledge becomes available, it will be possible to devise strategies to improve strains. If features such as a broader host range, improved speed of kill, reduced dosages, and increased resistance to harmful environmental factors could be improved, biopesticides may become better. Other possibilities to improve strains are somatic hybridization and genetic modification, but the latter is not accepted in Europe.

A novel approach for getting better results is to search for organisms or strains of organisms that are able to grow and reproduce on foliage or roots. Koike et al. (2004) found that strains of L. muscarium with an epiphytic character are able to colonize the leaf surface and give a higher mortality of whitefly over time than strains without this characteristic. This feature could be improved by designing a formulation that stimulates epiphytic growth. Similar examples are epiphytic bacteria like Pseudomonas syringae that colonize the leaf surface and thereby give an effective protection from frost injury (Hirano and Upper 2000), and root colonizing antagonistic fungi that are able to protect plant roots from pathogenic fungal diseases as demonstrated with T. harzianum (Harman 2006).

Further, there is a wide range of recent technological innovations that could be used to improve pathogen formulations. Shelf-life of biopesticides is critical and new packaging technology may help improve shelf-life. New materials such as oxygen absorbers, carbon dioxide absorbers, and water absorbing gels could stabilize the environment in the package. Packaging under inert gases may prevent degradation of the propagules and result in a longer shelf-life. Additives with a certain function could be used to improve efficacy. These could be compounds that enhance virulence (enzymes such as chitinases, proteases and lipases, or stimulators such as chitin and activity enhancers) or that increase survival (UV blockers, etc). Other additives could be used as growth stimulants or compounds that improve secondary pick-up of spores. The latter may be achieved by compounds with sticky features such as oils and gums; by behaviour modifying substances such as attrac-tants (sugars), repellents (garlic oil), alarm pheromones (P-farnesene for aphids) or by low doses of insecticides that increase insect movement and thereby increase the chance for pick-up of spores. This approach proved to be effective when 1% of imidacloprid, a sub-lethal dose, was used in combination with L. muscarium. The chemical dramatically increased aphid movement and as a result secondary pickup of spores increased and led to high mortality (Roditakis et al. 2000). Similar effects have been reported in trials studying mortality of vine weevil larvae with M. anisopliae and low, sub-lethal doses of imidacloprid and fipronil (Shah et al. 2007). New technologies, such as the Exosect technology (www.Exosect.com), may improve the efficacy of biopesticides through an improved adherence of particles, i.e. spores, by formulating them with electrostatic wax powder or magnetic metallic powders. Combinations of functional additives may give synergistic effects.

Technical innovations in the downstream and formulation processes offer another type of potential improvements. Protection of propagules by encapsulating techniques has been studied with bacterial cells of Pseudomonas fluorescens and mycelium of the nematophagous fungus Hirsutella rhossiliensis. Hydrogel capsules improved storage possibilities and efficacy (Patel et al. 2005). The hydrocapsule technology has been further developed by ARS, Inc., USA (www. ars-fla.com/hydrocapsules/html), and applications for biopesticides have been suggested. Propagules can be protected during formulation processes with different drying and coating technologies. New equipment and technologies together with new compounds enlarge the possibilities by methods such as spray-drying, agglomeration, fluidized-bed granulation, freeze-drying, encapsulation and (film-)coating. With these processes, various types of formulations can be made, starting with either particles or liquids, and resulting in dry flowables, water dispersible granules or capsules. A new formulation technique has been developed to coat propagules with water-soluble compounds and then to dissolve the particles in oil. This can be applied to the targets, such as locusts, where the formulation provides protection of the pathogens against heat and solar radiation (patent US 2004/0038825).

According to Hynes and Boyetchko (2006) there is a high priority for formulation development with a multi-disciplinary approach to optimize biopesticide-pest interaction. They made a plea for a better understanding of the ecology of the interaction. This would certainly be a new area for investigations that may lead to better formulations. The authors also observed that researchers in formulation have focused on individual biopesticides rather than on developing general principles on groups of pathogens. This concurs with my experience. The ecology of the interactions should be studied more, and this should be done in institutional research because of its fundamental character. If pathogenicity factors could be elucidated further, then efficacy could be improved by better adapted formulations, and if application rates could be reduced, biopesticides would become more efficacious and more competitive with chemicals. This would be highly advantageous to both the producer and the user. Budgets should be made available for this kind of academic research in order to improve biopesticides.

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