Appl Microbiol Biotechnol (2003) 61:413–423 DOI 10.1007/s00253-003-1240-8

MINI-REVIEW

P. A. Shah · J. K. Pell

Entomopathogenic fungi as biological control agents

Received: 30 October 2002 / Revised: 2 January 2003 / Accepted: 3 January 2003 / Published online: 18 March 2003
Springer-Verlag 2003

Introduction The Order Insecta contains nearly one million described species (May 2000) which comprise approximately 67% of the world’s described fauna and flora. Insects are central to the performance of many ecosystem processes. However, it is in their role as herbivores that conflicts arise with agricultural production due to direct consumption of cultivated crops and indirect damage by plant virus transmission or spoilage of potential yield. Natural enemies such as predators, parasitic wasps and flies, as well as pathogens have long been studied for exploitation in biological control and integrated pest management (IPM) strategies. For the purposes of this review, we will concentrate on examples of work with entomopathogenic fungi, which illustrate the principles or strategies which can be used to reduce losses by insect pests. Classification of entomopathogenic fungi Entomopathogenic fungi are found in the divisions Zygomycota, Ascomycota and Deuteromycota (Samson et al. 1988), as well as the Chytridiomycota and Oomycota, which were previously classified within the Fungi (Table 1). Many of the genera of entomopathogenic fungi currently under research either belong to the class Entomophthorales in the Zygomycota or the class Hyphomycetes in the Deuteromycota. It is important to mention that fungal infections occur in other arthropods as well as insects and/or species which are not pests of cultivated crops. For example, Gibellula species infect spiders and several species of Cordyceps and Erynia infect ants. Further information on the biology and P. A. Shah ()) · J. K. Pell Plant and Invertebrate Ecology Division, Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK e-mail: [email protected] Tel.: +44-1582-763133 Fax: +44-1582-760981

ecology of entomopathogenic fungi can be obtained from Steinhaus (1949, 1964), Samson et al. (1988), Evans (1989), Bałazy (1993) and Eilenberg (2002). General biology of entomopathogenic fungi Most, if not all, entomopathogenic fungi have life cycles which synchronise with insect host stages and environmental conditions. In broad terms, the differences between Hyphomycetes and Zygomycetes, particularly Entomophthorales, are described below. However, as with all general principles, there are exceptions, with features of the two groups forming a continuum. Species, and sometimes isolates within a species, can behave very differently. For example, insect host range, infection levels, germination rates and temperature optima can vary between species and isolates (see e.g. Sierotzki et al. 2000; Pell et al. 2001; Shaw et al. 2002). Members of the Hymphoycetes are generally considered to be opportunistic pathogens infecting many species in a range of insect orders and host death is commonly associated with toxin production overwhelming host defence responses (Roberts 1981; Samson et al. 1988). In contrast, other groups of fungi are thought to have evolved into higher parasitic forms. For example, infection and host death by Entomophthorales tends to occur due to tissue colonisation with little or no use of toxins (Humber 1984). One of the best examples of a highly evolved insect pathogenic fungus is Strongwellsea castrans infecting flies. Infection does not interfere with insect feeding and movement but conidia are discharged, and thereby dispersed, from a cavity in the abdomen of infected insects over a long period of time prior to death (Pell et al. 2001). In general, Entomophthorales have biotrophic relationships with their insect hosts with little or no saprophytism, while Hyphomycetes can be hemibiotrophic with well-defined parasitic phases within insect hosts and saprophytic phases on death of their hosts. The origin of the entomopathogenic lifestyle may have arisen several times from a common saprophytic ancestor

414 Table 1 Outline classification of fungal entomopathogenic genera and examples of insect hosts Phylum/ Class/ Ordera

Genus

Examples of insect hosts

Commercial productsb

Oomycota Chytriomycota Zygomycota Zygomycetes

Lagenidium Coelomomyces Conidiobolus, Entomophaga Entomophthora, Erynia, Neozygites, Pandora Zoophthora Cordyceps, Hypocrella, Torrubiella Aschersonia, Beauveria Hirsutella, Metarhizium

Mosquitoes Mosquitoes Aphids, flies, caterpillars

L. giganteum registered in USA

Entomophthorales Ascomycota Deuteromycota Hyphomycetes Moniliales a b

Scale insects, butterflies and moths, beetles Scale insects, whiteflies, aphids, grasshoppers and locusts, butterflies and moths, beetles

Several products based on B. bassiana, B. brongniartii, M. anisopliae, P. fumosoroseus or V. lecanii

Nomuraea, Paecilomyces, Tolypocladium, Verticillium

Members of Oomycota are now classified within the kingdom of Straminipila, not within the Fungi Copping (1998); Shah and Goettel (1999); Wraight et al. (2001)

inhabiting soil and leaf litter (Humber 1984; Samson et al. 1988; Evans 1989; Spatafora and Blackwell 1993). The greatest radiation into different host groups occurred within the Clavicipitaceae (Ascomycotina), and involved multiple inter-kingdom jumps between animals (e.g. insects), fungi and plants (Nikoh and Fukatsu 2000; Artjariyasripong et al. 2001; Spatafora and Sung 2002). Hyphomycete species exist as separate asexual (anamorph) and sexual (teleomorph) forms. All known genera of entomopathogenic Hyphomycetes now have proven teleomorphs in the Clavicipitales, and life cycle stages for Hyphomycetes may have become simplified in agricultural situations because of a superabundance of insect hosts (Evans 2003; H.C. Evans, personal communication). The loss of a sexual state in some Hyphomycetes, such as Syngliocladium acridiorum in Africa, could have been triggered by evolutionary adaptations to deforestation and desertification (Evans and Shah 2002). Fungal infection and transmission Asexually produced fungal spores or conidia are generally responsible for infection and are dispersed throughout the environment in which the insect hosts are present. When conidia land on the cuticle of a suitable host, they attach and germinate, initiating cascades of recognition and enzyme activation reactions both by the host and the fungal parasite (Samson et al. 1988). Invasion of the insect body and circulatory system (haemolymph) occurs once the fungus has passed through the cuticle of the external insect skeleton. Structures and processes for the invasion of insect tissues are similar to plant pathogens, including the formation of germ tubes, appresoria and penetration pegs (Samson et al. 1988). With entomophthoralean fungi, unicellular yeast-like cells with chitinous walls (hyphal bodies) spread throughout the insect obtaining nutrients, leading to the death of the host by physiological starvation about 3–7 days after

infection. Some entomophthoralean species initially produce rounded protoplasts, which either lack sugar-rich residues in outer cell layers or mask their presence, in order to avoid detection by insect haemocytes (Samson et al. 1988; Glare and Milner 1991; Pell et al. 2001). With hyphomycete fungi, circulation within the insect haemolymph and toxin production is carried out by yeast-like cells which resemble hyphal bodies but are termed blastospores with this fungal group (Samson et al. 1988). On death of the insect host, the fungus emerges from the dead host and sporulation or conidiogenesis usually occurs on the outside of the cadaver. Sporulation can occur internally when ambient humidity precludes external sporulation. For example West African grasshoppers infected with Metarhizium anisopliae var. acridum (= M. flavoviride) exhibited sporulation on the internal surfaces of the desiccated hosts. With entomophthoralean species, cadavers are attached to foliage by fungal rhizoids, which emerge through the ventral surface or mouthparts of the cadaver. Specialised attachment structures ensure that the fungus remains in the environment of new hosts for further transmission. Conidia dispersal Conidia of Hyphomycetes such as Metarhizium and Beauveria spp. are hydrophobic and are passively dispersed from infected cadavers. Entomophthoralean conidia are actively discharged under hydrostatic pressure with the exception of Massospora spp. After discharge entomophthoralean conidia are carried on wind currents or by co-occurring insects (e.g. Hemmati et al. 2001; Roy et al. 2001). If primary conidia of entomophthoralean species fail to find suitable host substrates on which to germinate, then most form higher order conidia which can either be actively discharged, e.g. Erynia or Pandora spp., or form passively held secondary conidia (capilliconidia) borne on long stalks (capilliconidiophores), e.g. Zooph-

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thora spp. With Neozygites spp., the primary conidia are not infective but serve to disperse the infective secondary conidia produced on capilliconidiophores. Conidia of some entomophthoralean species are discharged while the host insect is still alive, e.g. flies infected with S. castrans and thrips infected with Entomophthora thripidum (Steinhaus 1964; Pell et al. 2001).

encounter associations, and both examples involve entomophthoralean fungi. Firstly Entomophaga maimaiga in USA, established serendipitously in the early 1900s, and secondly, Z. radicans, in which an Israeli isolate of the fungus was released in Australia. Entomophthora maimaiga and gypsy moth

Methods for persistence When host numbers are low and/or unfavourable environmental conditions occur, most entomophthoralean species produce resting spores which can arise from meiosis (zygospores) or mitosis (azygospores) and persist in soil for long periods of time (Wilding and Brady 1984; Glare and Milner 1991). Resting spores have not been recorded for the important aphid pathogen Pandora (=Erynia) neoaphidis. The mechanism by which P. neoaphidis overwinters is uncertain but may include spherical hyphal bodies inside insect cadavers, thickwalled conidia (loricoconidia) in the soil, or by cycling in small populations of overwintering hosts (Feng et al. 1991, 1992; Nielsen et al. 1998). Hyphomycete fungi may also form overwintering structures based on compressed hyphae (sclerotia) or thick-walled resting spores (chlamydospores). Infection by Beauveria spp. in soil environments may be improved by the radiation of modified hyphal strands away from cadavers (Keller and Zimmerman 1989).

Use of fungi for insect biological control Entomopathogenic fungi, in common with other insect natural enemies, can be employed under three broad biological control strategies, namely classical biological control, augmentation or conservation. The emphasis for presenting the selected examples was to concentrate on recent or ongoing programmes which illustrate the development and uptake of these three strategies. Recent reviews by Roberts and Hajek (1992), Burges (1998), Fuxa (1998), Jackson et al. (2000) and Butt et al. (2001) provide further information and case studies. Classical biological control Classical biological control is generally accepted to be the use of natural enemies against a host which is exotic in an area and has established without its full guild of natural enemies. Surveys are made in the centre of origin of the insect pest to identify suitable candidate natural enemies which are then released in its newly expanded range (Samways 1981). In general, successful classical biological control programmes provide long-term sustainable and economical control of insect pests. With entomopathogenic fungi, there are only two clear examples of classical biological control exploiting old-

Larvae of the gypsy moth, Lymantria dispar, feed on the leaves of many trees, most commonly oaks and aspens. It was accidentally introduced near Boston in north-eastern USA during the 1860s and within 10 years it had spread dramatically. Attempts to introduce E. maimaiga from Japan were made in the early 1900s, by placing infected cadavers onto tree trunks, but the releases were considered to be unsuccessful at the time (Hajek et al. 1990). In 1989, gypsy moth caterpillars were found infected by E. maimaiga in the USA. This fungus was similar to E. maimaiga from Japan but it is still unclear whether it was derived from releases carried out in the 1900s or from accidental introductions made later, possibly when timber containing infected L. dispar was imported into the USA (Hajek et al. 1995). Since 1989, E. maimaiga has been widely documented spreading throughout forests in north-eastern USA, keeping L. dispar at small population sizes (Elkinton et al. 1991; Hajek et al. 1996). Fungal spread has been due to a combination of airborne movement of conidia as well as human manipulation. The fungus cannot be easily cultured on artificial media so two methods were developed to release E. maimaiga in areas where the insect had just become established. Firstly, soil containing resting spores was collected from the bases of trees which had large numbers of infected cadavers. Secondly, infected cadavers collected from natural fungal outbreaks (epizootics) have been hand-collected for redistribution (Hajek et al. 1998a, 1998b). Since there have been concerns about the transport of pathogenic microorganisms in the soil, the latter method is currently used even though it is highly labour-intensive. At present, E. maimaiga is distributed over much of the area infested by L. dispar in north-eastern USA, but there is still a demand to establish E. maimaiga in new outbreak areas of the gypsy moth (Pell et al. 2001). Current research studies are investigating methods for mass production, storage, germination and infectivity of laboratory-produced resting spores to be used when new outbreaks occur (Hajek et al. 2002). Zoophthora radicans and spotted alfalfa aphid The spotted alfalfa aphid, Therioaphis trifolii f. maculata, was introduced into Australia in 1977 and quickly became an important pest of legume plants present in pastures used for grazing cattle. Six species of fungi were isolated from this aphid in Australia but the entomophthoralean species Z. radicans was not recorded (Milner et al. 1980).

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In Israel, Z. radicans was known to cause dramatic epizootics in populations of the spotted alfalfa aphid (Kenneth and Olmert 1975) and an isolate of Z. radicans was imported from Israel for release in Australia. The particular isolate was chosen because of climatic similarities between the area of origin of the isolate and the site for initial releases in Australia. In 1979, experimental releases were made at four sites using three methods. These involved the placement of laboratory-infected but still living aphids onto lucerne (alfalfa), the addition of dead, infected aphids, or the positioning of sporulating Petri dish cultures over lucerne plants infested with living aphids. The high humidity necessary for infection was obtained by inverting large plastic bins over the treatments and misting with water (Milner et al. 1982). Within 5 weeks, infections caused by Z. radicans were observed within the release sites despite the lack of rainfall during this period. At one site, infection could be detected up to 100 m from the release point, and a threshold of 9 h with more than 90% relative humidity (RH) seemed to be needed for effective transmission and dispersal of infection. Formation of resting spores enabled the fungus to persist between seasons. Spotted alfalfa aphid is no longer a serious pest problem, partly due to Z. radicans but also because of the importation and large-scale releases of the parasitic wasp Trioxys complanatus, which were not infected by Z. radicans (Pell et al. 2001). Generally, isolates of Z. radicans are more likely to infect insect species which are closely related to the original host species than other insects (Glare and Milner 1991). Augmentation In many situations natural enemies are present in indigenous pest populations, but they are either too few or active too late to limit crop damage. In these cases the natural enemies can be augmented. There are two approaches to augmentation; inoculation and inundation. In an inoculative approach the fungus is applied, often in small amounts, early in the season of the crop, with the expectation that it will repeatedly cycle (i.e. establish epizootics) in pest populations and spread over a period of time, thereby maintaining the pest population below the economic threshold. Inundative augmentation involves applying the fungus, often in large amounts, for rapid short-term control with no expectation of secondary infection (Weiser et al. 1976). In this way, the fungus is used in a similar way to a chemical insecticide. The terms “mycopesticide” or “mycoinsecticide” have been used to describe this approach. For fungi, augmentation usually involves adding in vitro-produced mycelia or conidia in aqueous suspensions to a field or glasshouse crop, often in combination with synthetic materials, which are formulation components to enhance persistence and/or infectivity. Overviews of mass production methods and recent advances in the formulation of microbial insecticides have been presented by Bradley et al. (1992) and Burges (1998), respectively.

Hyphomycete fungi have great potential as inundative biocontrol agents, since they are relatively easy to massproduce and formulate for use with conventional spray application equipment. Several commercial products are available for insect control in different agricultural operations (see Table 1; Copping 1998; Shah and Goettel 1999; Wraight et al. 2001). Inundation Three examples are given on the use of commercial or semi-commercial products containing isolates of hyphomycete fungi. Verticillium lecanii is used in Europe for control of aphids and related insects in glasshouses; Beauveria bassiana is available for use against a wide range of insect pests and largely sold in North America; finally, Metarhizium anisopliae var. acridum has recently gained approval for use against locust and grasshopper pests in Africa. Verticillium lecanii against aphids in Europe Verticillium lecanii has been reported causing natural epizootics in aphid and scale populations in tropical and sub-tropical regions and was the first fungus studied and developed for use as an inundative mycoinsecticide in glasshouses. It is available in the form of two products manufactured by Koppert Biological Systems in the Netherlands which contain different isolates as their active ingredient; “Vertalec” against aphids and “Mycotal” against whiteflies and thrips. The products are registered in Denmark, Finland, the Netherlands, Norway and the UK, and registration is pending for France, Spain and Turkey. Aphids on chrysanthemum plants were the initial market for “Vertalec” when it was introduced in 1981 and good efficacy against a number of different aphid species has been demonstrated (Hall 1981; Milner 1997; Burges 2000; Yeo et al. 2003). “Vertalec” is produced by liquid fermentation as blastospores which are formulated with a nutrient source in a wettable powder (Milner 1997). Nutrient formulations allow satellite colonies to grow on leaf surfaces, increasing effective coverage 40-fold (Hall and Papierok 1982). Control can be enhanced by applying the fungus in conjunction with aphid alarm pheromone, sublethal doses of the insecticide imidacloprid, and as part of autodissemination strategies (Hockland et al. 1986; Roditakis et al. 2000; Hartfield et al. 2001). “Vertalec” and “Mycotal” are used exclusively in glasshouses, where the ambient humidity can be modified to favour infection (Milner 1997). Recent advances by Koppert in formulation technology have resulted in an adjuvant based on an emulsifiable vegetable oil, called “Addit”, which can enhance activity of “Mycotal” at low humidities (W. Ravensberg, personal communication). Unfortunately it is not compatible with “Vertalec” but formulation studies continue in order to improve fungal

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efficacy at low humidities and allow application of the products to field crops (W. Ravensberg, personal communication). Milner (1997) suggested that the small market size for “Vertalec” is partly due to its humidity requirements, which limit its use in open-field crops. Beauveria bassiana against insect pests in North America Since the late 1980s, considerable effort has been focused on the development of B. bassiana as a commercial mycoinsecticide by Mycotech, which resulted in three formulated products for insect control sold primarily in North America. In 2000, Mycotech was taken over by Emerald BioAgriculture, who continue to market these fungal-based formulations. Research and development work by Mycotech concentrated on B. bassiana strain GHA. A solid-culture process was patented to mass-produce aerial conidia of B. bassiana (Bradley et al. 1992). Field and laboratory testing of early formulations was assisted by the provision of experimental samples to university and governmentfunded researchers in the USA (e.g. Poprawski et al. 1999; Vandenberg et al. 1998). In this way, a considerable amount of information was accumulated, which aided eventual registration and clarified recommended uses for the B. bassiana product. “Mycotrol” was fully registered in 1999 by the Environmental Protection Agency of the USA, and can be used on a large variety of tree and field crops for control of grasshoppers, whiteflies, thrips, aphids and many other insect pests. Two other formulated products are also available; “BotaniGard” is recommended for use in glasshouses, while “Mycotrol O” contains formulation ingredients which can be used by organic farmers in the USA. All three products are seen as direct replacements for synthetic insecticides, with frequencies of applications and timings similar to conventional insecticides (e.g. Shelton et al. 1998; Wraight et al. 2000). Conidia in the “Mycotrol” formulation are infective even after more than 12 months’ storage at 25C (Wraight et al. 2001). The integrated use of B. bassiana with chemical insecticides is under investigation, as this could improve resistance management strategies and minimise environmental side-effects caused by synthetic insecticides. For example, brassica crops may be sprayed between five and nine times in a growing season, but the replacement of early-season insecticide sprays with B. bassiana to control beetle and caterpillar pests can be effective and economical (Poprawski et al. 1997; Vandenberg et al. 1998). Non-target insects, such as insect predators and parasitic wasps, could be affected by B. bassiana but environmental assessment studies in cotton showed this to be a low and acceptable risk (Jaronski et al. 1998).

Metarhizium anisopliae against locusts and grasshoppers in Africa Locusts and grasshoppers can be devastating pests of crops and pasture grasses in many parts of the world. Between 1985 and 1989, there were outbreaks of the Desert locust, Schistocerca gregaria, and several grasshopper species in many parts of Africa, leading to widespread applications of chemical insecticides for their control. In 1990, a collaborative research programme was initiated between research institutes in the UK, the Netherlands, and the Republics of Benin and Niger to investigate the possibility of using entomopathogenic fungi as biological control agents. The programme was largely prompted by international concern on adverse environmental impacts caused by insecticide applications used during conventional control campaigns (Prior and Greathead 1989). The main intention of the work was to produce formulations of hyphomycete fungi which could be applied using spinning-disc ultra-low-volume sprayers, normally used in tropical countries for insecticide applications. During the course of the work, M. anisopliae var. acridum was found to be an important natural pathogen of locusts and grasshoppers (Shah et al. 1997; Driver et al. 2000). Formulation, mass production and field studies concentrated on an isolate of this fungus found infecting grasshoppers in Niger, West Africa. Many technical issues had to be overcome and an overview of the advances made by the programme has recently been given by Lomer et al. (2001). Mass production of conidia was possible by inoculating sterile rice in plastic bags and tubs, modified from methods previously used in Latin America (Mendona 1992). Aerial conidia were extracted from rice and dried to a low moisture content (<5%) for storage. Infective conidia can be used for spraying even after they have been kept for more than 12 months at 30C in foil sachets with silica gel (Moore et al. 1996; Jenkins et al. 1998). Experimental formulations contained a mixture of kerosene, groundnut oil and dried conidia. Field trials progressed in a tiered fashion from demonstrating infection in small plots using hand-held sprayers, to large-scale operational trials where population reduction was caused by applying conidia through vehicle-mounted sprayers (e.g. Bateman et al. 1992; Lomer et al. 1993; Langewald et al. 1999). Currently, the Metarhizium-based mycoinsecticide is supplied in sachets containing dried conidia which should be mixed with diesel oil or kerosene before spraying (Bateman et al. 1998). Infection and death of 70–90% of treated locusts or grasshoppers occurs within 14–20 days after application, without any detrimental effects on non-target organisms (Lomer et al. 2001). A patented product, “Green Muscle”, was commercially available after 12 years of research involving at least 40 scientists and costing $17 million (D. Dent, personal communication). “Green Muscle” is recommended for locust and grasshopper control by the Food and Agricul-

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ture Organisation of the United Nations (Lomer et al. 2001).

studies were carried out with B. brongniartii (Jung et al. 2002).

Inoculation

Autodissemination of Z. radicans against the diamondback moth

Two examples are presented. Firstly, Beauveria brongniartii, a hyphomycete species primarily used against larvae of cockchafer beetles in Europe and, secondly, an example on the combined use of Zoophthora radicans with semiochemicals for the control of the diamondback moth, Plutella xylostella. Beauveria brongniartii against cockchafers in Europe Many species of scarab beetles, especially the European cockchafer, Melolontha melolontha, are sporadic pests of grasslands, forests and orchards in central Europe. Population swarms may occur every 27–45 years, and the most serious damage is caused by the larvae, which feed on plant and tree roots. A research programme on fungal control of cockchafers was started in the early 1970s, focusing on the use of B. brongniartii, which is specific towards cockchafers. Approximately 300 isolates were found from M. melolontha, many of which showed a divergence in characteristics such as growth rates and virulence as well as effects on host insects from different geographical origins (Keller 1992; Keller et al. 1999). Beauveria brongniartii forms blastospores in liquid culture which can be inoculated onto barley seeds to produce mycelium and aerial conidia. In long-term field trials conducted in Switzerland, blastospores were tested against swarming adult females at the edges of forests with the aim that they could carry the fungus into breeding sites. Helicopter applications of blastospores in aqueous suspensions were made. The formulation contained skimmed milk, which acted as a sticker and protectant against ultra-violet radiation. In several trial sites where blastospores were applied in the 1970s or 1980s, numbers of adults and larvae declined quite substantially due to fungal infection over a 6–9-year period (Keller 1992; Keller et al. 1997). Conidia grown on barley kernels were also applied against larvae in an orchard and natural meadow using a tractor-mounted seed drill, which pressed the kernels 3– 5 cm into the soil to the depth at which cockchafer larvae feed. Infection by B. brongniartii was greater in the treated areas than the control and a decline in larval numbers was found 1 year after applications were made (Keller 1992; Keller et al. 1999). Currently, conidia grown on barley kernels are sold commercially under the trade names of “Engerlingspilz” and “Beauveria Schweizer”. The use of conidia on barley kernels is preferred, since the products can be stored for over a year at 2C while the blastospores are more unstable and need to be used within 4 weeks of production (Keller 1992). A recent European project, BIPESCO, has been completed during which different production, formulation and application

The hypothesis for autodissemination is that insects can be attracted by a host-specific semiochemical into an inoculation device, or trap, where they are contaminated with fungal conidia. Conidia can then be dispersed by these insects once they leave the trap and before they become infected. The aim is to establish disease epizootics earlier than would happen naturally by regular inoculation of the environment by contaminated moths. This method is target-specific, reduces the amount of fungus required and protects it while it is in the trap (Pell et al. 1993a). Over the last 13 years at Rothamsted, laboratory and field studies have been made to develop and evaluate this technique with the fungus Z. radicans against the diamondback moth, P. xylostella, which is an important worldwide pest of brassicas. Fundamental ecological studies have quantified fungal virulence, persistence and transmission between hosts, providing a foundation for trap design and small-scale field evaluation in Malaysia and Kenya. (Pell et al. 1993a, 1993b; Furlong et al. 1995; Furlong and Pell 1997, 2001). Recent proof of concept trials are currently being extended within Australia (Vickers et al. 2001), as well as Cuba and Mexico (J.K. Pell, unpublished data). As a strategy, autodissemination has also proved useful with other entomopathogenic fungi and for integration with other insect natural enemies (Vega et al. 2000). Conservation This strategy involves the modification of farming practices to enhance the activity of an entomopathogen population (Fuxa 1998). Biological control through conservation seeks to identify effective indigenous natural enemies and adopt management practices which conserve and promote them in the field. Management practices which favour entomopathogenic fungi may include provision of increased moisture, e.g. by irrigation, reduction in pesticide use and provision of overwintering sites of alternative hosts. In a looser definition, we can also include the development of “inaction thresholds” which determine the population size of the fungus in addition to the pest to determine whether the fungus can control the pest population without the requirement for insecticides. Here we describe two examples where conservation has been used or is under development.

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Neozygites fresenii and Aphis gossypii in the USA Widespread infections of cotton aphids by N. fresenii have been documented since 1989 in the cotton-growing areas of middle and southern USA (Steinkraus et al. 1991, 1995). In 1993, a diagnosis service was started in Arkansas which identified the presence of N. fresenii in cotton aphid populations, and this can be used by farmers to decide whether insecticide applications are necessary or not. Currently, this service covers the states of Alabama, Georgia, Louisiana and Mississippi and is extending into South Carolina and Florida (Steinkraus et al. 1996, 1998; Steinkraus and Boys 1997; D. Steinkraus, personal communication). Aphid samples from cotton fields are collected by farmers, placed in 70% ethanol and mailed to the University of Arkansas where they are examined microscopically for the presence of N. fresenii. If 15% or more of the aphids show infection then it is highly likely that aphid populations will decline naturally due to the fungus within 1 week. If 50% of aphids are infected, then there is a strong likelihood that aphid numbers will decline within a few days (Steinkraus et al. 1996, 1998). The diagnosis service can now provide detailed information via a dedicated internet web site (http:// www.uark.edu/misc/aphid/) so that farmers are aware of the seasonal progression of N. fresenii in their area. At the 15% level, farmers are increasingly deciding not to apply insecticides, thus saving themselves time and money, as well as preserving beneficial insects and reducing environmental contamination (Pell et al. 2001). Field margins as refugia for entomophthoralean fungi Non-crop plants can act as alternative hosts for insects and their associated fungal pathogens. In Switzerland, P. neoaphidis and Conidiobolus obscurus were reported to multiply in economically unimportant aphid species in meadows adjacent to annual crops, then cause reductions in pest aphid populations in adjacent annual crops (Keller and Suter 1980). Powell et al. (1986) found that entomophthoralean fungi were more common at the edges of fields, since alternative aphid hosts were present and the weed canopy afforded a better environment for transmission than the wheat crop alone. Aphid-pathogenic species also overwinter in aphid hosts in hedges and forest borders (Keller 1998). At Rothamsted, studies on the effects of habitat manipulation are being conducted, looking at the possible role of managed field margins as refugia for aphidpathogenic fungi, particularly P. neoaphidis (Shah et al. 2001). An experimental field margin containing wildflowers and grasses has been sown and the margin and adjacent wheat crop have been sampled to identify potentially useful combinations of plants and aphids for entomopathogenic fungi. Detailed analyses are being made of the spatio-temporal distribution of P. neoaphidis and other entomophthoralean species in the margin and

the adjacent cereal crop. Molecular techniques are also being developed in order to distinguish among isolates of P. neoaphidis (Tymon et al. 2002), so as to determine the spatial structure of P. neoaphidis and the potential for dispersal between aphid hosts in margins and adjacent crops. To date, aphids that are highly likely to represent alternative hosts for fungi include Microlophium carnosum on perennial stinging nettles (Urtica dioica) and Sitobion spp. on the annual grass Yorkshire fog (Holcus lanatus) (P.A. Shah, unpublished data).

Conclusions When considering the strategies within which entomopathogenic fungi can be used in biological control, it is sometimes difficult to apply the same terms that have been developed and applied to the use of predators and parasitoids in biological control. This partly reflects our increasing understanding of fungal taxonomy from molecular studies, which can be an important consideration for the use of indigenous or exotic isolates against pest species. It is also important to note that many biological control strategies benefit from being used together, or in conjunction with other conventional and cultural methods in IPM. For example, classical and inoculative approaches can always be complemented by conservation strategies to enhance their activity, as happens with E. maimaiga for control of the gypsy moth (Pell et al. 2001). In theory, mycoinsecticides used inundatively have no requirement for secondary cycling, but long-term control can be enhanced when cycling does occur e.g. M. anisopliae against locusts and grasshoppers (Lomer et al. 2001). In determining whether the use of entomopathogenic fungi has been successful in pest management, it is necessary to consider each case individually, and direct comparisons with chemical insecticides are usually inappropriate. Gelernter and Lomer (2000) concluded that for any microbial control agent to be successful, technical efficacy was essential but had to be combined with at least two other criteria from among the following: practical efficacy (easy and cheap uptake), commercial viability (profitability), sustainability (long term control) and/or public benefit (safety). The safety of entomopathogenic fungi towards humans, the environment and non-target organisms is clearly an important criterion for consideration and each insect–fungus system must again be considered on a caseby-case basis. However, existing research suggests that there are minimal effects of entomopathogenic fungi on non-targets, and they offer a safer alternative for use in IPM than chemical insecticides (Goettel and Hajek 2000; Pell et al. 2001). The use of inundative mycoinsecticides may not be as sustainable as non-inundative strategies but they are more likely to be commercially successful, depending on the value of the target market. Even so, in 1995 commercial microbial products including Bacillus thuringiensis represented less than 1% of total insecticide sales and there

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continues to be a decrease in the number of private companies offering microbial products commercially (Georgis 1997; Gelernter 1997). Identifying an appropriate market is essential for entomopathogenic fungi. These can be in niche markets, e.g. V. lecanii in glasshouses. However, fungal products may not necessarily be sold directly to farmers but may be provided by aid agencies in resource-poor countries, such as M. anisopliae in Africa (Gelernter and Lomer 2000). Technical efficacy, and to a lesser extent practical efficacy, is essential for success and major advances have been made in the production, formulation and application of hyphomycete fungi as mycoinsecticides. In this respect the development of entomophthoralean fungi as mycoinsecticides has been beset by technical difficulties. Obstacles mainly relate to mass-production and the size and stability of propagules for storage and formulation (Wilding and Latteur 1987; Gray and Markham 1997; Pell et al. 2001; Wraight et al. 2001). Limited research on several species of Entomophthorales may have overcome some of these problems (McCabe and Soper 1985; Pell et al. 1998; Shah et al. 1998, 2000, 2002) but there are still significant hurdles involving large-scale in vitro production, as well as appropriate formulation and application technology. These problems would require significant financial investment in research and development, which need to be strongly linked with the identification of appropriate markets for any eventual product based on an entomophthoralean fungus. However, entomophthoralean fungi are extremely well suited to classical, inoculative and conservation approaches which are not necessarily limited by technical problems of production and formulation. Fundamental studies on fungal ecology and epidemiology are necessary to support these strategies (Pell et al. 2001). Successes with entomopathogenic fungi are often based on considerable, multidisciplinary financial investment in research and development from industry, aid agencies and governments. When commercial interests are absent, especially in the development of classical, inoculative and conservation strategies, then long-term government support is essential (Gelernter and Lomer 2000). Most entomopathogenic fungi are best used when total eradication of a pest is not required, but instead insect populations are controlled below an economic threshold, with some crop damage being acceptable. In addition entomopathogenic fungi have an essential role in IPM if they can be used in conjunction with other strategies for sustainable pest control. The North American Plant Protection Organization considers IPM to be the basis for pest control and biological control to be the basis for IPM (Dorworth 1997). There is no single criterion that guarantees the successful uptake of fungal biological control agents, and the difficulties that need to be overcome include scientific, economic, social and political aspects. However, entomopathogenic fungi have considerable potential to become major components in

sustainable IPM, provided that there is continued investment in research, technology transfer and education. Acknowledgements The authors thank J. Mitchell (University of Portsmouth, UK), D. Dent and H.C. Evans (both CABI Biosciences, UK) for advice and additional information. P.A.S. was sponsored by the Biotechnology and Biological Sciences Research Council, UK (BBSRC) through the Sustainable Arable LINK Programme with support from the following industrial partners: Home-Grown Cereals Authority, Horticulture Development Council, Processors’ and Growers’ Research Organisation, Sainsbury’s plc, British Potato Council, and the National Institute for Agricultural Botany. J.K.P. was funded by the Department for Environment, Food and Rural Affairs, UK (DEFRA). Rothamsted Research receives grant-aided support from BBSRC.

References Artjariyasripong S, Mitchell JI, Hywel-Jones NL, Jones EBG (2001) Relationship of the genus Cordyceps and related genera based on parsimony and spectral analysis of partial 18S and 28S ribosomal gene sequences. Mycoscience 42:503–517 Bałazy S (1993) Entomophthorales. In: Flora of Poland, Fungi (Mycota), vol XXIV. Instytut Botaniki, Krakw, Poland Bateman RP, Godonou I, Kpindu D, Lomer CJ, Paraso A (1992) Development of a novel bioassay technique for assessing mycoinsecticide ULV formulations. In: Lomer CJ, Prior C (eds) Biological control of locusts and grasshoppers. CAB International, Wallingford, pp 255–262 Bateman RP, Neethling D, Oosthuizen F (1998) Green Muscle handbook for central and southern Africa. LUBILOSA/Biological Control Products SA Bradley CA, Black WE, Kearns R, Wood P (1992) Role of production technology in mycoinsecticide development. In: Leatham GF (ed) Frontiers in industrial mycology. Chapman and Hall, New York, pp 160–173 Burges HD (1998) Formulation of microbial biopesticides: beneficial microorganisms, nematodes and seed treatments. Kluwer Academic, Dordrecht, The Netherlands Burges HD (2000) Techniques for testing microbials for control of arthropod pests in greenhouses. In: Lacey LA, Kaya HK (eds) Field manual of techniques in invertebrate pathology: application and evaluation of pathogens for control of insects and other invertebrate pests. Kluwer Academic, Dordrecht, The Netherlands, pp 505–526 Butt TM, Jackson C, Magan N (2001) Fungi as biocontrol agents: progress, problems and potential. CABI International, Wallingford Copping LG (1998) The BioPesticide manual, 1st edn. British Crop Protection Council, Farnham, Surrey Dorworth CE (1997) Two models for the development of fungal biological control agents as instruments of ecological management. In: Chapela IH, Palm ME (eds) Mycology and sustainable development: expanding concepts, vanishing borders. Parkway, Boone, N.C., pp 211–224 Driver F, Milner RJ, Trueman JWH (2000) A taxonomic revision of Metarhizium based on a phylogenetic analysis of rDNA sequence data. Mycol Res 104:134–150 Eilenberg J (2002) Biology of fungi from the order Entomophthorales, with emphasis on the genera Entomophthora, Strongwellsea and Eryniopsis. The Royal Veterinary and Agricultural University, Copenhagen, Denmark Elkinton JS, Hajek AE, Boettner GH, Simons EE (1991) Distribution and apparent spread of Entomophaga maimaiga (Zygomycetes: Entomophthorales) in gypsy moth (Lepidoptera: Lymantriidae) populations in North America. Environ Entomol 20:1601–1605 Evans HC (1989) Mycopathogens of insects of epigeal and aerial habitats. In: Wilding N, Collins NM, Hammond PM, Weber JF

421 (eds) Insect–fungus interactions. Academic Press, London, pp 205–238 Evans HC (2003) Use of Clavicipitalean fungi for the biological control of arthropod pests. In: White JF, et al (eds) Clavicipitalean fungi. Marcel Dekker, New York Evans HC, Shah PA (2002) Taxonomic status of the genera Sorosporella and Syngliocladium associated with grasshoppers and locusts (Orthoptera: Acridoidea) in Africa. Mycol Res 106:737–744 Feng MG, Johnson JB, Halbert SE (1991) Natural control of cereal aphids (Homoptera: Aphididae) by entomopathogenic fungi (Zygomycetes: Entomophthorales) and parasitoids (Hymenoptera: Braconidae and Encyrtidae) on irrigated spring wheat in southwestern Idaho. Environ Entomol 20:1699–1710 Feng MG, Nowierski RM, Klein RE, Scharen AL, Sands DC (1992) Spherical hyphal bodies of Pandora neoaphidis (Remaudire and Hennebert) Humber (Zygomycetes: Entomophthorales) on Acyrthosiphon pisum Harris (Homoptera: Aphididae): a potential overwintering form. Pan-Pacific Entomol 68:100–104 Furlong MJ, Pell JK (1997) The influence of environmental factors on the persistence of Zoophthora radicans conidia. J Invert Pathol 69:223–233 Furlong MJ, Pell JK (2001) Horizontal transmission of entomopathogenic fungi by the diamondback moth. Biol Control 22:288–299 Furlong MJ, Pell JK, Ong PC, Syed AR (1995) Field and laboratory evaluation of a sex pheromone trap for the autodissemination of the fungal entomopathogen Zoophthora radicans (Entomophthorales) by the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae). Bull Entomol Res 85:331–337 Fuxa JR (1998) Environmental manipulation for microbial control of insects. In: Barbosa P (ed) Conservation biological control. Academic Press, San Diego, pp 255–289 Gelernter WD (1997) Resistance to microbial insecticides: the scale of the problem and how to manage it. In: Evans HF (ed) Microbial insecticides: novelty or necessity? British Crop Protection Council Symposium 68:201–214 Gelernter WD, Lomer CJ (2000) Success in biological control of above-ground insects by pathogens. In: Gurr G, Wratten S (eds) Biological control: measures of success. Kluwer Academic, Dordrecht, The Netherlands, pp 297–322 Georgis R (1997) Commercial prospects of microbial insecticides in agriculture. In: Evans HF (ed) Microbial insecticides: novelty or necessity? British Crop Protection Council Symposium 68:243–252 Glare TR, Milner RJ (1991) Ecology of entomopathogenic fungi. In: Arora DK, Ajello L, Mukerji KG (eds) Handbook of applied mycology, vol 2: humans, animals, and insects. Marcel Dekker, New York, pp 547-612 Goettel MS, Hajek AE (2000) Evaluation of non-target effects of pathogens used for management of arthropods. In: Wajnberg E, Scott JK, Quimby PC (eds) Evaluating indirect ecological effects of biological control. CABI Publishing, Wallingford, pp 81–97 Gray SN, Markham P (1997) A model to explain the growth kinetics of the aphid-pathogenic fungus Erynia neoaphidis in liquid culture. Mycol Res 101:1475–1483 Hajek AE, Humber RA, Elkinton JS, May B, Walsh SRA, Silver JC (1990) Allozyme and RFLP analyses confirm Entomophaga maimaiga responsible for 1989 epizootics in North American gypsy moth populations. Proc Nat Acad Sci USA 87:6979-6982 Hajek AE, Humber RA, Elkinton JS (1995) Mysterious origin of Entomophaga maimaiga in North America. Am Entomol 41:31–42 Hajek AE, Elkinton JS, Witocosky JJ (1996) Introduction and spread of the fungal pathogen Entomophaga maimaiga along the leading edge of gypsy moth spread. Environ Entomol 25:1235–1247 Hajek AE, Bauer L, Wheeler MM, McManus ML (1998a) Distribution of resting spores of the Lymantria dispar pathogen Entomophaga maimaiga in soil and on bark. BioControl 43:189–200

Hajek AE, Tatman KM, Wanner PH, Wheeler MM (1998b) Location and persistence of gypsy moth, Lymantria dispar, cadavers containing Entomophaga maimaiga azygospores. Mycologia 90:754–760 Hajek AE, McNeil JR, Filotas MJ (2002) Using Entomophthorales for control: maturation, dormancy and survival of laboratoryproduced Entomophaga maimaiga. Proceedings of the XXXIV Annual Meeting of the Society for Invertebrate Pathology, Noordwijkerhout, The Netherlands Hall RA (1981) The fungus Verticillium lecanii as a microbial insecticide against aphids and scales. In: Burges HD (ed) Microbial control of pests and plant diseases 1970–1980. Academic Press, London, pp 483–498 Hall RA, Papierok B (1982) Fungi as biological agents of arthropods of agricultural and medical importance. Parasitology 84:205–240 Hartfield CM, Campbell CAM, Hardie J, Pickett JA, Wadhams LJ (2001) Pheromone traps for the dissemination of an entomopathogen by the damson-hop aphid, Phorodon humuli. Biocontrol Sci Technol 11:401–410 Hemmati F, Pell JK, McCartney HA, Deadman ML (2001) Airborne concentrations of conidia of Erynia neoaphidis above cereal fields. Mycol Res 105:485–489 Hockland SH, Dawson GW, Griffiths DC, Marples B, Pickett JA, Woodcock CM (1986) The use of aphid alarm pheromone ((E)ß-farnesene) to increase effectiveness of the entomophilic fungus Verticillium lecanii in controlling aphids on chrysanthemums. Proceedings of the Fourth International Colloquium of Invertebrate Pathology, Veldhoven, The Netherlands Humber RA (1984) Foundations for an evolutionary classification of the Entomophthorales (Zygomycetes). In: Wheeler Q, Blackwell M (eds) Fungus–insect relationships. Columbia University Press, New York, pp 166–183 Jackson TA, Alves SB, Pereira RM (2000) Success in biological control of soil-dwelling insects by pathogens and nematodes. In: Gurr G, Wratten S (eds) Biological control: measures of success. Kluwer Academic, Dordrecht, The Netherlands, pp 271–296 Jaronski ST, Lord J, Rosinska J, Bradley C, Hoelmer K, Simmons G, Osterlind R, Brown C, Staten R (1998) Effect of a Beauveria bassiana-based mycoinsecticide on beneficial insects under field conditions. Proceedings of the 1998 Brighton Conference – Pests and Diseases 2:651–657 Jenkins NE, Heviefo G, Langewald J, Cherry AJ, Lomer CJ (1998) Development of mass production technology for aerial conidia for use as mycopesticides. Biocontrol News Inform 19:21N– 31N Jung K, Gonschorrek J, Ruther J, Zimmerman G (2002) Field testing of new biocontrol strategies to decrease population density in Melolontha hippocastani, an important scarab species in Germany. Proceedings of the XXXV Annual meeting of the Society for Invertebrate Pathology, Iguassu Falls, Brazil Keller S (1992) The Beauveria-Melolontha project: experiences with regard to locust and grasshopper control. In: Lomer CJ, Prior C (eds) Biological control of locusts and grasshoppers. CAB International, Wallingford, pp. 279–286 Keller S (1998) The role of Entomophthorales in a sustainable agriculture. IOBC Bulletin 21:13–16 Keller S, Suter H (1980) Epizootiologische Untersuchungen ber das Entomophthora-Auftreten bei feldbaulich wichtigen Blattlausarten. Acta Oecol Oecol Appl 1:63–81 Keller S, Zimmerman G (1989) Mycopathogens of soil insects. In: Wilding N, Collins NM, Hammond PM, Weber JF (eds) Insect– fungus interactions. Academic Press, London, pp 239–270 Keller S, Schweizer C, Keller E, Brenner H (1997) Control of white grubs (Melolontha melolontha L.) by treating adults with the fungus Beauveria brongniartii. Biocontrol Sci Technol 7:105– 116 Keller S, Schweizer C, Shah P (1999) Differential susceptibility of two Melolontha populations to infections by the fungus Beauveria brongniartii. Biocontrol Sci Technol 9:441–446

422 Kenneth R, Olmert I (1975) Entomopathogenic fungi and their insect hosts in Israel: additions. Israel J Entomol 10:105–112 Langewald J, Ouambama Z, Mamadou A, Peveling R, Stolz I, Bateman R, Attignon S, Blanford S, Arthurs A, Lomer C (1999) Comparison of an organophosphate insecticide with a mycoinsecticide for the control of Oedaleus senegalensis (Orthoptera: Acrididae) and other Sahelian grasshoppers at an operational scale. Biocontrol Sci Technol 9:199–214 Lomer CJ, Bateman RP, Godonou I, Kpindou D, Shah PA, Paraso A, Prior C (1993) Field infection of Zonocerus variegatus following application of an oil-based formulation of Metarhizium flavoviride conidia. Biocontrol Sci Technol 3:337–346 Lomer CJ, Bateman RP, Johnson DL, Langewald J, Thomas MB (2001) Biological control of locusts and grasshoppers. Annu Rev Entomol 46:667–702 May RM (2000) The dimensions of Life on Earth. In: Raven PH, Williams T (eds) Nature and human society: the quest for a sustainable world. National Academy Press, Washington D.C., pp 30–45 McCabe D, Soper RS (1985) Preparation of an entomopathogenic fungal insect control agent. US Patent No 4530834 Mendona AF (1992) Mass production, application and formulation of Metarhizium anisopliae for control of sugarcane froghopper Mahanarva posticata in Brazil. In: Lomer CJ, Prior C (eds) Biological control of locusts and grasshoppers. CAB International, Wallingford, pp 239–254 Milner RJ 1(1997) Prospects for biopesticides for aphid control. Entomophaga 42:227–239 Milner RJ, Teakle RE, Lutton GG, Dare FM (1980) Pathogens of the blue green aphid Acyrthosiphon kondoi Shinji and other aphids in Australia. Aust J Bot 28:601–619 Milner RJ, Soper RS, Lutton GG (1982) Field release of an Israeli strain of the fungus Zoophthora radicans (Brefeld) Batko for biological control of Therioaphis trifolii (Monell) f. maculata. J Aust Entomol Soc 21:113-118 Moore D, DouroKpindou OK, Jenkins NE, Lomer CJ (1996) Effects of moisture content and temperature on storage of Metarhizium flavoviride conidia. Biocontrol Sci Technol 6:51– 61 Nikoh N, Fukatsu T (2000) Interkingdom host jumping underground: phylogenetic analysis of entomoparasitic fungi of the genus Cordyceps. Mol Biol Evol 17:629–638 Nielsen C, Hajek AE, Humber RA, Eilenberg J (1998) Soil: a natural source of entomophthoralean fungi infecting aphids. IOBC Bull 21:45–48 Pell JK, Macaulay EDM, Wilding N (1993a) A pheromone trap for dispersal of the pathogen, Zoophthora radicans Brefeld (Zygomycetes: Entomophthorales) amongst populations of the diamondback moth, Plutella xylostella L. (Lepidoptera: Yponomeutidae). Biocontrol Sci Technol 3:315–320 Pell JK, Wilding N, Player AL, Clark SJ (1993b) Selection of an isolate of Zoophthora radicans (Zygomycetes: Entomophthorales) for biocontrol of the diamondback moth Plutella xylostella (Lepidoptera: Yponomeutidae). J Invert Pathol 61:75–80 Pell JK, Barker ADP, Clark SJ, Wilding N, Alderson PG (1998) Use of a novel sporulation monitor to quantify the effects of formulation and storage on conidia production by dried mycelia of the entomopathogenic fungus Zoophthora radicans. Biocontrol Sci Technol 8:13–21 Pell JK, Eilenberg J, Hajek AE, Steinkraus DC (2001) Biology, ecology and pest management potential of Entomophthorales. In: Butt TM, Jackson C, Magan N (eds) Fungi as biocontrol agents: progress, problems and potential. CAB International, Wallingford, pp 71–153 Poprawski TJ, Carruthers RI, Speese J, Vacek DC, Wendel LE (1997) Early-season applications of the fungus Beauveria bassiana and introduction of the hemipteran predator Perillus bioculatus for control of Colorado potato beetle. Biol Control 10:48–57 Poprawski TJ, Parker PE, Tsai JH (1999) Laboratory and field evaluation of hyphomycete insect pathogenic fungi for control

of brown citrus aphid (Homoptera: Aphididae). Environ Entomol 28:315–321 Powell W, Dean GJ, Wilding N (1986) The influence of weeds on aphid-specific natural enemies in winter wheat. Crop Prot 5:182–189 Prior C, Greathead DG (1989) Biological control of locusts: the potential for the exploitation of pathogens. FAO Plant Prot Bull 37:37–48 Roberts DR (1981) Toxins of entomopathogenic fungi. In: Burges HD (ed) Microbial control of pests and diseases, 1970–1980. Academic Press, London, pp 441–464 Roberts DR, Hajek AE (1992) Entomopathogenic fungi as bioinsecticides. In: Leatham GF (ed) Frontiers in industrial mycology. Chapman and Hall, New York, pp 144–159 Roditakis E, Couzin ID, Balrow K, Franks NR, Charnley AK (2000) Improving the secondary pick up of insect pathogen conidia by manipulating host behaviour. Ann Appl Biol 137:329–335 Roy HE, Pell JK, Alderson PG (2001) Targeted dispersal of the aphid pathogenic fungus Erynia neoaphidis by the aphid predator Coccinella septempunctata. Biocontrol Sci Technol 11:99–110 Samson RA, Evans HC, Latg JP (1988) Atlas of entomopathogenic fungi. Springer, Berlin Heidelberg New York Samways MJ (1981) Biological control of pests and weeds. (Studies in biology no 132) Edward Arnold, London Shah PA, Goettel MS (1999) Directory of microbial control products and services, 2nd edn. Division on Microbial Control, Society for Invertebrate Pathology Shah PA, Kooyman C, Paraso A (1997) Surveys for fungal pathogens of locusts and grasshoppers in Africa and the Near East. Mem Entomol Soc Can 171:27–35 Shah PA, Aebi M, Tuor U (1998) Method to immobilise the aphidpathogenic fungus Erynia neoaphidis in an alginate matrix for biocontrol. Appl Environ Microbiol 64:4260–4263 Shah PA, Aebi M, Tuor U (2000) Infection of Macrosiphum euphorbiae with mycelial preparations of Erynia neoaphidis in a greenhouse trial. Mycol Res 104:645–652 Shah PA, Tymon A, Pell JK (2001) Conservation biocontrol with mycopathogens: Erynia neoaphidis and agricultural field margins. Proceedings of the XXXIV Annual Meeting of the Society for Invertebrate Pathology, Noordwijkerhout, The Netherlands Shah PA, Aebi M, Tuor U (2002) Effects of constant and fluctuating temperatures on sporulation and infection by the aphid-pathogenic fungus Pandora neoaphidis. Entomol Exp Appl 103:257–266 Shaw KE, Davidson G, Clark SJ, Ball BV, Pell JK, Chandler D, Sunderland KD (2002) Laboratory bioassays to assess the pathogenicity of mitosporic fungi to Varroa destructor (Acari: Mesostigmata), an ectoparasitic mite of the honeybee, Apis mellifera. Biol Control 24:266–276 Shelton AM, Vandenberg JD, Ramos M, Wilsey WT (1998) Efficacy and persistence of Beauveria bassiana and other fungi for control of diamondback moth (Lepidoptera: Plutellidae) on cabbage seedlings. J Entomol Sci 33:142–152 Sierotzki H, Camastral F, Shah PA, Aebi M, Tuor U (2000) Biological characteristics of selected Erynia neoaphidis isolates. Mycol Res 104:213–219 Spatafora JW, Blackwell M (1993) Molecular systematics of unitunicate perithecia ascomycetes: the Clavicipitales-Hypocreales connection. Mycologia 85:912–922 Spatafora JW, Sung G-H (2002) Evolution of inter-kingdom hostjumping of Cordyceps and the Clavicipitaceae. Proceedings of the 7th International Mycological Congress, Oslo Steinhaus EA (1949) Principles of insect pathology. McGraw-Hill, New York Steinhaus EA (1964) Microbial diseases of insects. In: DeBach P (ed) Biological control of insect pests and weeds. Chapman and Hall, London, pp 515–547 Steinkraus DC, Boys GO (1997) Update on prediction of epizootics with extension-based sampling service. Proceedings of the

423 Beltwide Cotton Conferences. National Cotton Council of America, Memphis, Tenn. Steinkraus DC, Kring TJ, Tugwell NP (1991) Neozygites fresnii in Aphis gossypii on cotton. Southwestern Entomol 16:118–122 Steinkraus DC, Hollingsworth RG, Slaymaker PH (1995) Prevalence of Neozygites fresenii (Entomopthorales: Neozygitaceae) on cotton aphids (Homoptera: Aphididae) in Arkansas cotton. Environ Entomol 24:465–474 Steinkraus DC, Boys GO, Hollingsworth RG, Bacheler JS, Durant JA, Freeman BL, Gaylor MJ, Harris FA, Knutson A, Lentz GL, Leonard BR, Luttrell R, Parker D, Powell JD, Ruberson JR, Sorenson C (1996) Multistate sampling for Neozygites fresenii in cotton. Proceedings of the Beltwide Cotton Research Conferences, Nashville, Tenn. Steinkraus DC, Boys GO, Bagwell RD, Johnson DR, Lorenz GM, Meyers H, Layton MB, O’Leary PF (1998) Expansion of extension-based aphid fungus sampling service to Louisiana and Mississippi. Proceedings of the Beltwide Cotton Conferences, San Diego, Calif. Tymon A, Shah PA, Pell JK (2002). Molecular studies on intraspecific variation in the aphid pathogenic fungus Erynia (= Pandora) neoaphidis. Proceedings of the XXXV Annual Meeting of the Society for Invertebrate Pathology, Iguassu Falls, Brazil Vandenberg JD, Shelton AM, Wilsey WT, Ramos M (1998) Assessment of Beauveria bassiana sprays for control of diamondback moth (Lepidoptera: Plutellidae) on crucifers. J Econ Entomol 91:73–85 Vega FE, Dowd PF, Lacey LA, Pell JK, Jackson DM, Klein MG (2000) Dissemination of beneficial microbial agents by insects. In: Lacey LA, Kaya HK (eds) Field manual of techniques in

invertebrate pathology. Kluwer Academic, Dordrecht, The Netherlands, pp 153–177 Vickers R, Pell JK, White A, Furlong MJ (2001) Proof-of-concept trials for control of DBM by auto-dissemination. Proceedings of the IV international workshop on control of diamondback moth and other cruciferous pests. Melbourne, Australia Weiser J, Bucher GE, Poinar GO Jr (1976) Host relationships and utility of pathogens. In: Huffaker CB, Messenger PS (eds) Theory and practice of biological control. Academic Press, New York, pp 169–185 Wilding N, Brady BL (1984) CMI descriptions of pathogenic fungi and bacteria, set 82, nos 811–820. Commonwealth Agricultural Bureaux, The Cambrian News (Aberystwyth), UK Wilding N, Latteur G (1987) The Entomophthorales: problems relative to their mass production and their utilisation. Meded Facult Landbouw Rijksuniv Gent 52:159–164 Wraight SP, Carruthers RI, Jaronski ST, Bradley CA, Garza CJ, Galaini-Wraight S (2000) Evaluation of the entomopathogenic fungi Paecilomyces spp. and Beauveria bassiana against the silverleaf whitefly, Bemisia argentifolii. J Invert Pathol 71:217– 226 Wraight SP, Jackson MA, Kock SL de (2001) Production, stabilization and formulation of fungal biocontrol agents. In: Butt TM, Jackson C, Magan N (eds) Fungi as biocontrol agents: progress, problems and potential. CAB International, Wallingford, pp 253–287 Yeo H, Pell JK, Alderson PG, Clark SJ, Pye BJ (2003) Laboratory evaluation of temperature effects on the germination and growth of entomopathogenic fungi and on their pathogenicity to two aphid species. Pest Manage Sci 59:156–165