Bioassays of entomogenous fungi

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Bioassays of Entomogenous Fungi
T.M. Butt1and M.S. Goettel2
1
School of Biological Sciences, University of Wales, Swansea,
UK;
2
Lethbridge Research Centre, Agriculture and Agri-Food
Canada, Alberta, Canada
Introduction
Many entomogenous fungi are relatively common and often induce epi-
zootics and are therefore an important factor regulating insect populations.
Most species attacking terrestrial insects belong to the Hyphomycetes and
Entomophthorales while those attacking aquatic insects are generally from
the Chytridiomycetes and Oomycetes. The host is usually invaded through
the external cuticle, although infection through the digestive tract occurs
with some species. Spores attach to the cuticle, germinate, and penetrate the
integument by means of a combination of physical pressure and enzymatic
degradation of the cuticle. The mycelium then ramifies throughout the host
haemocoel. Host death is usually due to a combination of nutrient deple-
tion, invasion of organs and the action of fungal toxins. Hyphae usually
emerge from the cadaver and, under appropriate conditions, produce spores
on the exterior of the host.
The importance of entomogenous fungi as biological control agents has
been reviewed by Latge and Moletta (1988), McCoy et al. (1988), McCoy
(1990), Ferron et al. (1991), Roberts and Hajek (1992), Tanada and Kaya
(1993), and Hajek and St Leger (1994). Examples of common entomogenous
fungi, including those of commercial importance, are given in Table 4.1, but
a more detailed list is provided by Roberts (1989).
The search for commercially viable entomogenous fungi for use in inte-
grated pest management programmes entails several steps. Fungal species
and isolates must first be obtained from diseased insects or the environment,
and identified. They must then be evaluated under laboratory conditions to
identify the most promising candidates. Concomitantly, several problems
© CAB
International
2000.
Bioassays of Entomopathogenic Microbes and
Nematodes
(eds A. Navon and K.R.S. Ascher) 141
4

have to be addressed. The selected isolate must be economically mass pro-
duced, have adequate storage properties, and it must be efficacious under
field conditions. Formulation is an important factor that can affect many of
these properties. For instance, formulations can improve storage time and
field efficacy by protecting against desiccation and harmful UV radiation.
Some formulations can enhance fungal virulence by improving spore
attachment to the host surface, diluting the fungistatic compounds in the
epicuticular waxes and stimulating germination. Rapid germination and
infection are a hallmark of virulent isolates. Finally, the inoculum must be
targeted effectively because mortality is dose related.
Well designed bioassays are central to the successful development of
entomogenous fungi. There exists a wide range of attributes among fungal
isolates and species (Table 4.2). Bioassays are the tools for identifying the
following key parameters: (i) host range, (ii) virulence, (iii) ecological com-
petency (i.e. performance under field conditions), (iv) conditions imped-
ing/enhancing epizootics, and (v) barriers to infection.
The development of bioassays requires a thorough understanding of
both host and pathogen requirements. Failure to understand these can lead
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Table 4.1. Some common entomogenous fungi and their hosts.
Entomogenous fungus Invertebrate host
Division Zygomycotina
Conidiobolus obscurus
Aphids
Entomophaga aulicae
Lepidopteran insects
Entomophaga grylli
Orthopteran insects
Entomophthora muscae
Dipteran insects
Entomophthora thripidum
Thrips
Erynia neoaphidis
Aphids
Massospora cicadina
Cicada
Neozygites fresenii
Aphids
Zoophthora radicans
Certain Hemiptera and Lepidoptera
Division Deuteromycotina
Aschersonia aleyrodis
Whiteflies, scales
Beauveria bassiana
Wide host range
Beauveria brongniartii
Cockchafers and sugarcane borer
Culicinomyces spp.
Mosquitoes
Hirsutella thompsonii
Spider mites, citrus mites
Metarhizium album
Homopteran insects
Metarhizium anisopliae
Wide host range
Metarhizium flavoviride
Orthopteran insects
Nomuraea rileyi
Lepidoptera
Paecilomyces farinosus
Coleoptera, Lepidoptera
Paecilomyces fumosoroseus
Wide host range
Tolypocladium cylindrosporum
Mosquitoes
Verticillium lecanii
Wide host range

to inconsistent results, high control mortality, and poor assessment of fun-
gal virulence. The production, formulation and application methods
employed can also influence fungal viability, virulence and efficacy.
Methods for isolation, cultivation and storage of entomogenous fungi and
important aspects of host–pathogen relationships are briefly reviewed,
focusing on specific factors which could influence the results of laboratory
and field-based bioassays. The methods used for bioassay of these fungi
against insects are discussed and examples are used to illustrate the differ-
ent methods used, augmenting the recent reviews on bioassay techniques
by Goettel and Inglis (1997), Kerwin and Petersen (1997) and Papierok and
Hajek (1997).
Isolation of Entomogenous Fungi
Details on initial handling and diagnosis of diseased insects have been
recently reviewed by Lacey and Brooks (1997). Pathogens can be retrieved
directly from the surface of cadavers if the fungus has already sporulated.
Most Hyphomycetes can be scraped directly off the cadaver (Goettel and
Inglis, 1997), while insects infected with entomophthoralean fungi may be
positioned to shower their conidia directly on to a nutrient surface
Bioassays of Entomogenous Fungi
143
Table 4.2. Comparison of the attributes of specialist and generalist insect
pathogenic fungi.
Specialist Generalist
Narrow host range Wide host range
Mostly biotrophs Mostly hemibiotrophs
Usually produce relatively few, Produce copious small conidia
large conidia
May produce more than one type Produce single type of spore
of spore
Conidia may be forcibly discharged Conidia usually passively dispersed
from conidiophores
Conidia coated in mucilage Hydrophobic conidia
Few conidia required to cause Many conidia required to cause rapid
rapid infection infection
Subtilisins not detected One or more subtilisins secreted
Little evidence of toxicosis Toxins may play important role in host
death
Colonize haemocoel as protoplasts or Colonize haemocoel as blastospores or
thin-walled hyphal bodies filamentous hyphae
Induce spectacular epizootics Natural epizootics usually less obvious
Diseased insects usually located on Diseased insects mostly found in the soil
aerial parts of plant
Difficult to culture Easy to culture

(Papierok and Hajek, 1997). If sporulation or external hyphal growth has
not yet taken place, diseased insects can be incubated in a humid chamber
such as a Petri dish lined with moist filter paper to encourage sporulation.
Sporulating cadavers can be placed whole or dabbed on a selective medium
for isolation of the pathogen.
Most selective media contain either a fungicide and/or antibiotics which
encourage growth of entomogenous fungi and discourage growth of sapro-
phytic fungi and bacteria. Some entomopathogenic fungi can be isolated
indirectly from the soil by live baiting with insects such as larvae of Galleria
spp. (Zimmermann, 1986), or directly by extraction using an aqueous solu-
tion, often in conjunction with a selective medium (e.g. Beilharz et al., 1982;
Appendix 4.1) or discontinuous density gradients (Hajek and Wheeler,
1994). Aquatic fungi can be baited using a variety of substrates such as
hemp seed (Kerwin and Petersen, 1997).
Once isolated, many fungi, especially from the Hyphomycetes, can be
maintained in vitro on several media. Conidia and mycelium should be
stored in cryovials under nitrogen, or freeze-dried and stored in sterile glass
ampoules (Humber, 1997). Freshly harvested conidia can also be air dried
and stored in a desiccator at 4°C or room temperature. Several hyphomycete
fungi (e.g. Verticillium lecanii, Metarhizium anisopliae) can be stored as
conidia bound to silica gel at 240°C. More details on isolation and storage
of entomopathogenic fungi are given by Goettel and Inglis (1997), Humber
(1997), Kerwin and Petersen (1997) and Papierok and Hajek (1997).
Production and Formulation
Once isolates have been identified, the next step is the production of stable,
non-attenuated inoculum for use in evaluation bioassays. Minor changes in
production, storage or formulation can greatly influence bioassay results.
The amount of inoculum required for most bioassays is minimal and can
sometimes even be obtained from cadavers. Although this is sometimes the
only source of inoculum in fastidious fungi which do not readily grow on
artificial media, it is preferable to obtain inoculum cultured on an artificial
medium. The method of culture will largely depend on fungal species and
the type of propagule required. More details on laboratory-scale production
of entomopathogenic fungi are given by Goettel and Inglis (1997), Kerwin
and Petersen (1997) and Papierok and Hajek (1997). More recent general
reviews on mass production and formulation are those of Bartlett and
Jaronski (1988), Baker and Henis (1990), Auld (1992), Bradley et al. (1992),
Goettel and Roberts (1992), Feng et al. (1994), Jenkins and Goettel (1997)
and Moore and Caudwell (1997).
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Attenuation of virulence
Successive subculturing on artificial media often results in attenuation of vir-
ulence. Therefore, when possible, large quantities of inoculum should be
produced using the initial isolate and stored (e.g. as dry conidia) for use in
successive bioassays and studies. The rate of attenuation clearly depends on
the isolate and species of pathogen.
Some fungal pathogens retain their virulence even after prolonged cul-
ture in vitro (e.g. Culicinomyces clavisporus, Sweeney, 1981; Beauveria
bassiana, Samsinakova and Kalalova, 1983; V. lecanii, Hall, 1980). In con-
trast, some isolates rapidly loose virulence after only a few subcultures on
artificial media. For instance, Nagaich (1973) noted that an isolate of V.
lecanii pathogenic to aphids lost its virulence after the second or third sub-
culturing. Lagenidium giganteum progressively lost the ability to form
oospores and zoospores and to infect Aedes aegypti larvae after prolonged
culture on a sterol-free agar medium (Lord and Roberts, 1986).
Virulence of attenuated isolates can often be regained with passage
through an appropriate host. This has been demonstrated with several
pathogens including M. anisopliae (Fargues and Robert, 1983), Nomuraea rileyi
(Morrow et al., 1989), Paecilomyces farinosus (Prenerová, 1994), B. bassiana
(Hall et al., 1972; Wasti and Hartmann, 1975), Lagenidium giganteum (Lord
and Roberts, 1986) and Conidiobolus coronatus (Hartmann and Wasti, 1974).
The effects of culture history on virulence poses a special problem if
bioassays are used to compare virulence among isolates obtained from var-
ious sources and culture collections, as the precise culture history of such
isolates is seldom known. In an attempt to address this problem, each iso-
late can first be passed through an insect host prior to culture on artificial
media and use in bioassays. Vidal et al. (1997) first passed 30 isolates of
Paecilomyces fumosoroseus through nymphs of Bemisia argentifolii, then
carried out bioassays to compare their virulence against this host. Fargues et
al. (1997b) passed isolates through a non-host prior to bioassays against a
host; isolates of Metarhizium flavoviride were first passed through the wax-
moth, Galleria mellonella, by injecting conidia into seventh-instar larvae
prior to use in bioassays against the desert locust, Schistocerca gregaria.
Although the waxmoth is not known to be a natural host of this pathogen,
it was felt that growth and sporulation of the fungus on a natural substrate
would help restore virulence. Although original, the utility of this technique
in comparative bioassays of fungal isolates has not yet been determined.
Further studies in this regard are warranted.
Production of infection propagules
There are four general methods for the production of fungal propagules on
artificial media: (i) surface culture on solid media, (ii) fermentation on
Bioassays of Entomogenous Fungi
145

semi-solid media, (iii) submerged fermentation and (iv) diphasic fermenta-
tion. Although production on solid media is considered as the most expen-
sive, it is also the simplest and usually suffices for the production of the
relatively small amounts of inoculum required for laboratory bioassays. The
production methods of some important fungi are summarized in Table 4.3.
Because few generalizations can be made regarding culture and production
of propagules of the more fastidious fungi, we focus our attention here on
those fungi that are more amenable.
Surface culture on solid media
Most facultative entomogenous fungi will grow on one or more defined or
semi-defined agar-based medium (e.g. Czapek-Dox, Sabouraud) or on natural
substrates (e.g. wheat, bran, rice, egg yolk, potato pulp). Specialist fungi are
usually fastidious on artificial media and are usually best maintained on their
respective hosts. A few can be grown in vitro but require a complex medium.
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T.M. Butt and M.S. Goettel
Table 4.3. Production and storage information on selected entomogenous fungi.
Production Form of
Pathogen method Media inoculum
Aschersonia aleyrodis
1, 3 PDA or chopped millet DM, B, C
Beauveria bassiana
1, 3 Most nutrient agar and liquid media DM, B, C
Beauveria brongniartii
1, 3 Most nutrient agar and liquid media DM, B, C
Coelomomyces
spp. 2 Host rearing medium S, infected
copepods
Culicinomyces
clavisporus
1, 3 Most nutrient agar and liquid media DM, B, C
Entomophaga
spp. 1, 2 Sabouraud dextrose, egg yolk, C
milk agar (SEMA)
Erynia neoaphidis
1, 2, 3 SEMA DM, C
Hirsutella
spp. 1, 3 Most nutrient agar and liquid media Submerged
C, B, C
Lagenidium giganteum
1 Different solid or liquid media Z
that include a sterol
Metarhizium anisopliae
1, 3 Most nutrient agar and liquid media DM, B, C
Metarhizium flavoviride
1, 3 Most nutrient agar and liquid media DM, B, C
Nomuraea rileyi
1, 3 Most nutrient agar and liquid media DM, B, C
Paecilomyces farinosus
1, 3 Most nutrient agar and liquid media DM, B, C
Tolypocladium
spp. 1, 3 Most nutrient agar and liquid media DM, B, C
Verticillium lecanii
1, 3 Most nutrient agar and liquid media DM, B, C
Zoophthora radicans
1, 2, 3 SEMA DM, C
Production method is: 1, surface or submerged culture; 2, live host; 3, semi-solid or
diphasic culture. Form of inoculum: C, conidia; DM, dry mycelium; B,
blastospores; Z, zoospores; S, sporangia.

For example, Lagenidium giganteum can be cultivated on simple medium but
requires sterols to induce oosporogenesis (Kerwin et al., 1991).
Entomophthoralean fungi grow well on Sabouraud dextrose or maltose agar
fortified with coagulated egg yolk and milk (Papierok, 1978; Wilding, 1981).
Petri dishes and autoclavable plastic bags are recommended for small-
and larger-scale production, respectively. However, other containers such as
pans, glass bottles and inflated plastic tubing have been used (Samsinakova
et al., 1981; Goettel, 1984; Jenkins and Thomas, 1996). Agar-based media are
usually used for routine culture. Alternatively, cheaper substrates such as
rice or shelled barley can be used in autoclavable bags or other containers,
especially when larger amounts of inoculum are required (Aregger, 1992;
Jenkins and Thomas, 1996). Once the fungus has sporulated, conidia are
harvested either by washing off using water or a buffer, direct scraping from
the substrate surface (e.g. agar), or by sieving (e.g. rice). For some ento-
mophthoralean fungi, the forcibly discharged conidia are allowed to shower
directly on the host (Papierok and Hajek, 1997).
To obtain conidia virtually free of nutritive substrate contamination,
non-cellulolytic fungi can be grown on a semi-permeable membrane such
as cellophane (Goettel, 1984). Pans containing a nutritive substance such as
bran are lined with the cellophane, placed in sterile bags, autoclaved, inoc-
ulated and incubated. After sporulation has taken place, the membrane with
the adhering sporulating fungus is lifted from the nutritive substrate. Conidia
can then be scraped from the cellophane surface.
Fermentation in semi-solid media
Production of fungi on semi-solid media involves impregnation of small par-
ticles with nutrients. Typically wheat bran is mixed with an inorganic sub-
stance such as vermiculite, although other substances can be used to
provide a large surface area for growth. The mixture is then steam sterilized
and the moisture content adjusted to 50–70%. The fermentation process
takes place either in a bin or a rotating drum through which sterile, moist
air is passed. Primary inoculum is usually grown in liquid medium. Toward
the end of the fermentation cycle, the moist air is replaced by dry air to
reduce the moisture content of the bran and to encourage sporulation. The
temperature is controlled by regulating the circulating air temperature.
More recently, nutrient-impregnated membranes have been shown to
reduce production costs of M. anisopliae conidia (Bailey and Rath, 1994). A
range of membranes impregnated with skimmed milk were screened includ-
ing blotting paper, fly screen, hessian, and gauze-type fabrics. Sporulation
was profuse on Superwipe (an absorbent fibrous material) soaked in
skimmed milk (20 g l21) supplemented with sucrose (2 g l21) or dextrose
plus potassium nitrate. Spores could be washed off in a similar way to
removal of conidia from grain.
Bioassays of Entomogenous Fungi
147

Submerged and diphasic fermentation
Submerged fermentation can be used for production of blastospores and
submerged conidia of selected isolates of entomogenous fungi. Dimorphic
filamentous fungi like M. anisopliae, B. bassiana, Beauveria brongniartii, V.
lecanii, Paecilomyces farinosus and Nomuraea rileyi produce relatively thin-
walled blastospores in submerged culture that are infectious but difficult to
preserve (Adamek, 1965; Samsinakova, 1966; Blanchere et al., 1973; Ignoffo,
1981). Blastospores are produced in relatively large quantities during the log
phase of growth. Most often they are spherical, oval or rod-shaped single
cells which usually germinate within 2–6 h. Although several species of
entomogenous fungi produce blastospores, there is considerable intraspe-
cific variation. Some isolates produce blastospores more readily than others.
The culture medium has a profound influence on blastospore production.
There are several recipes for blastospore production (Appendix 4.2).
Blastospores sometimes are indistinguishable from submerged conidia.
For example, some isolates of M. flavoviride, M. anisopliae, and Hirsutella
thompsonii will produce conidia-shaped cells in submerged culture occa-
sionally from phialide-like structures (van Winkelhoff and McCoy, 1984;
Jenkins and Prior, 1993; T.M. Butt, unpublished observations). Van
Winkelhof and McCoy (1984) noted that of 14 isolates of H. thompsonii only
one produced true conidia. The others produced conidia-like cells.
Diphasic fermentation entails growth of fungi in liquid culture to the
end of log phase followed by surface conidiation on a nutrient or inert car-
rier. This method has been developed for mass production of B. bassiana
(Bradley et al., 1992) and M. flavoviride (Jenkins and Thomas, 1996). A sim-
ilar approach was used in the production of dry marcescent entomphtho-
ralean mycelium (McCabe and Soper, 1985).
Dry marcescent mycelium
The development of the dry marcescent process (McCabe and Soper, 1985)
provides a convenient method for production of fungi, especially fastidious
species like Zoophthora radicans. This process entails the production of the
mycelium by submerged fermentation, harvesting by filtration, coating the
harvested mycelium with a protective layer of sugar solution and then dry-
ing under controlled conditions. When hydrated, the mycelium quickly
sporulates to produce infectious conidia. The dry marcescent process has
been used successfully as a source of inoculum for M. anisopliae (Pereira
and Roberts, 1990; Krueger et al., 1992), C. clavisporus (Roberts et al., 1987),
B. bassiana (Rombach et al., 1988), Z. radicans and Erynia neoaphidis
(Wraight et al., 1990; Li et al., 1993).
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Effects of culture conditions on virulence and ecological fitness
Culture conditions can greatly influence the virulence, longevity and ecolog-
ical fitness of the resultant propagules. For example, St Leger et al. (1991)
found that levels of enzymes on conidia from infected Manduca sexta larvae
were higher than those cultured on an agar medium. Papierok (1982) found
that conidia of four isolates of Conidiobolus obscurus produced in vitro were
less virulent against aphids than those produced in vivo. Hallsworth and
Magan (1994a, b) found that B. bassiana, M. anisopliae and P. farinosus
accumulate polyols when grown on media with increasing ionic solute con-
centration and with different carbohydrate types at different concentrations.
Inoculum with high reserves of polyols was shown to germinate and grow
more rapidly at much lower water activities (Aw0.90 = 90% RH) than those
with small reserves of these polyols (Hallsworth and Magan, 1994c, 1995).
Furthermore, in bioassays with G. mellonella larvae at different RHs, conidia
with large amounts of glycerol and erythritol were more virulent than coni-
dia grown on rich nutrient substrates (Hallsworth and Magan, 1994c). Culture
conditions can also influence thermal tolerance. Increasing the sucrose con-
tent of the growth medium from 2 to 8% resulted in a reduction of thermal
tolerance by conidia of M. flavoviride (McClatchie et al., 1994).
Postharvest storage
The postharvest storage conditions greatly affect fungal viability and effi-
cacy. Conidial moisture content is an important factor with respect to tem-
perature tolerance and viability. Zimmermann (1982) showed that the
tolerance of M. anisopliae for high temperatures increases with increasing
desiccation, whilst Daoust and Roberts (1983) showed that at 37°C, two iso-
lates of M. anisopliae retained most viability after long-term storage at either
0 or 96% RH. Drying conidia in the presence of desiccating agents like sil-
ica gel and CaCl2appears to improve their viability but direct contact with
the desiccant can be detrimental (Daoust and Roberts, 1983).
Moore et al. (1995) found that dried conidia stored in oil formulations
remained viable longer than those stored as a dried powder, especially if
stored at relatively low temperatures (10–14°C compared with 28–32°C).
Addition of silica gel to oil-formulated conidia appears to prolong their shelf
life. Undried conidia of M. flavoviride lose viability rapidly, with germina-
tion dropping below 40% after 9 and 32 weeks at 17°C and 8°C, respec-
tively. After 127 weeks in storage, germination remained at over 60 and 80%
for the dried formulations at 17°C and 8°C, respectively (Moore et al., 1996).
These conidia were found to have retained virulence similar to that of
freshly prepared formulations. Furthermore, conidia dried to 4–5% moisture
content showed greater temperature tolerance than conidia with a higher
moisture content (McClatchie et al., 1994; Hedgecock et al., 1995).
Bioassays of Entomogenous Fungi
149

Formulation
Formulations can greatly improve the efficacy of entomopathogens both in
protected and field crops. The type of formulation and selection of additives
for a given formulation are critical to their stability. The basic components
of most formulations include, in addition to the active ingredient (i.e. fun-
gal spore), one or more of the following: a carrier, diluent, binder, disper-
sant, UV protectants and virulence-enhancing factors (Moore and Caudwell,
1997).
The most widely used carriers are oil and water. Because of their
hydrophobic nature, conidia of some hyphomycete fungi readily suspend in
oils, but oil itself can be toxic, especially when applied against small insects.
Oils are reasonably effective in sticking spores to insect and plant surfaces
(Inglis et al., 1996a). In contrast, surfactants (e.g. Tween) need to be added
to water to ensure conidial suspension, but these are toxic to conidia if used
at high concentrations (e.g. >0.1% v/v). Incorporation of humectants (e.g.
Silwet) can improve infection by providing moisture for germination and
infection.
Recent studies show that more than 60% of the fungal inoculum can be
removed from leaf surfaces by rain (Inglis et al., 1995c; T.M. Butt, unpub-
lished observations). Compounds increasing adhesion of spores to insect
and plant surfaces need to be evaluated. Equally important, the formulation
must not interfere with the infection process, and at best it should enhance
disease transmission.
Photoinactivation has emerged as one of the major environmental fac-
tors affecting persistence and thus efficacy of entomogenous fungi.
Ultraviolet radiation can sterilize surfaces of plants and insect cuticle
(Carruthers et al., 1992; Inglis et al., 1993). Incorporation of UV blockers
(e.g. Tinopal) in formulations can offer some protection against harmful UV
radiation (Inglis et al., 1995b).
Other Factors Affecting Virulence
How the culture, storage and formulation of fungi can influence their via-
bility, virulence and field efficacy has been summarized in the previous sec-
tion. In this section, other factors which could influence the results of
laboratory and field bioassays are considered.
Most entomopathogenic fungi gain entry to the haemocoel by pene-
trating the host cuticle using a combination of hydrolytic enzymes and
mechanical force (Goettel et al., 1989; St Leger et al., 1989a, b; Butt et al.,
1990, 1995; Schreiter et al., 1994). The speed of kill, and to some extent the
host range, are influenced by the number of infection propagules in contact
with the cuticle. Mortality is dose related. There are vulnerable sites on the
cuticle, such as the intersegmental membranes and sites under the elytra of
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certain beetles. Basking in the sun, preening and ecdysis reduce the amount
of viable inoculum on the insect surface. Handling of insects, rearing con-
ditions and insect vigour influence their susceptibility to fungal infection.
Fungal pathogens are greatly affected by abiotic factors such as temperature,
light and humidity.
Dose-related mortality
Susceptibility of most insects is dependent on spore dosage. It is presumed
that a threshold exists whereby a certain number of propagules are neces-
sary to overcome the host, however, the exact nature of this relationship has
not been determined. A positive correlation between the number of infec-
tive spores and mortality by mycosis has certainly been established for most
insect/pathogen combinations, but there are exceptions. For instance,
Goettel et al. (1993) reported a negative correlation between dose and mor-
tality at concentrations greater that 104ascospores of Ascosphaera aggregata
per leaf-cutting bee, Megachile rotundata, larva. Therefore, care must be
taken when interpreting results of very high application rates in some sys-
tems as the possibility of self-inhibition exists.
The dose–mortality relationship is the principal component in many
bioassay designs (Chapter 7). Insects are treated at several increasing doses
and the LD50 and its fiducial limits are then used to compare virulence or
‘potency’ against other isolates. The slope of the dose–mortality curve is also
very useful when comparing virulence amongst different isolates.
Vulnerable sites on the cuticle
Not all areas of the insect cuticle are equally vulnerable to penetration by
propagules of entomopathogenic fungi. The intersegmental membranes
(Wraight et al., 1990), areas under the elytra (Butt et al., 1995) and the buc-
cal cavity (Schabel, 1976) can be preferential sites of infection. Therefore,
the location where the inoculum lands on the cuticle can also influence the
probability of infection and the speed of kill. Consequently, targeting of the
inoculum is an important consideration in the development of bioassay pro-
tocols.
Insect behaviour may affect ultimate sites of penetration. For instance,
results of laboratory studies demonstrated that the most sensitive sites for
penetration of Beauveria brongniartii on larvae of the cockchafer,
Melolontha melolontha, were the mouth and anus (Delmas, 1973). However,
Ferron (1978) found that in larvae of the same species collected in nature,
the most frequent sites of infection occurred on the membranes between the
head capsule and thorax or between the segments on appendages. This
apparent contradiction is possibly due to the larval behaviour of burrowing
Bioassays of Entomogenous Fungi
151

in soil; particles continuously scrape infectious inoculum off the exposed
cuticle whereas the intersegmental membrane is protected from this
mechanical action. This is a good example of how results obtained from
laboratory bioassays must be treated with caution if used to predict the sit-
uation in the field.
Ecdysis and developmental stage
Not all stages in an insect’s life cycle are equally susceptible to infection by
entomogenous fungi. Pupal stages are often the most resistant stage, while
adults can be the most susceptible. For instance, larvae of the thrips,
Frankliniella occidentalis were found less susceptible to V. lecanii and M.
anisopliae than adults, while later instars were less susceptible than earlier
instars (Vestergaard et al., 1995). Larvae of Ostrinia nubilalis were found to
be most susceptible to infection by B. bassiana when exposed as first-instar
larvae, while fourth instars were most tolerant (Feng et al., 1985). Fransen
et al. (1987) found that older instars of Trialeurodes vaporariorum were less
susceptible to Aschersonia aleyrodis, while adults were seldom infected.
Within adult stages, there could also be differences in susceptibility between
different sexes and forms such as aphid alates and apterae (Oger and
Latteur, 1985).
The time of inoculation prior to ecdysis, and the length of the inter-
moult period are important factors that may significantly affect bioassay
results. Moulting may remove the penetrating fungus prior to the coloniza-
tion of the insect, if it occurs shortly after inoculation (Vey and Fargues,
1977; Fargues and Rodriguez-Rueda, 1979). In contrast, Goettel (1988) found
that larvae of the mosquito, Aedes aegypti, were more susceptible to
Tolypocladium cylindrosporum during their moulting period.
Effect of diet on susceptibility
Successful infections are also dependent upon the host diet. For example,
some insects maintained on artificial diet can be more susceptible to infec-
tion than insects fed a natural diet (Boucias et al., 1984; Goettel et al., 1993).
Likewise, laboratory-reared insects can be more susceptible than field-col-
lected ones (Bell and Hamalle, 1971). Insects which have been starved can
also differ in their susceptibility compared with well-fed ones (Milner and
Soper, 1981; Butt, unpublished observations).
Tritrophic interactions between host plants, insect pests and ento-
mopathogens have been reported for fungi. The pathogenicity of the ento-
mogenous fungus B. bassiana mediated by host plant species has been
reported for both the Colorado potato beetle (Hare and Andreadis, 1983)
and the chinch bug (Ramoska and Todd, 1985). Presumably, larvae growing
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on more favourable plant species are better able to mount a successful
defensive reaction to pathogens (Hare and Andreadis, 1983) or have a
shorter intermoult period (see previous section). For chinch bugs, adults
feeding on wheat or artificial diet and inoculated with B. bassiana demon-
strated higher mortality and greater fungal development than adults feeding
on maize or sorghum. These data were interpreted as showing that insects
are benefiting from the fungistatic secondary chemicals in maize and
sorghum (Ramoska and Todd, 1985).
An association has also been demonstrated between volatiles and fun-
gal development. Crucifers contain glucosinolates, nitrogen- and sulphur-
containing secondary metabolites. These are hydrolyzed by an enzyme to
release biologically active compounds which, in addition to playing a major
role in defending the plants from herbivores and fungal pathogens (Chew,
1988), also appear to interfere with the infection processes of insect-patho-
genic fungi (Inyang et al., 1999).
Sublethal Effects and Other Attributes
Measurement of the effectiveness of a pathogen against a host insect must
be based on many factors, in addition to virulence. Not all insects treated
with a fungus succumb to infection. Sublethal effects of entomopathogenic
fungi have been insufficiently studied. It is usually presumed that those
insects that do not succumb to infection do so at no expense. However,
this is not necessarily so. For instance, Fargues et al. (1991) demonstrated
that the fecundity of the Colorado potato beetle, Leptinotarsa decemlineata,
surviving treatment was much lower than in beetles that were not treated.
This study demonstrates that survival does not necessarily come without its
price.
Many attributes of a pathogen are important in determining its ecologi-
cal fitness. There exists a wide range of tolerance among fungal isolates to
environmental factors such as sunlight (Fargues et al., 1996) and tempera-
ture (Fargues et al., 1997a, b) biotic attributes such as speed of germination
(Papierok and Wilding, 1981) and ability to sporulate on the host cadaver
(Hall, 1984). In addition to virulence, these are some of the important
aspects that need to be considered when determining the effectiveness of
an isolate for development as a microbial control agent. Determination of
sublethal effects and other attributes is an important, yet much neglected
area, which warrants further study.
Bioassay Procedures
Use of bioassay to assess the effects of entomopathogenic fungi in insects is
essentially limitless. This, combined with the fact that there is a vast array of
Bioassays of Entomogenous Fungi
153

entomopathogenic fungi with a great variety of hosts, means that there are
no standardized bioassay methods as far as entomopathogenic fungi are
concerned. Consequently, bioassays must be tailored according to host,
pathogen and bioassay objective.
Bioassays can be used to determine and quantify host–pathogen rela-
tionships and the effect of biotic and abiotic parameters on these. Bioassays
of entomopathogenic fungi have been used extensively in five important
applications: (i) determination of virulence, (ii) comparison of virulence
among isolates, (iii) determination of host range, (iv) determination of epi-
zootic potential, and (v) studies on effects of biotic and abiotic factors such
as host age, host plant, temperature, humidity and formulation.
The objective of a bioassay must be well defined before a bioassay pro-
tocol is adopted. Although bioassay procedures must be as efficacious as
possible, they must also be designed to address the objectives and provide
as meaningful results as possible to meet these. Choice, rearing and devel-
opmental stage of the host, infective propagule, formulation and inoculation
method, conditions of post-inoculation incubation, method of mortality
assessment (including mortality in controls), bioassay design and statistical
analyses must all be carefully considered.
Special care must be taken when bioassays with non-target organisms
(NTO) are used for risk assessment. It is common for entomopathogenic
fungi to infect hosts in the laboratory which are never infected in the field.
For instance, Hajek et al. (1996) demonstrated that data on host range of
Entomophaga maimaiga from laboratory bioassays gave poor estimates for
predicting non-target impact; the host range under field conditions was
much narrower than that predicted from laboratory results. Laboratory
assays demonstrated an LD50 of 2.2 3105conidia of B. bassiana per honey
bee worker, whereas subsequent whole-hive exposures resulted in less than
1% mortality (M.R. Loeser, S.T. Jaronski, and J.M. Bromenshenk, cited in
Goettel and Jaronski, 1997). The US Environmental Protection Agency (EPA)
now accepts that infectivity tests with caged honey bees can be misleading
and recommends that 30-day whole-hive tests be used instead (Goettel and
Jaronski, 1997). Development of laboratory bioassays which better simulate
the environment to which bees are exposed (e.g. internal hive temperatures
are commonly held between 32 and 36°C) may provide a better and
cheaper alternative to whole-hive assays.
Low mortalities in the field can be obtained even after application of
highly virulent propagules. Inglis et al. (1997a) obtained low efficacy after
applying conidia of B. bassiana on to native rangeland against grasshop-
pers, despite excellent targeting. Results of laboratory assays demonstrated
high virulence, and high levels of infection were observed in caged field-
collected grasshoppers maintained under glasshouse conditions with simi-
lar temperature and humidity to those experienced in the field. Subsequent
studies revealed the importance of thermoregulation; grasshoppers bask in
the sun, elevating their temperature to levels that prevent disease progress
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(Inglis et al., 1996b, 1997b). The development of a bioassay design whereby
grasshoppers were allowed to thermoregulate allowed for more meaningful
prediction of virulence under field conditions (Inglis et al., 1996b, 1997b, c).
In bioassays designed to predict performance of the pathogen under
field conditions, as many pertinent environmental (e.g. temperature, pho-
toperiod) and other (e.g. inoculation method) parameters as possible must
be taken into account and incorporated into the bioassay design.
Unfortunately most bioassays are performed under static conditions (e.g.
constant temperature, RH, photoperiod). Although such assays may be use-
ful in comparing activity of different isolates, they often provide misleading
information as far as performance under field conditions is concerned.
However, bioassays performed under constant conditions studying single
parameters can be very useful in identifying the pertinent parameters that
need to be considered.
All bioassays should include a non-treatment control in order to moni-
tor survival of insects under the post-inoculation incubation conditions. Such
control insects should be treated with a carrier used for application of the
inoculum. In dose–mortality assays, control mortality is then corrected for in
the statistical analyses (Chapter 7). If bioassays are used for host range or
safety to non-target organisms, it is imperative that a known susceptible host
is also treated in parallel with the non-target organisms (i.e. positive con-
trol). Otherwise negative results are difficult to interpret unless evidence is
provided regarding the virulence of the inoculum against a susceptible host
under the same bioassay conditions.
Choice of sample size and range of doses is usually difficult when deal-
ing with fungal pathogens due to the great variability in responses between
different isolates and hosts. For dose–mortality assays, preliminary bioassays
should be first conducted using a wide range of doses and relatively small
numbers of hosts. A range of doses that would result in mortalities between
25 and 75% should then be chosen. The choice of sample size may be more
problematic and will depend very much on the pathogen–host system. For
instance, Oger and Latteur (1985) determined that, in bioassays of Erynia
neoaphidis against the pea aphid, Acyrthosiphon pisum, the factor that most
affected precision was the number of replicated assays. They found that a
sample size of ten aphids for each of 10–20 doses replicated three or four
times gave an adequate precision for comparative assays. However, more
commonly, five doses should suffice, especially if at least three or four of
the doses fall in the 25–75% mortality range. As in any scientific study, the
whole bioassay must be repeated at a later date, preferably using another
batch of insects and inoculum preparation in order to ensure reproducibil-
ity of results and thereby substantiate the conclusions. More discussion on
choice of doses, sample size, bioassay design and repetition of experiments
is presented in Chapter 7.
Bioassays of Entomogenous Fungi
155

Inoculation
Method of inoculation is influenced primarily by the form of the inoculum
and the size and fragility of the insect. Inoculum is most commonly admin-
istered to the surface of the cuticle either through direct methods such as
dusting, dipping or spraying or through indirect methods such as the use of
baits. Whatever the inoculation method, it is imperative that the viability of
the inoculum be determined as close to the treatment time as possible.
Otherwise, it is not possible to determine if lack of efficacy is due to low
viability of the inoculum. If at all possible, viability assessments of the for-
mulated product should be made. It may be necessary to compare viabili-
ties between the active ingredient and formulated product in order to
determine if the formulation adjuvants have a detrimental effect. Methods
for viability assessments are summarized by Goettel and Inglis (1997).
Entomophthoralean fungi differ from hyphomycete fungi in several
characteristics relevant to the development of bioassays. The former usually
produce comparatively few, large, forcibly discharged, sticky conidia. The
latter generally produce numerous, small, dry conidia. The methods for
inoculating insects with entomophthoralean fungi are usually limited to
either showering conidia on to anaesthetized insects or the host’s food
source such as leaf surfaces. The inoculum may be showered from mycosed
cadavers, sporulating cultures or marcescent mycelium (Papierok and Hajek,
1997). In contrast, inoculum of hyphomycete fungi may be applied by the
methods noted above.
The most important factor in choosing an inoculation method is to
ensure presentation of a precise dose which will reduce variability and help
ensure repeatable results. Consequently, crude methods of inoculation such
as allowing an insect to walk on the surface of a sporulating culture should
be avoided, although such methods may be useful in certain studies whose
aims are, for instance, to establish new host records per se. It is preferable,
however, if the amount of inoculum administered to each insect can be con-
trolled as precisely as possible. This is usually accomplished through enu-
meration of the inoculum and administering a precise dose to each insect.
In situations where it is difficult or impractical to determine the precise dose
being administered, it is common practice to obtain estimates of the dose by
recovering the propagules after application of the inoculum, either through
washing or homogenizing the insects, and then estimating propagule con-
centrations or through direct enumeration or spread plating (Goettel and
Inglis, 1997). Details on methods for enumeration of propagules are pre-
sented by Goettel and Inglis (1997) for Hyphomycetes, Kerwin and Petersen
(1997) for water moulds, and Papierok and Hajek (1997) for
Entomophthorales.
Introduction of inoculum through injection can be used in situations
where the importance of the cuticular barrier is not an issue (i.e. immuno-
logical assays). A tuberculin syringe attached to a motorized microinjector is
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T.M. Butt and M.S. Goettel

usually used to treat many insects rapidly and effectively. The inoculum can
be introduced per os or directly into the haemocoel by piercing the inter-
segmental membrane. Aquatic insects are usually treated by introducing
known numbers of propagules into their rearing medium. Specific methods
of inoculation are described in the examples presented below.
Bioassay chambers
A wide range of bioassay chambers has been used by various workers for
disparate insect species. Bioassay chambers are usually chosen according to
availability, price, convenience, ease of cleaning and requirements of the
host. With entomopathogens, it is important that the chambers be ade-
quately decontaminated prior to reuse. An alternative is to use disposable
containers. Some commonly used bioassay chambers include inexpensive
plastic or polystyrene coffee cups, ice cream cartons, cigar boxes, glass jars,
plastic bins, buckets or bowls, portable cages, and nylon/cotton fine-mesh
sleeves.
For assays with small insects, it is often possible to use single leaf peti-
oles or excised leaves in small bioassay chambers such as Petri dishes. In
such systems, it is important to delay leaf senescence as long as possible, by
providing a nutrient or water source for the plant tissue. For instance, stems
of single leaf petioles can be immersed in water (Fig. 4.1), kept wet with
moistened cotton wool placed on parafilm (Mesquita et al., 1996; Fig. 4.2)
or placed directly on to a nutritive substrate. Vidal et al. (1997) cut out
3.5-cm diameter discs from ornamental sweet-potato leaves, disinfected
them in a series of alcohol and sodium hypochlorite solutions, and placed
them in small Petri plates containing a KNOP medium (in g l21water: 0.25
KCl, 0.25 KH2PO4, 0.25 MgSO4, 0.02 FeSO4, 10 agar) (Fig. 4.3).
Choice of bioassay chamber is critical in field-cage bioassays. Although
these best ‘mimic’ field situations, the type of bioassay chamber can greatly
influence the climate within. Even screened cages provide shading and pro-
tection from wind. In a field-cage experiment using screened cages, Inglis
et al. (1997b) found minimal differences in temperature and relative humid-
ity within and outside the cages, but there was approximately 55% shading
within the cage due to the mesh screening.
Post-inoculation incubation conditions
Conditions of humidity, temperature and light can greatly influence bioas-
say results. Consequently, after inoculation, the insects should be incubated
under controlled environmental conditions or transferred to field bioassay
cages. Controlled environmental conditions are usually maintained using
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157

environment chambers or incubators. Insects can often be pooled in assay
chambers according to treatment group; however, it is preferable to incu-
bate cannibalistic insects such as grasshoppers singly. The conditions
chosen will vary according to the objectives of the bioassay, but they
generally should be favourable for survival of non-inoculated insects.
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T.M. Butt and M.S. Goettel
Fig. 4.1. Bioassay chamber containing single leaf with its water supply. Photo by
courtesy of Lerry Lacey.
Fig. 4.2. Ventilated bioassay chamber containing single blades of barley leaves.
Moisture is provided by soaked cotton battens placed on pieces of Parafilm. Photo
by courtesy of Antonio Mesquita and Lerry Lacey.

Bioassays must often be run for 1–2 weeks or even longer with slower-
acting pathogens such as fungi, where LT50s of 4–6 days are common. With
such long time spans, control mortalities can often be problematic.
Meaningless results are obtained if control mortalities are too high. Control
mortalities are usually accounted for in the statistical analyses (Chapter 7),
but as a general rule, results must be suspect if control mortalities are higher
than 15–20%. If control mortalities greater than 20% are unavoidable, results
must be interpreted with caution.
For some fungi, humidities approaching saturation must be maintained
in order to obtain infection (Papierok and Hajek, 1997). Saturated and near-
saturated conditions are usually provided using water agar or saturated fil-
ter paper within the bioassay chambers. Precise conditions of humidity can
be maintained in the bioassay chambers by continuously circulating air
through a humidifying medium of saturated salt solution (Fargues et al.,
1997b). Sealed chambers without air circulation should be avoided as the
aerial humidity occurs at equilibrium only at the solution/air interface.
Temperature is one of the most easily controlled factors. Most bioassays
comparing virulence among isolates use only one constant temperature,
however, the results obtained could provide misleading information. For
instance, Fargues et al. (1997b) compared the virulence of four isolates of
M. flavoviride at four constant temperatures. No significant differences in vir-
ulence occurred among these isolates at temperatures of 25, 30 and 40°C. In
contrast, there were significant differences in virulence among isolates at
35°C. This demonstrates the importance of using several temperatures when
Bioassays of Entomogenous Fungi
159
Fig. 4.3. Ventilated bioassay chamber containing an excised sweet-potato leaf disc
placed on a nutritive agar medium. Photo by courtesy of Claire Vidal and Lerry
Lacey.

screening for the most virulent isolates. Cyclical conditions of temperature
approximating as much as possible the natural conditions should be con-
sidered for such screenings.
Mortality assessments
Mortality assessments should be made daily. In addition to computing
median lethal doses, this allows computation of median lethal times, which
can be very useful in making comparisons between different treatments
(Chapter 7). If insects are not being incubated singly, dead insects must be
removed as soon as possible and certainly prior to sporulation to prevent
horizontal transmission and loss due to cannibalism. Incubation time varies
according to the insect and fungus being evaluated. Generally, incubation
should continue as long as insects continue to succumb to the pathogen.
Mycosis is usually verified by incubating dead insects at high RH (e.g.
in Petri dishes containing moistened filter paper or water agar) to allow for
fungal colonization and sporulation on the cadaver. It is important to also
incubate cadavers from control insects to determine residual infection lev-
els or if accidental contamination occurred.
Some Examples
Here we provide specific examples to illustrate some of the many different
bioassay methods used with an array of fungal pathogens and insect hosts.
It is not our intent to provide a detailed evaluation or critique on the meth-
ods used. Each bioassay needs to be adapted to the specific needs of the
host/pathogen combination and according to the objectives of the bioassays
themselves. We have divided the sections according to inoculation method.
Spray
Spray bioassays are used extensively, especially against small and fragile
insects that are otherwise difficult to treat. Although sophisticated stationary
and track sprayers are available for this purpose, simpler and less expensive
systems can be developed using an artist’s air-brush. Drop size and distrib-
ution must be carefully monitored.
Honey bees with
M. flavoviride
As mentioned previously, laboratory assays for testing safety against honey
bees can provide misleading results (Goettel and Jaronski, 1997). The assay
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presented here was used to determine the safety of an oil formulation of
Metarhizium flavoviride to adult honey bee workers (Ball et al., 1994).
Similar protocols were used by Vandenberg (1990) and Butt et al. (1994) to
test the safety of B. bassiana and M. anisopliae to honey bees.
1. Combs containing mature honey bee pupae were removed from colonies
and maintained overnight in an incubator at 35°C. Groups of newly
emerged bees (less than 18 h old) were transferred to small cages, supplied
with concentrated sucrose, water and pollen, and maintained at 30°C for 1
week before use.
2. For the application of conidial suspensions, bees were briefly anaes-
thetized with carbon dioxide and transferred to spray cages made from
12.5 cm square perspex 6 mm thick with a hole 10 cm in diameter in the
centre. The bees were sandwiched between two layers of 0.71 mm galva-
nized wire mesh with an aperture of 2.46 mm, 25 bees to each cage.
3. The bees were allowed to recover at room temperature and oil formula-
tions of conidia were applied from a rotary atomizer attached to a track
sprayer in a room maintained at 30°C. Sprays were calibrated to simulate
field dose levels equivalent to twice and 20-fold expected field application
rates. A solution of fluorescent tracer Uvitex OB (Ciba-Geigy) in Ondina oil
was used to determine the volume of formulation deposited on the bees.
The controls consisted of six cages of bees which were not sprayed and
eight cages of bees sprayed with the oil carrier alone. Locust positive con-
trols were also treated in parallel.
4. Immediately after treatment, bees were returned to their original cages
without anaesthetization and maintained at 30°C. Bees that died within 24
h of transfer were omitted from the assay.
5. Cages were checked daily for 14 days and dead bees removed and incu-
bated at room temperature on moist filter paper in plastic Petri dishes.
Conidia of the fungus appeared within a few days over the surface of
infected individuals.
6. A field dose killed 11% of the bees, twice field dose killed 30%, while a
20-fold dose killed 87%.
Whiteflies with
Aschersonia aleyrodis
Spore suspensions are often sprayed directly on to leaves containing the
host. The assay presented here was used by Fransen et al. (1987) to study
differential mortality of different life stages of the glasshouse whitefly,
Trialeurodes vaporariorum treated with conidia of Aschersonia aleyrodis.
Difficulties with this protocol can be encountered when used with highly
mobile insects, as the insects may differentially pick up inoculum post-appli-
cation depending on their mobility.
1. Spores of A. aleyrodis were obtained from a 3-week culture grown on
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161

coarse cornflour at 25°C. Spores were suspended in sterile distilled water
and enumerated in a counting chamber.
2. Two millilitres containing 4 3106spores ml21were applied with a Potter
spray tower at 34.5 kPa to the underside of a cucumber leaf bearing the
whiteflies. Ten leaves per age class were treated with the spore suspension
and two leaves were treated with distilled water as a control.
3. Spore viability was determined by spraying a spore suspension on to a
water agar plate and counting the number of germlings 24 h after spraying
at 25°C.
4. After the water had evaporated from the leaves, plants were covered with
plastic bags to ensure saturated moisture conditions for the first 24 h at 20°C
and 16 h photoperiod. Thereafter, the bags were removed and the plants
were kept at 70 ± 10% RH.
5. Adult whiteflies had to be anaesthetized before inoculation, but difficul-
ties were encountered. If adults did not revive quickly, they drowned in the
spore suspension. To alleviate this problem, adults were exposed directly to
spores on the surface of a sporulating culture for 24 h prior to transfer to
leaves in clip cages.
6. Disease progress was recorded at 3- to 4-day intervals until 90% of the
survivors had developed into adults.
7. It was found that older instars were less susceptible and that adults were
seldom infected.
European corn borer with
B. bassiana
Larger insects are often sprayed directly and then transferred to rearing con-
tainers. Using the bioassay method described here, Feng et al. (1985) deter-
mined age-specific dose–mortality effects of B. bassiana on the European
corn borer (ECB), Ostrinia nubilalis.
1. Larvae were collected from overwintering sites (field corn stubble), trans-
ferred to a meridic diet and maintained for at least three generations to elim-
inate weak and diseased individuals.
2. Three isolates of B. bassiana obtained from a culture collection were first
passaged through ECB larvae. Conidial suspensions were tower-sprayed on
to larvae and incubated at 26°C. Conidia were then harvested from cadav-
ers and inoculated on to Sabouraud dextrose agar (SDA). After incubation
at 26°C for 20 days, conidia were harvested, dried and stored in vials at 4°C
until use.
3. Spore suspensions were prepared and applied to groups of 20 newly
moulted larvae of each instar in individual Petri dishes using a spray tower.
Dose was assessed by spraying SDA Petri plates and counting colony-form-
ing units (CFU) cm22that developed. Five doses of between 9.3 3102and
2.9 3106CFU cm22were applied for each isolate.
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4. The Petri dishes in which the larvae were sprayed were covered. Larvae
were fed an artificial diet with all fungicides removed, and incubated at 26°C
for 24 h, after which time they were transferred to uncontaminated diet
without fungicides and incubated for a further 24 h. After 48 h, standard arti-
ficial diet was provided. The larvae were examined daily for mortality.
5. The experiment was replicated four or five times using different batches
of larvae and new conidial suspensions.
6. First-instar larvae were found most susceptible, there was little difference
between 2nd, 3rd and 5th instars while 4th instars were most tolerant.
Immersion
Insects are often dosed by immersion into suspensions of spores for a spec-
ified time. Although this is usually a quick and convenient dosing method,
precise measurement of dose is difficult. In order to ensure that each insect
from a treatment group receives similar doses, groups of insects should be
simultaneously dipped. Care must be taken that each insect remains pre-
cisely the same amount of time in the dipping suspension. If insects are
dosed singly, then a separate suspension should be prepared for each
insect. Otherwise, each subsequent insect dipped could receive less inocu-
lum, especially since conidia of many entomogenous fungi are hydropho-
bic and therefore adhere preferentially to the insect cuticle.
Aphids with
Verticillium lecanii
The difficulty of immersing small insects for a specified time can be over-
come by draining off the inoculum rather than dipping the insects into the
inoculum per se. This method was used by Hall (1976) to bioassay
Verticillium lecanii against the aphid, Macrosiphoniella sanborni (see
below). It can be adapted for use against almost any insect which can with-
stand submergence for a short period. However, this method is difficult to
use when attempting to study effects of different carriers and formulations,
as the inoculation method does not represent that which would be expected
under operational conditions.
This method has been adopted for use with many insects and fungi.
Some examples include bioassay of B. bassiana against larvae of the cur-
culionid weevils in the genera Sitona, Hylobius,Diaprepes, Chalcodermus
and Pachnaeus (McCoy et al., 1985) and bioassays of M. anisopliae against
beetle and aphid crucifer pests (Butt et al., 1992, 1994).
1. Conidia were obtained from a single spore isolate of V. lecanii grown on
SDA at 23°C for 7 days and then stored at 217°C. Conidia for bioassay were
Bioassays of Entomogenous Fungi
163

produced by spread plating a spore suspension obtained from storage on to
SDA and incubating at 23°C. After 7 days, spores were harvested using a
bent glass rod and phosphate buffer containing 0.02% Triton X100 as a wet-
ting agent. The spore suspension was purified by filtering through cheese-
cloth, centrifuging and washing four times in phosphate buffer. The
concentration of spores was determined using a haemocytometer. A viabil-
ity assessment was performed by incubating three drops of a suspension
containing approximately 106spores ml21on a thin layer of sterile SDA on
a glass slide and incubating for 12 h at 23°C before examining using phase-
contrast microscopy.
2. Adult alate aphids, obtained from a stock culture maintained on potted
chrysanthemum plants, were transferred to chrysanthemum leaf discs in
breeding cells. After 7 days, apterous progeny from the alatae were trans-
ferred to fresh leaf discs and were used for bioassay 8 days later.
3. Batches of mature aphids were placed in glass Petri dishes. Each batch
was transferred on to filter paper in a 7.5-cm diameter Büchner funnel.
Thirty millilitres of spore suspension were then gently poured over the
aphids. After 2 s, the suspension was quickly drained by suction.
4. After inoculation, treated insects were placed singly on leaf discs in high
humidity bioassay chambers, incubated at 20°C for 6 days, and examined
daily for mortality. Dead insects were examined microscopically for signs of
mycosis.
5. The LC50was found to be 2.3 3105spores ml21. It was noted that aphids
tolerated transfer to the assay cells much better if preconditioned in breed-
ing cells than direct transfer from plants.
Dusting
Dusting is sometimes used to inoculate insects. Whereas some workers lit-
erally dust the insects others may simply allow healthy insects to walk over
a sporulating culture (Bidochka et al., 1993). Dusting allows inoculation of
large numbers of insects at once. Care must be taken to ensure that the
insects can withstand this procedure. If at all possible, attempts should be
made to quantify the amount of inoculum received by each insect. Because
it may be possible that death could be caused by suffocation due to obstruc-
tion of tracheal passages, controls should consist of killed spores plus the
carrier. The great variance in dosage acquired should be noted from the
example below. For this reason, generally, this method should be avoided
for dose–mortality assays unless precautions are taken to ensure that the
variation in the amount of dose administered within a dosage group is min-
imized.
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Chinch bug with
B. bassiana
Ramoska and Todd (1985) used a dusting method to study the effects of
host plant on virulence of B. bassiana towards the chinch bug, Blissus leu-
copterus leucopterus. Although dose–mortality assays were not used, dosage
levels received by each insect were estimated.
1. A culture of B. bassiana was grown on Sabouraud maltose agar for 3
weeks at 27°C. Conidia were harvested, dried, sieved and stored at 4°C until
used.
2. Chinch bugs were inoculated by placing 25 adults at a time in a Petri dish
containing dry conidia. Petri plates were then shaken to ensure full cover-
age of the insects. Insects were then removed to incubation chambers.
3. In order to quantify dose levels, two insects from each batch were
removed and immersed in 10 ml of 5% v/v aqueous Tween 20 and vigor-
ously agitated. Conidial density in the suspension was calculated using a
haemocytometer. Dosage levels ranged from 0.9 3105to 1.8 3108conidia
per insect.
4. Inoculated bugs were transferred on to host plant seedlings and a 43-cm,
ventilated, clear plastic collar was placed over the plant which served to
cage the insects. Twenty-five insects were placed into each of four replicate
chambers.
5. After 2 weeks, the test chambers were dismantled and mortality was
assessed. Dead bugs were transferred to Petri dishes containing moist paper
towelling to assess fungal growth on the cadavers.
6. Results showed that feeding on sorghum and maize resulted in greater
tolerance to the fungus compared with insects feeding on other food
sources.
Japanese beetle with
B. bassiana
and
M. anisopliae
Although dusting is generally not recommended for dose–mortality assays
due to the high variability of inoculum received by insects in a dosage
group, as seen in the example above, this inoculation method has been
used successfully in dose–mortality assays with some insects. In the exam-
ple presented here, Lacey et al. (1994) determined LT50 and LC50 estimates
for isolates of B. bassiana and M. anisopliae in adults of the Japanese bee-
tle, Popillia japonica.
1. One isolate each of B. bassiana and M. anisopliae was cultured on SDA,
harvested with a rubber spatula, dried in an incubator overnight at 30°C, and
then passed through a 250 µm sieve. Spore viability was determined by plat-
ing 100 µl of the conidial suspensions on SDA and counting the number of
colonies formed after 48 h. Spore counts were estimated as 2 3107conidia
mg21for B. bassiana and 3.6 3107conidia mg21for M. anisopliae.
Bioassays of Entomogenous Fungi
165

2. Adult Japanese beetles were field-collected using baited traps and held
briefly in the laboratory prior to treatment. One hundred individuals were
counted out into 140-ml polystyrene cups, which were closed with perfo-
rated screw caps.
3. Five dosages ranging from 0.5 to 10 mg conidia were weighed out and
added to the cups containing the adults. In a later modification of the pro-
cedure, lower dosages were used in which talcum powder at a ratio of 990
mg to 10 mg conidia was added as a carrier. Five replicate cups were pre-
pared for each dosage. The cups were periodically rotated end-over-end for
a 1-h period to help distribute the conidia.
4. Thirty beetles from each treatment were then removed and divided
among three holding cages, which consisted of 950-ml plastic containers
with perforated lids. Water and humidity was provided by dental wicks
which protruded through the bottom of the cage into 100 ml reservoirs
below. Adults were incubated at 22–24°C, checked for mortality, and pro-
vided with fresh blackberry leaves daily for up to 8 days. Four replicate tests
were conducted on each of four separate dates.
5. Dead beetles were transferred to 950-ml plastic tubs containing 500 g
moistened sterilized soil, incubated at 22–24°C for 1 week and then were
examined for fungal outgrowth.
6. A dose–mortality response was obtained with LC50 estimates of 0.7 mg
conidia per 100 adults for M. anisopliae and 0.026 mg conidia per 100 adults
for B. bassiana.
Direct deposition on to individual insects
A precise droplet of inoculum can be placed directly on to the surface of
the insect. This method can be used with larger insects that can tolerate
handling. Often, some form of immobilization of the insect is required. Due
to the hydrophobic nature of the cuticle, it is sometimes difficult to admin-
ister a drop of aqueous inoculum precisely. In such cases, it may be help-
ful to choose an inoculation site that would absorb the droplet by capillarity.
Cocoa weevil with
B. bassiana
Prior et al. (1988) used the direct deposition method to compare the viru-
lence of water and oil formulations of B. bassiana against the cocoa weevil
pest, Pantorhytes plutus. Other examples of direct deposition bioassay
include studies with M. anisopliae against flea beetles (Butt et al., 1995).
1. B. bassiana was cultured for 2 weeks at 28°C on 2% malt agar or on
autoclaved brown rice or oat grain in 250-ml conical flasks. Formulations
were prepared by adding 100 ml of either filtered coconut oil or distilled
166
T.M. Butt and M.S. Goettel

water with 0.01 ml of Tween 80 into the cereal cultures, stirring and filter-
ing through a metal strainer. Serial dilutions were then made as necessary
and conidial concentrations determined using a haemocytometer. Conidial
viability was verified by streaking on to malt agar plates.
2. Adult weevils were field-collected, individually secured by pressing the
dorsal abdomen lightly on to Blue tak®adhesive, and inoculated by apply-
ing 1 µl to the mouthparts. A Hamilton syringe was used to apply the oil
formulation and an Agla microsyringe for the water formulation. For the
water formulation, it was necessary to retain the insects secured until the
drop of inoculum had dried. Otherwise the drop would run off.
3. Inoculated insects were transferred to 1-l plastic containers and fed every
3–4 days with pieces of cocoa stem. Mortality was checked daily and dead
insects were transferred to plastic cups containing damp tissue for verification
of fungal outgrowth. Only insects that showed visible outgrowth of the fun-
gus were included in the analyses to determine the LD50.
4. The oil formulation was found to be much more effective than the water
formulation. LD50 estimates were 1.2 3103conidia per insect for the oil for-
mulation and 4.3 3104for the water formulation.
Subterranean termite with
B. bassiana
and
M. anisopliae
Lai et al. (1982) used the direct drop deposition method in bioassays to
determine the virulence of six isolates of entomogenous fungi to the sub-
terranean termite, Coptotermes formosanus.
1. Cultures of the fungi were kept on SDAY (SDA with yeast). Virulence was
maintained by passage through termites every 3 months. For bioassay, 0.1
g of spores were scraped off 20-day-old culture plates. Conidia were then
suspended in 10 ml of 0.1% Tween 80 to a final dilution of 1:100 using a
magnetic stirrer. The suspension was filtered through two layers of
Kimwipes®. A 0.5 µl aliquot of the spore suspension was placed on a micro-
scope slide and the number of conidia in this drop counted under a phase-
contrast microscope (Ko et al., 1973). Dilutions were then performed as
required.
2. Foraging termite workers were obtained from a field colony. One hun-
dred workers were weighed in groups of ten in glass vials and the mean
body weight was used to determine the dosage as expressed by the num-
ber of conidia per mg body weight.
3. Termites were anaesthetized with CO2 for 10 s then transferred to a 100
320 mm Petri dish lined with filter paper.
4. A Hamilton microsyringe was used to apply 0.5 µl inoculum to the sur-
face of the prothoracic area. This volume was enough to cover the insect
without runoff. Mortality was reduced by avoiding direct contact with the
syringe on the termite.
Bioassays of Entomogenous Fungi
167

5. Insects were kept in inoculation chambers at 25°C and 56% RH with the
filter paper and applicator sticks changed every 4 days to prevent secondary
infection by saprophytes such as Rhizopus spp.
6. Thirty foraging workers were treated at each dosage level and caged in
groups of ten. Three groups constituted a single replication and the exper-
iment was repeated three times. Control groups were inoculated with the
carrier alone.
7. Inoculated termites were incubated at room temperature for 15 days.
Mortality data at 12 days was used for the probit analysis as control mortali-
ties drastically increased thereafter. Samples of dead termites were homoge-
nized on a microscope slide and examined for the presence of hyphal bodies.
8. Estimates of LD50 and LT50 revealed differences in virulence among iso-
lates. Overall, isolates of M. anisopliae appeared more virulent than those
of B. bassiana.
Inoculation of soil
Insects which either inhabit or are associated with soil during a part of their
life cycle can be exposed to inoculum contained on the surface or within
the soil. Soil texture, humidity and microbial flora can affect conidial viabil-
ity and virulence and need to be considered. A discussion of procedures
and precautions is presented by Goettel and Inglis (1997).
Pecan weevil with
B. bassiana
Champlin et al. (1981) applied an aqueous suspension of B. bassiana coni-
dia to the surface of soil to compare virulence of mutants against the pecan
weevil, Curculio caryae.
1. Five B. bassiana mutants were obtained by ultraviolet irradiation of a
wild-type culture of B. bassiana. Conidia from 14- to 21-day-old cultures
grown on SDA + 3% yeast extract were obtained by washing with sterile
0.03% Triton X-100. Conidia were washed twice in sterile distilled water and
the concentration estimated spectrophotometrically at 540 nm.
Concentrations were determined by plating appropriate dilutions on SDA
and counting CFUs.
2. Large plastic cups (14.5 cm deep, 11.5 cm diameter) containing auto-
claved soil–sand mixture (10 : 1) were inoculated with 10 ml of spore sus-
pension distributed over the entire 95 cm2surface in a dropwise manner
using a pipette. After allowing the solution to be absorbed into the soil (to
an estimated depth of c. 1.3 cm) 25 4th-instar larvae of field-collected pecan
weevils were allowed to burrow down into the soil in each cup. Four
dilutions of each mutant were prepared.
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T.M. Butt and M.S. Goettel

3. Cups were covered with Parafilm in which ten holes were punched, and
incubated at 25°C. After 7 days, 5 ml of sterile water was added to each cup
to maintain moisture. Two to three replicate treatments were performed for
each mutant strain. Mortality was assessed 21 days post-inoculation. The
percentage of insects that were mummified was used in the LC50 assess-
ments.
4. The mutants exhibited different degrees of virulence with LC50s ranging
from 9.7 3106to 1.0 3109conidia ml21.
Ovipositing grasshoppers with
B. bassiana
Conidia can be incorporated directly into the soil to bioassay virulence
against insects which burrow or oviposit into soil (Fig. 4.4). Inglis et al.
(1995a, 1998) used this method to determine the effects of conidial concen-
tration, soil texture and soil sterilization on virulence of B. bassiana to
ovipositing adults and emerging nymphs.
1. Conidia of B. bassiana were obtained commercially. Numbers of conidia
g21were determined using serial dilutions in water and a haemocytometer.
Conidia were mixed uniformly into autoclaved sand at a concentration of
108conidia g21dry-weight sand. Water was added to obtain a water content
of 9.2% (w/w) and 880 g of sand was added into each of three plastic con-
tainers (10 310 37 cm) per treatment. A 5 mm layer of dry sterile sand
Bioassays of Entomogenous Fungi
169
Fig. 4.4. Grasshopper ovipositing into
B. bassiana
-augmented sand. Photo by
courtesy of Doug Inglis.

was then placed on the surface. The moisture level within each cup was
monitored daily by weighing and readjusted to 9.2% as necessary.
2. Two cores of sand were removed from each container using a 3.5-mm
diameter cork borer. The sand was vortexed for 30 s at high speed in 10 ml
of 0.05% Tween 80 in phosphate buffer. The suspension was diluted and
spread on to a selective medium. The number of CFU was then determined
after incubation in the dark at 25°C for 5–6 days.
3. A minimum of 30 virgin females and 20 virgin males of a non-diapausing
laboratory strain of Melanoplus sanguinipes were placed into cages, 40 cm
square. The cages had holes in the bottom so that the containers of the
inoculated soil could be inserted with the surface of the sand being level
with the cage bottom. The cages were maintained at a 25/20°C day/night
temperature regime with a 16:8 h (light:dark) photoperiod.
4. Adults copulated and the pronota of the first seven females per cage to
oviposit were marked with paint. The duration of each oviposition period
was recorded, and upon completion, these adults were sacrificed, and the
extent of abdominal infestation with conidia was quantified by excising the
abdomens, sealing the cut end with molten Parafilm, and washing in 5 ml
of buffer in 20 ml vials, vigorously shaken at 300 rpm for 2 h on a rotary
shaker. The spore suspension was diluted, plated on selective medium and
the number of CFU determined.
5. The remaining adults were maintained within the cages on a diet of bran
and wheat leaves. At the end of 7 days, containers of sand were replaced
with freshly inoculated sand for a further 7 days. At the time of removal,
populations of conidia within the sand were assessed as described previ-
ously. Dead insects were removed daily and cadavers were surface steril-
ized in 0.5% sodium hypochlorite with 0.1% Tween 80 followed by two
rinses in sterile water. The presence of hyphal bodies in the haemolymph
or outgrowth of B. bassiana in cadavers held under moist conditions was
recorded.
6. Each egg container was incubated at 25/20°C day/night with a 16:8 h
(light:dark) photoperiod and nymphal emergence was recorded daily. At the
time of first nymphal emergence, densities of viable conidia were enumer-
ated as previously described. Ten newly emerged nymphs per replicate
were collected and anaesthetized in CO2. Nymphs were homogenized and
the CFU were determined on a selective medium.
7. Remaining nymphs were maintained in cages on a diet of wheat
seedlings for a period of 10 days. Dead nymphs were removed daily and
placed on moistened filter paper within Petri plates. After 10 days, egg pods
were sifted from the sand and the number of unhatched eggs per female
was determined.
8. All experiments were repeated and analysed as completely randomized
designs. Extensive mortality attributed to the fungus occurred in ovipositing
females, associated males and in emergent nymphs.
170
T.M. Butt and M.S. Goettel

Inoculation through contact with contaminated substrates
Insects are sometimes inoculated by allowing them to walk over the surface
of a substrate, such as filter paper, which was pretreated with a known con-
centration of inoculum. Although such methods are an improvement over
allowing insects to walk over sporulating cultures, there are still difficulties
in ensuring that each insect receives precise and equitable doses. For
instance, when using this method, it would be expected that the more
mobile insects would acquire more propagules than the less active individ-
uals. Nevertheless, this inoculation method may have utility in some situa-
tions.
Aphids with
M. anisopliae
Butt et al. (1994) used spore-impregnated filter paper to assay M. anisopliae
against Lipaphis erysimi and Myzus persicae. Similar methods have also
been used to assay pathogens against thrips (Butt, unpublished) and corn
earworm, Heliothis zea larvae (Champlin et al., 1981).
1. Conidia from 8–12-day-old sporulating cultures of two M. anisopliae iso-
lates were harvested in a 0.03% solution of Tween 80 and diluted to the
desired concentrations.
2. Myzus persicae and Lipaphis erysimi were placed for 15 s on filter paper
impregnated with conidia by vacuum filtration of a 10 ml conidial suspen-
sion of 1 3107or 1 31010 conidia ml21. Aphids were then transferred to a
healthy Chinese cabbage leaf in a ventilated perspex box (5.5 311.5 3
17.5 cm) lined with moist tissue paper. Control insects were treated similarly
with 0.03% Tween 80. The aphids were incubated at 23°C in a 16:8 h
(light:dark) photoperiod and humid conditions were maintained for the first
24 h by placing the boxes between wet paper towels.
3. Mortality of both M. persicae and L. erysimi was 100% within 4 days post-
inoculation at 1 3107or 1 31010 conidia ml21with little or no control mor-
tality (0–3%). The earliest deaths were recorded on the first day after
inoculation and sporulation occurred 1 to 2 days after death. Young, healthy
aphids which contacted mycosed insects also succumbed to the M. aniso-
pliae isolates.
Bait
Inoculum can be incorporated directly into the diet and presented to the
insects as a bait. Although this method is most often used with fungi that
infect through the gut (e.g. Ascosphaera aggregata), it can also be used as
Bioassays of Entomogenous Fungi
171

a method for inoculation of fungi that invade through the external integu-
ment. While feeding, insects contaminate their mouthparts and body with
the pathogen propagules.
Leaf-cutting bees with
Ascosphaera aggregata
Ascosphaera aggregata is one of the few species of entomogenous fungi
which infects the host through the gut. Consequently, a bioassay method
has been developed whereby the inoculum is introduced on an artificial diet
to study the susceptibility of different ages of larvae of leaf-cutting bees,
Megachile rotundata, to this fungus (Vandenberg, 1992). In a later study, a
similar bioassay technique was used to demonstrate that larval susceptibil-
ity was much reduced when larvae were fed a natural diet (Goettel et al.,
1993; Fig. 4.5). This study demonstrates the importance in choice of diet if
results are used to predict events under natural conditions.
1. A pollen/agar-based diet was prepared and dispensed asceptically into
wells of sterile flexible microtitre plates. Sections of 16 wells were cut and
placed in 60 315-mm sterile plastic Petri dishes. Eggs were obtained from
field-collected bee cells and were transferred to the sterile diet.
2. Ascospores were obtained by scraping field-collected cadavers and stored
at 220°C. Inoculum was prepared by suspending spores in sterile buffer
and grinding between two microscope slides to break up the spore balls.
172
T.M. Butt and M.S. Goettel
Fig. 4.5. Ninety-six well microtitre plate used for bioassay with leaf-cutting bees.
Artificial media are separated with empty wells to help prevent possible cross-
contamination. Photo by courtesy of Grant Duke.

Appropriate dilutions were made and concentrations determined using a
haemocytometer.
3. Bees were inoculated within 1 h of inoculum preparation by applying 2
µl of the spore suspension to the diet surface adjacent to the mouthparts of
the newly emerged larvae. Larvae were inoculated at 1, 2, 3 or 5 days of
age. A total of 15 assays were carried out.
4. Larvae were checked daily for mortality. Unhatched eggs and larvae
which died within the first 24 h were not included in the analyses.
Uncertain diagnoses were verified by microscopy or fungal isolation into
pure culture.
5. A dose–mortality relationship was found. There was an increase in LD50
with increasing age. The estimated LD50 values ranged between 120 and
1698 spores per bee, depending on age of larvae at time of inoculation.
Grasshoppers with
B. bassiana
A leaf surface treatment bioassay has been used successfully to inoculate
numerous fungi against several insect hosts (Ignoffo et al., 1983 and refer-
ences therein; Inyang et al., 1998). Inglis et al. (1996a) used an oil-bait
bioassay method to compare the virulence of several isolates of B. bassiana
against the grasshopper, M. sanguinipes. In subsequent studies, this method
was used to demonstrate the effect of bait substrate and formulation on vir-
ulence of this fungus (Inglis et al., 1996c). It was demonstrated that the effi-
cacy of this method depends on the extent to which nymphs become
surface-contaminated with conidia during ingestion.
1. Conidia of several isolates of B. bassiana were obtained from cultures
grown in the dark at 25°C on potato dextrose agar (PDA ) for 7–10 days.
Conidial viability was assessed on PDA amended with 0.005% Benlate
(Dupont), 0.04% penicillin and 0.1% streptomycin after 24 h incubation at
25°C. Conidia were scraped from the surface of the PDA and suspended in
sunflower oil. Conidial densities were determined using a haemocytometer
and adjusted as necessary to obtain final concentrations of 1 3105, 3.2 3
104, 1 3104, 3.2 3103and 1 3103viable conidia.
2. Nymphs hatched from eggs laid by field-collected adults were reared on
a diet of bran and wheat leaves. Third-instar nymphs were individually
placed in sterile 20-ml vials stoppered with a sterile polyurethane foam plug,
and starved for 12 h.
3. Five-microlitre aliquots of conidial suspensions were pipetted on to 5-mm
diameter lettuce discs. A control consisted of oil applied to the discs alone.
The inoculated discs were then pierced in the centre with a pin and sus-
pended approximately 2 cm into the vial from the foam plug, and presented
to the starving nymphs (Fig. 4.6). Nymphs were held at 25°C under incan-
descent and fluorescent lights for 12 h. Nymphs that underwent
Bioassays of Entomogenous Fungi
173

ecdysis or did not consume the disc after this period were excluded from the
experiment.
4. Groups of 12 to 15 nymphs per treatment were transferred to 21 328 3
15 cm Plexiglass containers equipped with a perforated metal floor to
reduce contact with frass (Fig. 4.7). Cages were incubated at a 25/20°C
day/night and 16 : 8 h (light : dark) photoperiod regime and the nymphs
were maintained on a diet of wheat leaves. Alternatively, in some assays,
grasshoppers were kept singly in plastic cups (Inglis et al., 1996b; Fig. 4.8).
5. The experiment was arranged as a randomized complete block design
with four blocks conducted in time. The total number of nymphs per iso-
late–dose combination ranged from 46 to 61 nymphs. Nymphs that died and
subsequently produced hyphal growth of B. bassiana on moistened filter
paper were considered to have died of mycosis.
6. The oil-bait bioassay method facilitated the rapid inoculation of grasshop-
per nymphs. Within 1 h, 350 nymphs could be easily inoculated using this
method. A dose–mortality relationship was demonstrated and substantial dif-
ferences in virulence between isolates were found.
174
T.M. Butt and M.S. Goettel
Fig. 4.6. Inoculation method for grasshoppers. An inoculated leaf disc is pinned to
the inside of a foam plug and presented to a starved nymph within a shell vial.
Photo by courtesy of Doug Inglis.
Fig. 4.7. A Plexiglass bioassay chamber used to incubate groups of inoculated
grasshoppers. Photo by courtesy of Doug Inglis.

Inoculation using forcibly discharged conidia
Most fungi in the Entomophthorales produce forcibly-discharged conidia.
These conidia are usually relatively short-lived and it is often not possible
to harvest and enumerate them before using as inoculum. Consequently,
many bioassays with these fungi use methods to inoculate the host directly
from sporulating cultures or cadavers. In using such methods, much atten-
tion must be paid to the specific conditions that are required to induce
spore discharge (Papierok and Hajek, 1997). Depending on the species
involved, spores can be obtained from cultures maintained on agar medium,
sporulating cadavers or hydrated marcescent mycelium.
Potato leafhopper with
Zoophthora radicans
Wraight et al. (1990) used the forcibly discharged conidia of Zoophthora
radicans from cultures and infected cadavers to inoculate the potato
leafhopper, Empoasca fabae.
1. Dry mycelium of Z. radicans was prepared according to McCabe and
Soper (1985), milled and sieved to retain particles between 1 and 0.5 mm.
The mycelial particles were spread evenly on to water agar in Petri dishes
and incubated at 21–22°C for approximately 12 h to obtain abundant sporu-
lation.
2. Bioassay chambers consisted of 35-mm diameter plastic Petri dishes. Most
of the upper surface of the lid was excised, leaving a narrow strip across the
Bioassays of Entomogenous Fungi
175
Fig. 4.8. A plastic container used to incubate single grasshopper nymphs. Photo by
courtesy of Doug Inglis.

centre for attachment with a small screw to a flat Plexiglass base. A cowpea
leaf was then sandwiched between the base and modified lid. Each leaf was
misted with water and each chamber was covered with a matching, unmod-
ified lid.
3. Newly moulted, 5th-instar nymphs of E. fabae from a laboratory colony
on cowpea were anaesthetized with CO2 and randomly collected in groups
of five. Individuals were placed dorsal side up on the wet leaf surface in a
bioassay chamber. The chamber lid was then replaced with a lid containing
the sporulating fungus. The leafhoppers were continuously exposed to the
sporulating culture for 7 min. During this exposure period, the culture was
continuously rotated.
4. After inoculation, each group of insects was transferred to clean cham-
bers with fresh leaves. Each chamber was sealed in plastic bags and incu-
bated at 20–22°C and 90–100% RH.
5. The LD50 was estimated at 4.1 spores per leafhopper.
Aquatic insects
Bioassay of aquatic insects is usually accomplished by introducing the
inoculum directly into the water. However, use of high concentrations of
inoculum in static aqueous systems may have adverse effects on water
quality. Therefore, at times it is necessary to replenish the water depend-
ing on host species. Also, continuous exposure to propagules of some
fungi such as Tolypocladium cylindrosporum may not be ideal, because the
effective dose may vary according to length of exposure, as hosts continu-
ally reingest excreted conidia that remain viable (Goettel, 1987). This prob-
lem can be overcome by using a limited exposure time (Nadeau and
Boisvert, 1994).
Mosquitoes with
Culicinomyces clavisporus
Sweeney (1983) used a static bioassay method to determine the time–
mortality responses of mosquito larvae inoculated with Culicinomyces
clavisporus.
1. C. clavisporus was cultured in a broth of 1.25% corn steep liquor, 0.2%
glucose and 0.1% yeast extract for 7 days at 20–24°C. Conidia were sepa-
rated from the mycelium by filtration through fine gauze, then pelleted by
centrifugation followed by two washes with sterile water. Conidia were
counted using a haemocytometer and adjusted to the desired concentration.
2. Within 4–6 h of emergence, 5th-instar larvae of Anopheles hilli were
placed in groups of 40 into plastic trays (18 312 35 cm) with 200 ml of
water. Conidia were added on the following day.
176
T.M. Butt and M.S. Goettel

3. The trays were incubated at 25°C and larvae were fed daily with pow-
dered animal food pellets. Dead larvae were removed daily and the exper-
iments were terminated after 12 days.
4. Nine separate experiments were performed with six to eight concentra-
tions of conidia in each experiment. Five separate trays were dosed at each
concentration and five trays were kept as a control.
5. A dose–mortality relationship was found and time to death decreased
with increasing dose.
Mosquitoes with
Coelomomyces
Toohey et al. (1982) used a bioassay to determine the intermediate copepod
host in Fiji for a Coelomomyces sp. The fungus is a pathogen of mosquitoes
which requires an alternate host to complete its development.
1. Cultures of five species of copepods and three species of mosquito were
reared in the laboratory in rain water in transparent or opaque cups (6 3
10 cm).
2. Field- and laboratory-reared Coelomomyces-infected mosquito larvae
which had been dead for less than 24 h were used as the inoculum.
3. Inoculum and 150–200 copepods of various ages were placed into each
bioassay cup containing 200 ml of boiled treehole water. Ten to 12 days
later, 20 first-instar Aedes larvae were placed in each cup. Cups were exam-
ined three times a week and dead larvae, pupae and adults were removed
and examined microscopically for signs of infection. If infection was not
apparent, a second group of larvae were added. Crushed mouse chow was
added periodically for food.
4. Controls consisted of a set of three cups, one with only copepods, a sec-
ond only with inoculum and the third of both copepods and larvae. There
were at least five replicates for each copepod species tested and a total of
20 controls for all the species tested.
5. Only one species of copepod, Elaphoidella taroi, was found to be the
intermediate host.
Novel bioassay methods
As stated previously, bioassays must be adapted to suit the host, pathogen
and objectives of the bioassay. At times, the approach taken is very novel
and sometimes even controversial. Novel approaches must balance efficacy
and usefulness of the results.
Bioassays of Entomogenous Fungi
177

Silverleaf whitefly with
P. fumosoroseus, V. lecanii
and
B. bassiana
Landa et al. (1994) used a novel approach to bioassay entomopathogenic
fungi against the whitefly, Bemisia argentifolii. The bioassay is based on
rapid characterization of the growth rate and development of the fungi on
whitefly nymphs. It can be used in determining effects of environmental fac-
tors, adjuvants and pesticides on development of these fungi in whiteflies.
This method could be adapted for use with many other small insects.
1. Isolates of P. fumosoroseus, V. lecanii and B. bassiana were cultured on
PDA at 25°C in constant light for 7–10 days. Conidia were harvested by rins-
ing the cultures with 0.05% aqueous Tween 80. The suspension was mixed
using a vortex mixer and conidia were enumerated using a haemocytome-
ter and adjusted to a final concentration of 1.0 3107conidia ml21.
2. Test materials (adjuvants, pesticides) were then added to the conidial sus-
pensions. Drops of the test suspension were then placed on sterile micro-
scope slides, 30 drops in three rows per slide. Laboratory-reared, early
4th-instar nymphs were singly placed in the centre of each drop and a total
of 25 nymphs were placed on each slide. The remaining five drops were
used as controls.
3. The slides were dried in a laminar airflow hood, placed in plastic Petri
dishes with a sterile wet filter paper on the bottom and incubated for 7 days
at 25°C under constant light. Each fungus was assayed using ten slides.
4. The influence of the bioassay protocol on the development of nymphs
was assessed. Early 4th-instar nymphs were incubated on microscope slides
in the diluted Tween 80 only for 7 days. The number of emerged adults was
assessed daily.
5. A rating system, named the Fungus Growth Development Index (FGDI),
was used to assess the degree of fungal development on the insect host.
Ratings were made at either daily intervals or at 24, 72 and 120 h. An FGDI
of 0.5 represented the first sign of viability of conidia, 1.5 for colonization
of the host and 2.5 for initial sporulation on the cadaver.
Semi-field and field-based assays
Field trials are essential to demonstrate that fungal isolates identified as vir-
ulent in laboratory trials are efficacious in the field. Initial trials may be done
using small cages enclosed in gauze or potted plants enclosed in a nylon
sleeve which allow for easy monitoring of insect pests. Trials may be done
in ‘walk in’ cages containing potted plants to ease the collection of insects
and assess the efficacy of the pathogen under field conditions. However,
most small-scale trials are done in randomized plots (3 m 33 m) alongside,
178
T.M. Butt and M.S. Goettel

or occasionally within, a growing crop. Although it may be more difficult to
find the target insect there are various ways of assessing the impact of the
pathogen. For example, insects may be collected randomly within each plot
and incubated in humid chambers favouring fungal development. This
could reveal more about targeting of the pathogen and its potential impact
on the pest population. Alternatively, incubating healthy insects with plant
parts collected from trial sites can reveal a considerable amount about the
persistence (distribution on the plant and viability) of inoculum under field
conditions. Assessing plant damage, or the number of larvae infesting leaves
or flowers in control and treated plots is another way of assessing the
impact of the pathogen. Fewer larvae would be expected to be found in
plots where the pathogen was deployed. Field trials may not only be con-
ducted on growing field crops but also rooted cuttings (Dorschner et al.,
1991).
Field trials against subterranean pests are technically more difficult for
several reasons. First, the soil is a natural reservoir of many insect-patho-
genic fungi so it would not be surprising to find target pests in control plots
killed by fungi related to introduced pathogens. Second, targeting of the
pathogen is not easy. Most often, inoculum is applied as a drench or
ploughed into the soil using specialized equipment, but some workers have
even used helicopters to treat large areas of pasture (e.g. Keller et al., 1989).
Field bioassay of
B. bassiana
against grasshoppers
Inglis et al. (1997b) used a field cage bioassay to study the influence of envi-
ronmental conditions on mycosis of grasshoppers caused by B. bassiana.
1. Conidia of B. bassiana, obtained from Mycotech Corp., were suspended
in 1.5% (w/v) oil emulsion amended with 4% clay and applied to 12 ha of
rangeland at a rate of 112 l ha21. Grasshoppers were collected in sweep
nets immediately after treatment and placed in cages (41 361 348 cm)
(Fig. 4.9), 100 hoppers per cage. Treatments consisted of cages: (i) placed
in a glasshouse located at the laboratory, (ii) exposed to full sunlight, (iii)
shaded from sunlight by a black plastic screen, and (iv) protected from UVB
radiation by a UVB-absorbing plastic film (Fig. 4.9). Field cages were
arranged as a randomized complete block with four sub-blocks, each con-
taining three cage treatments per sub-block.
2. Grasshoppers were maintained on a diet of wheat seedlings and range-
land grasses. Cadavers were removed daily and assessed for mycosis by
placing on moist filter paper.
3. Higher prevalence and more rapid development of the disease were
observed in grasshoppers kept in shaded cages than in cages exposed to
full sunlight or protected from UVB radiation.
Bioassays of Entomogenous Fungi
179

Honey bee mediated infection of pollen beetle
(Meligethes aeneus)
by
M. anisopliae
Butt et al. (1998) evaluated dissemination of fungal inoculum by honey bees
against pollen beetles in oilseed rape (= canola) using field-caged insects.
This method has also been shown to control seed weevil (Ceutorhynchus
assimilis) and has the potential to control most floral pests including thrips
(T.M. Butt, unpublished observations).
1. Trials were carried out in winter oilseed rape between late April and late
May, and in spring oilseed rape between mid-June and late July.
2. Nine insect-proof cages (2.7 32.7 31.8 m high) were erected over the
flowering crop infested with adult pollen beetles. Small colonies of honey
bees were placed in the corner of each of six of the cages; each consisted
of about six British Standard combs of bees and brood housed in a single
British Standard Modified National hive body. Three of the hives had mod-
ified entrances containing an inoculum dispenser similar to that used by
Peng et al. (1992). This consisted of a Perspex tray to contain the inoculum,
through which the bees walked on leaving the hive. Bees returned to the
hive via an entrance below the dispenser to prevent inoculum being
brought into the hive. Inoculum was replenished at 48-h intervals. The three
treatments (bees without inoculum, bees with inoculum, and no bees or
inoculum) were randomized.
3. Ninety pollen beetles were collected from each cage at intervals of 3–6
days in winter rape and 7 days in spring rape, and were placed in groups
of 30 in ventilated Perspex boxes (5.5 311.5 317.5 cm) lined with moist
180
T.M. Butt and M.S. Goettel
Fig. 4.9. Field cages used to study the effects of solar radiation and shade on
virulence of
B. bassiana
against grasshoppers under field conditions. Photo by
courtesy of Doug Inglis.

tissue paper and incubated at 23°C and 16:8 h (light:dark) photoperiod.
Three freshly cut inflorescences of rape were placed in each box as food.
Mortality was recorded daily for 14 days. Dead beetles were removed and
placed in a Petri dish lined with moist filter paper to encourage external
conidiation of the fungus.
4. The first mortalities due to M. anisopliae were 3–5 days and 2–6 days
after the sample was taken in winter and spring rape, respectively. The final
mortalities for samples 1 and 2 were approximately 60% on winter rape and
99% and 69%, respectively, on spring rape. These results suggest that honey
bees are effective in delivering conidia of M. anisopliae to flowers of oilseed
rape and in the subsequent control of pollen beetles.
Checklist of Bioassay Preconditions and Requirements
There are several aspects which need to be checked to ensure effective
bioassays with fungal pathogens.
1. It is important to ensure that the pathogen:
• has not lost virulence during culturing,
• inoculum is viable and percentage germination is determined,
• application method is satisfactory.
2. The target insect must be:
• healthy,
• not overcrowded or stressed,
• isolated if carnivorous or cannibalistic.
3. The bioassay chamber must:
• allow survival of control insects,
• not contain harmful substances, such as formaldehyde in food.
4. All bioassays should have:
• large enough sample size and enough replicates per treatment to
make the results meaningful,
• the assays repeated at least once,
• field plots which are randomized,
• internal environments in the field cages which approximate to the
external environment,
• sampling procedures which reflect the field fitness of pathogens.
Concluding Remarks
Bioassays are central to the successful development of fungi as microbial
control agents. Although useful in providing valuable information on the
Bioassays of Entomogenous Fungi
181

insect–pathogen–environment interactions, the validity of bioassay results
depend on the bioassay design, execution, analysis and interpretation of
results. The ultimate challenge is to develop bioassays that can be used to
predict field efficacy. It is therefore imperative that pertinent environmental
parameters be incorporated into bioassay designs. For instance, knowledge
of an LD50 or LT50 obtained from comparative laboratory assays of numer-
ous isolates under static conditions provides minimal useful information as
far as predicting the potential efficacy of a strain under field conditions is
concerned.
Bioassay designs must be constantly improved to provide more mean-
ingful information. The advent of increasingly sophisticated equipment such
as incubators, environmental monitoring and inoculum application devices
has allowed for the development of more complex bioassay designs which
provide more pertinent results. Computerized statistical analyses have made
it possible to model environmental parameters and process data with greater
ease. As our understanding of the pertinent parameters important in fungal
epizootiology increases, bioassays must be adapted so that they will provide
information applicable for prediction of efficacy under field conditions.
We have provided some of the important parameters that need to be
considered in the development and execution of a bioassay with an ento-
mopathogenic fungus. We have also provided numerous examples of bioas-
says to illustrate the many methods and bioassay designs that have been
used with an array of fungal and target species combinations. It is hoped
this provides the reader with adequate information that should stimulate and
facilitate the design of novel and pertinent bioassays which will provide use-
ful information for the understanding of fungal biology, host–pathogen
interactions, epizootiology and ultimately aid in the development of these
microorganisms as microbial control agents of pest insects.
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Appendix 4.1: Selective Media for Isolation of Entomogenous
Fungi
Veen’s agar medium (1 l) (Veen and Ferron, 1966)
35 g Mycological agar (Difco) or 10 g Oxoid neutralized soya peptone, 10 g
dextrose, 15 g No. 1. agar (or Bacto-agar), 1 g chloramphenicol (store 4°C),
and 0.5 g cycloheximide (= Actidione; store 4°C). Add 1 l distilled water, stir,
and cover. Autoclave for 10–15 min at 18–20 psi. Cool to c. 52°C and pour
plates in laminar flow cabinet.
Oatmeal dodine agar (Beilharz et al., 1982)
1. Antibiotic stock solution: add 4 g penicillin G (Sigma) and 10g strepto-
mycin sulphate (Sigma) to 40 ml sterile distilled water under sterile condi-
tions. Store at 4°C.
2. Crystal violet stock solution: add 0.1 g crystal violet (Sigma) to 200 ml dis-
tilled water. Store in the dark.
3. Add 17.5 g oatmeal agar (Difco) and 2.5 g agar (Fisons) slowly to 0.5 l
distilled water while stirring vigorously and heat to boil.
4. Add 0.5 ml of the fungicide dodine (N-dodecylguanidine monoacetate;
Cyprex 65WP, American Cyanamid Co.) and 5 ml crystal violet stock solu-
tion to the medium.
5. Autoclave for 20 min at 15 psi.
6. Allow medium to cool to 50–55°C and add 2 ml of antibiotic stock solu-
tion under sterile conditions.
7. Swirl flask well to ensure thorough mixing of compounds and pour while
warm. There should be enough media for twenty 9-cm diameter Petri
dishes.
Selective agar medium (1 l) (Kerry et al., 1993)
37.5 mg carbendazim, 37.5 mg thiabendazole, 75 mg rose bengal, 17.5 g
NaCl, 50 mg each of streptomycin sulphate, aureomycin and chlorampheni-
col, 3 ml Triton X-100, and 17 g corn meal agar (Oxoid) in 1 l distilled water.
This medium is appropriate for selecting some Paecilomyces spp. and
Verticillium spp. from soil.
Paecilomyces lilicanus
medium (Mitchell et al., 1987)
To prepare 1 l of medium, mix the following: 39 g PDA, 10–30 g NaCl, 1 g
Tergitol, 500 mg pentachloronitrobenzene, 500 mg benomyl, 100 mg strep-
tomycin sulphate, and 50 mg chlorotetracycline hydrochloride.
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Wheat germ based selective agar medium (1 l) (Sneh, 1991)
1. Prepare an aqueous extract of wheat germ – mix 30 g wheat germ in 1 l
water, autoclave for 10 min and filter through four layers of cheesecloth.
2. Mix wheat germ extract (1 l) with 0.25 g chloramphenicol (heat stable) +
0.8 mg benlate (50% benomyl), 0.3 g dodine (65% n-dodecyl-guanidine
acetate), 10 mg crystal violet and 15 g agar.
3. Autoclave and pour into plates.
Copper-based selective agar medium (1 l) (Baath, 1991)
2% malt extract (Oxoid), 1.5% Agar (Difco) amended with 2–4 mg
CuSO4·5H2O per litre. Cordyceps militaris and Paecilomyces farinosus are
tolerant of high Cu levels (400 mg l21), followed by Metarhizium anisopliae
and Beauveria bassiana. Most other soil-borne fungi including
nematophagous species of Verticillium were less tolerant.
Bioassays of Entomogenous Fungi
193

Appendix 4.2: General Culture Media
Medium Ingredients g l–1
Straw agar medium Supernatant of boiled straw 40
Agar (Difco) 8
Aureomycin 0.05
Streptomycin 0.05
Chloramphenicol 0.05
Soya peptone medium Soya peptone 10
K2HPO40.3
MgSO4·7H2O 0.3
NaCl 0.15
CaCl2·6H2O 0.3
MnSO4·6H2O 0.008
CuSO4·5H2O 0.0002
FeSO4·7H2O 0.002
Minimum medium K2HPO40.3
MgSO4·7H2O 0.3
NaCl 0.15
CaCl2·6H2O 0.3
MnSO4·6H2O 0.008
CuSO4·5H2O 0.0002
FeSO4·7H2O 0.002
Agar 20.0
MC medium Potassium phosphate dibasic 36
Sodium phosphate heptahydrate 1.1
Magnesium sulphate heptahydrate 0.6
Potassium chloride 1
Glucose 10
Ammonium nitrate 0.7
Yeast extract 5
Agar 20
Sabouraud dextrose agar (SDA) Mycopeptone 10
Dextrose 40
Agar 15
Oatmeal agar (OA) Oatmeal 30
Agar 20
Potato dextrose agar (PDA) PDA (Oxoid) 39
Malt extract agar (MEA) Malt extract 30
Mycological peptone 5
Agar (technical grade) 15
Continued
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T.M. Butt and M.S. Goettel

Medium Ingredients g l–1
Sabouraud dextrose agar with Dextrose 40
yeast (SDAY) Neopeptone 10
Yeast extract 10
Agar 15
V8 Juice V8 200
CaCO33
Agar 20
Blastospore-producing medium Corn steep liquor 20
Sucrose 30
KH2PO42.26
Na2HPO4·12H2O 3.8
MgSO4·7H2O 0.123
FeSO4·7H2O 0.023
ZnSO40.020
K2SO40.174
CaCl2·2H2O 0.147
PYG with supplements Peptone 1.25
Glucose 3.0
Yeast extract 1.25
Agar 20
Vegetable oil (e.g. soybean, maize) 1–2 ml
Sterol (e.g. cholesterol, ergosterol) 0.01–0.1
Lecithin 0.05–0.1
CaCl2·2H2O 0.07
Blastospore-producing medium Glucose 25
Soluble starch 25
Corn steep 20
NaCl 5
CaCO35
Note: most solid media can be used as liquid media by excluding the agar.
Conversely, adding agar can convert a liquid medium to a solid medium. The pH of
most media ranges between 5 and 9 with most workers adjusting to pH 7 with 1 M
NaOH or HCl.
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