DEVELOPMENT OFBEAUVERIA BASSIANA FOR CONTROL

OF GRASSHOPPERS AND LOCUSTS

STBT,aN T. JARONSKI
Mycotech Corporation, Butte, Montana, USA 59702

and MeRr S. Gor,rrBr-

Agriculture and Agri-Food Canada Research Centre, Lethbridge, Alberta, Canada TlJ 48l

Abstract Memoirs of the Entomological Society of Canada l7l: 225 231 (.1991)
Recognition of the potential of Beauveria bassiana (Balsamo) Vuillemin as a control
agent of grasshoppers and locusts occurred as early as I 936, in South Africa. Field testing
of B. bassiana as an inundative control agent of grasshoppers and locusts has been
f acilitated by development of a solid substrate method for mass-production of the fungus

and has resulted in the registration of a strain against grasshoppers in the United States.
In some, but not all field trials, application has resulted in substantial reductions in
grasshopper populations. Numerous environmental constraints, including temperature
and ultraviolet (UV) radiation, may limit field efficacy of the fungus. Laboratory studies
suggest that low humidity does not limit the ability of the fungus to initiate disease.
Sunlight is the major cause of mortality of conidia on leaf surfaces. The incorporation of
UVB protectants in formulations can increase conidial survival; however, these have not
yet been evaluated for their effects on field ef1icacy of B. bassiana against insects.
Themoregulation by grasshoppers has been implicated in resistance to mycosis. Results
of laboratory studies indicate that grasshoppers infected with B. bassiana preferentially
seek temperatures between 40 and 42"C and these temperatures are inhibitory to disease
development. ln field-cage trials, a higher prevalence and more rapid development of
disease were observed in grasshoppers placed in shaded cages than in grasshoppers
placed in cages exposed to 1ull sunlight. In laboratory experiments simulating
grasshopper thermoregulation during daylight periods, application of both Metarhizium
Jlavoviride Gams and Rozsypal and B. bas.siana simultaneously resulted in a linal
prevalence of disease that was greater than M . .flavoviride alone in the hot temperature
envrronment, and equal to B. bassiana alone in the cool temperature environment.
Incorporation of sublethal levels of Dimilin with conidia of B. bassiana increased
efTicacy of the fungus against grasshoppers in laboratory and field trials. Once
environmental consfaints are better ouantified. it mav be nossible to overcome them
through improved lbrmulation. strain selection. generic orphenotypic manipulation. and
inoculum targeting. Ultimately, success of B. bassiana as a microbial control agent will
depend on our ability to overcome environmental and other constraints and/or to predict
its efficacy under various environmental conditions.

Jauonski, S.T., et M.S. Goettel. 1997. Pr6paration de Beauver ia basslana en vue de la lutte biologique
contre les criquets. Memoirs of the Entomoktgk:aL Society of Canada l7l: 225-23'/ .

R6sum6
Le potentiel de Beauyeria bassiana (Balsamo) Vuillemin comme agent de lutte conffe
les criquets a 6t6 reconnu dbs 1936 en Afrique du Sud. Les tests sur le pathogbne des
criquets en nature ont 6t6 facilit6s par la mise au point d'une m6thode de productron en
masse du champignon sur un substrat solide, ce qui a pemis l'enregistrement d'une
souche efficace conffe les criquets aux Etats-Unis. Dans la plupart des tests en nature,
pas dans tous, I'application du pathogbne a occasionn6 de fortes r6ductions des
populations de criquets. De nombreuses contraintes dcologiques, notamment la
temp6rature et les radiations ultra-violettes (UV), peuvent nuire d I'efficacit6 de
l'organisme sur le terain. Les dtudes en laboratoire semblent indiquer qu'une humidit6
faible ne diminue pas I'action pathogdne du champignon. La lumidre solaire semble 6tre
la principale cause de mortalit6 des conidies i la surface des feuilles. L'incorporation de
substances protecffices contre les rayons UVB dans les pr6parations peut am6liorer la

225

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 226 MEMoIRs oF THE E\ToMoLoctcAL socrETY oF cANADA No. 17 I

survie des conidies, mais l'effet de ces substances sur I'efficacit6 du pathogbne en nature
n'a pas encore 6t6 d6termin6. La r6gulation thermique a 6t6 invoqu6e comme facteur de
r6sistance d la mycose chez les criquets. Les r6sultats d'expdriences en laboratoire
indiquent que les criquets infect6s par B. hassiana manifestent une pr6f6rence pour les
temp6ratures de 40-42'C, temp6ratures qui ont un effet inhibiteur sur le d6veloppement
de la maladie. Dans des essais sur le terrain, la maladie s'est av6r6e plus fr6quente et a
6volu6 plus rapidement chez les criquets gard6s dans des cages d I'ombre que chez ceux
gard6s dans des cages mises en plein soleil. Dans des exp6riences de laboratoire destindes
dL simuter la r6gulation thermique des criquets ) ta Iumibre du jour, I'exposition aux deux

pathogdnes Metarhizium flavoviride Gams et Rozsypal et B. bassiana a enffain6 des

pathologies plus g6n6ralis6es que 1'exposition exclusive d M. Jlavoviride d haute
temp6rature et des pathologies de pr6valence 6gale d celle provoqu6e par I'exposition
exclusive d B. bassiana d temp6rature fraiche. L'incorporation de doses subl6tales de
Dimilin aux pr6parations de conidies de B. bassiana a augment6 l'efficacit6 du
champignon contre les criquets, aussi bien en laboratoire qu'en nature. Lorsque les
contraintes 6cologiques auront 6t6 mieux comprises, il sera plus facile de les circonvenir
par utilisation de prdparations am6lior6es et de souches plus efficaces, par tentative de
manipulations g6n6tiques ou ph6notypiques et par un meilleur choix de cibles pour
recevoir f inoculum. En fin du compte, le succbs de B. bassiana comme agent de lutte
microbienne d6pendra de notre aptitude d contourner les contraintes environnementales
ou autres et (ou) de notre capacit6 de pr6voir 1'efficacit6 du champignon dans diverses
conditions du milieu.
[Traduit par la R6daction]

Introduction
Fungi are among the most important microbial pathogens of grasshoppers (Goettel et
al. 1995). Beauyeria bassiana (Balsamo) Vuillemin is a common pathogen of Orthoptera
(Goettel 1992 Goeltet et al. 1995), although fungi in the Entomophaga grylli (Fres.) Batko
complex are most often noted for their epizootics in grasshoppers (Carruthers et al. 1997).
Beauveria bassiana has been repeatedly isolated from grasshopper populations in North
America (Goettel 1992; Moore and Erlandson 1988), Pakistan, Africa (Schaefet 1936;
Jaronski et al.1994; Shah et al. 1991), Brazil and Australia (Goettel 1992). Sporadic high
mortality among laboratory populations of the brown locust, Locustana pardalina (Walk.),
has also been attributed to this pathogen (Prinsloo 1962).
The Chinese recognized beauveriosis around 1000 AD and the fungus was first demon-
strated to be an insect pathogen in 1834 by Augustino Bassi, after whom the type species of
Beauveriawas named. Earlier attempts to induceB eaureria epizootics by releasing diseased
insects (Luger 1 888) or massive releases of B . bassiand conidia across the state of Kansas
(summarized by Billings and Glenn 1911), Illinois, Nebraska, Missouri, Ohio, and Okla-
homa (summarized by Steinhaus 1915') to control the chinch bug (B/isszs leucopterus Say)
were not successful. In the 1930s, a widespread epizootic among locusts in southern Africa
demonstrated Ihat B. bassianahad potential as a control agent of grasshoppers and locusts
(Schaefer 1936).
Beauyeria bassiana, along with other hyphomycetous entomopathogens such as
Metarhizium anisopliae (Metsch.) Sorokin, has been the focus of commercial-scale efforts
to use fungi to manage insect pests in the past 20-30 years, especially in the former USSR,
France, eastern Europe, and the Republic of China, but also in the United States and Canada.
Beauveria bassiana has been used with limited success against the Colorado potato beetle,
Leptinotarsa der:emlineata Say, in the former USSR and Ukraine since the 1960s; typical
rates exceed 2 x 7013 conidia per hectare, in conjunction with chemical insecticides. Codling
moth, Cyclia pomonella (L.), has been another principal target, in France as well as in the
fomer USSR. Several groups in the former Czechoslovakia have ongoing developmental
programmes using.B. bassiana for the control of Colorado potato beetle, codling moth, and

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 MICROBIAL CONTROL OF GRASSHOPPERS AND LOCUSTS 221

a variety of other insect pests. One product (Boverin@), a powder or dust formulation, has
been commercially produced for a number of years, albeit on a limited scale, by one or more
agricultural cooperatives and small enterprises.
By the late 1980s, several ministries in the former USSR had Beauyeria production
facilities, two of which were each capable of producing several tons of conidia per year.
Production, howeveq was not sufficient to meet the plans to treat over 17 000 000 ha of
potatoes to control the Colorado potato beetle in the late 1980s (M. Fedorenko, former USSR
MINMEDBIOPROM, pers. comm.). In China, B. bassiana has been produced for control
of thecomborer,Ostriniafurnacalis (Hbn.), sincethemid 1970s by agriculturalcommunes
under somewhat primitive conditions, as well as by regional institutes using more sophisti-
cated procedures (Feng et al. 1994).
American commercial efforts date from 1962, when Nutrilite Corp. attempted to
mass-produce B . bassiana, but failed to develop a commercial product. Abbott Laboratories'
pursuit of mass-production and commercialization of the fungus for use against a number
of insects ended in 1989. More recently, Mycotech Corporation has developed mass-
production technology for B. bassiana, and has been pursuing commercialization ofseveral
strains of B. bassiana (Bradley et al. 1992). Their first mycoinsecticide, Mycocide GH@,
was registered in 1995 in the United States for the control of grasshoppers, locusts, and
molrnon crickets, Anabris simplex Haldeman. Registration was also received shorlly there-
after for the control of Homoptera, particularly whitefly, aphids, and thrips in a variety of
crops.

Thxonomy
Because its perfect stage is presently unknown,.B. bassiana is placed in the Deuteromy-
cotina (Fungi Imperfecti) within the Class Hyphomycetes. It is characterized by the sympo-
dial development of conidia from ampilliform conidiogenous cells (Fig. I ). The genus has
been extensively treated by Macleod (1954) and de Hoog (1972). Currently, the genus
Beauveria contains several species: bassiana, brongniartii (Saccardo) Petch 1= tenella),
amorpha (von Hoehnel) Samson and Evans, yelata Samson and Evans, and caledonica
Bissett and Widden. Beauveria amorpha and B. velata are rarely isolated and are pathogenic
toward Coleoptera and Lepidoptera, respectively (Samson and Evans 1982), whereas
B. caledonica has been isolated from the soil only (Bissett and Widden 1988).
Recent analysis of key polymorphic isozymes has revealed that B. bassiana may well
be a complex of species with affinities, both within and between the morphological species
andother Beauveria species. St. Leger etal. (1992) placed 142 isolates of B. bassiana,two
B. brongniartii, one B. vermiconia, and a Tolypocladium cylindrosporum Gams in 47
genotypic classes based on polymorphic alleles for four enzymes , with 52o/o of their isolates
falling into three classes and the remaining 76 isolates distributed among the remaining 44
classes. These studies suggest that the most common genotypes have a worldwide distribu-
tion. Indications are that Beauveria populations are composed of many distinct clones
isolated by heterokaryon incompatibility (Couteaudiere et al. 1994).

Pathogenicity, Distribution, and Host Range

Beauveria bassiana is a ubiquitous fungus that is infectious to a wide variety of insects
from most orders (Goettel et al. 1990; Li 1988). However, different genotypes exhibit
differences in virulence to different insect host species. For instance, Mycotech strain
GHA is highly virulent to the grasshopper Melanoplus sanguinipes (Fab.) yet either non-
pathogenic or weakly virulent to many other hosts (Goettel and Jaronski 1991). At least one
strain may be endophytic within some hybrid lines of com (Bing and Lewis 1992) and the
fungus can be isolated from the phylloplane of many plants (S.T. Jtuonski, unpublished).
Soils from a wide variety of climates harbour B. bassiana (Harrison and Gardner 1991;

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 MEMOIRS OFTHE ENTO\4OLOCTCALSOCIETY OF CANADANO I7I

Frc I Conidiogenesis in Beaut'eria bassiana Conidia are fbmed sympodially on a rachis originating fiom ampul-
lilbrm conidiogenous cells. Bar = 5 p,m. Micrograph courtesy of G Douglas Inglis

Humber 1992; Ouedraogo 1993; Shah et al. 1991 , M.S. Goettel and D.L. Johnson, unpub-
lished) often at levels of several thousand colony-forming units (cfu) per gram of soil
(S.T. Jaronski, unpublished). It is unclear whether the fungus can grow saprophytically in
soil or is present only as contaminating conidla.
The interaction of B. bassiana with insect hosts has been recently reviewed by Hajek
and St. Leger (1993), St. Leger (1993), and Bidochka et al. (1997) and will not be dealt with
in great detail herein. In brief, when aerial conidia contact the cuticle of susceptible insects,
they germinate. Although it is generally believed that high humidity is a requirement for
infection, this is not necessarily so with several hosts, including grasshoppers (Ferron 1977;
Ramoska 1984; Marcandier and Khachatourians 1987; S.T. Jaronski, unpublished). Cer-
tainly, when conidia of Metarhizium flavoviride Gams and Rozsypal are applied to insects
in oil, ambient humidity does not seem to be imporlant in the initial gemination and
penetration of the host (Bateman et al. 1993). A short hypha emerging from the conidium
penetrates the cuticle by mechanical pressure and enzyme action. The fungus rapidly
proliferates within the insect's haemocoel, either by multiplication of yeast-like blastospores
or by hyphal growth. The host insect dies within a few days; time to death depends upon
dose as well as strain-specific pathogenicity. Soon after the insect dies, and when relative
humidity is high, the fungus produces mycelium that quickly coverthe surface of the cadaver
and give rise to conidiogenous cells and conidia, often appearing like a white powder
covering the insect. A number of secondary metabolites such as the cyclodepsipeptides,
beauvericin and bassianolide, and a dihydroxybenzoquinone, oosporein (Roberts 198 1), are
thought to repress saprophytic microorganisms in the insect cadaver, allowing B. bassiana
to complete its development and sporulate.

Sporulation and Mass-production

Beauyeria bassiana can produce three spore types in culture: aerial conidia, blasto-
spores, and submerged conidia. Aerial conidia are produced on insect cadavers and on solid

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 MICROBIALCONTROLOF GRASSHOPPERS AND LOCUSTS 229

substrates. Thin-walled, single-cell hyphal bodies, termed blastospores, along with varying
amounts of mycelium are produced in submerged culture by most isolates, especially when
amino nitrogen exceeds a critical level (Bidochka et al. 1987). These propagules are
smooth-walled, larger than conidia, and contain different wall surface carbohydrates. Blas-
tospores usually germinate much faster than either of the two other forms, 507o germination
being reached within 5-6 h.
Submerged conidia are produced either singly at the tips of conidiogenous cells arising
from mycelium, or directly on the tips of short germ tubes arising directly fiom blastospores
(Thomas et al. 1987; Hegedus et al. 1990). Low concentrations of phosphate in combination
with a simple, inorganic nitrogen source (a C:N ratio of 5:1 may be optimal) seem to induce
this conidial form, but the process may be isolate-specific (S.T. Jaronski, unpublished). The
hydrophobicity and lectin-binding characteristics of aerial and submerged conidia are very
similar and both types of spores ale virulent against grasshoppers (Hegedus et al. 1992).
There has been considerable speculation and hope that submerged conidia could serve
as an active ingredient in a commercial mycoinsecticide. That hope has not yet been realized
and aerial conidia seem to be the most suitable spores for commercialization (Jenkins and
Goettel 1997). The best reported yield of submerged conidia is 5.9 x l0rrper litre, compared
with I -1.5 x 1013 aerial conidia perkilogram in Mycotech's Solid Culture System@ (Bradley
et al. 1992; Jenkins and Goettel 1997). Efforts to improve the production efficiency of
submerged conidia have not been successful. The production ofB. bassiana has been dealt
with in more detail by Jenkins and Goettel (1991), as well as by Feng et aL (1994), and will
not be discussed further herein.

Persistence
A number of factors affect persistence of B. bassiana conidia in epigeal habitats.
Sunlight is the major cause of conidial mortality on the phylloplane. Inglis et al. (1993)
observed a steady decline in B. bassiana populations after application of conidia to crested
wheatgrass and alfalfa plots in Canada. The rate of decay was affected by plant species
(probably as affected by the morphology of the planr canopy), location within the canopy,
and spray formulation, although differences resulting from formulation were minimal;
compared with their original levels, B. bassiana conidial populations 16 days after applica-
tion were 170 on crested wheatgrass and 15-127o on alfalfa. In a subsequent study, Inglis,
Johnson, and Goettel (1991a) found that conidial persisrence on grasshopper nymphs was
similar to that of conidia on leaf surfaces. Daoust and Pereira (1986) observed a conidial
half-life on cowpea foliage in Brazil of l-2 days, with almost complete disappearance of
viable conidia by 1 week after application. S.T. Jaronski (unpublished) determined the
halflife of B. bassiana conidia on the upper surfaces of foliage in southem Califomia to be
1.7 days on melon and I day on broccoli. After 1 week, conidial populations on broccoli
were less than l%o of original levels and in melons, l}a/o. The slopes of the conidial
degradation were significantly steeper on the latter plant, suggesting a possible role of the
plant surl'ace in the rate of degradation. Survival on the undersides of melon leaves is greater,
with conidial half'-lives of 5-9 days and quarter-lives of 7-16 days (S.T. Jaronski and
J.C. Lord, unpublished). Gardner et al. (1977) measured persistence in soybean indirectly,
by infection rates among fall armyworm, Spodoptera /iugiperda (J.E. Smith), and found the
half-life of Beauveria to be 4.2 days.
Rainfall also removes conidia from foliage, but Inglis, Johnson, and Goettel (1995)
observed only a 567o reduction in conidial levels on alfalfa and wheat after exposure to
113 mm of simulated rainfall in t h (conidia were applied in a water suspension). In the field,
the rapid deactivation of conidiaby solarradiation may obscure the eff'ect of conidialremoval
by rain (Inglis et al.1993; Inglis, Goettel, and Johnson 1995; Inglis, Johnson, and Goertel
1995). The use of UVB protectants in formulations can increase conidial persistence on

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 230 MEMotRs oF THE ENToMoLocICAL socIETY oF cANADA No. 171

phylloplanes (Inglis, Goettel, and Johnson 1995; Moore and Caudwell 1997); however, these
have not yet been evaluated for their effects on field efficacy of B . bassiana against insects
or for their commercial economics.
Persistence of B. bassiana in the soil depends on many biotic and abiotic factors. The
soil microflora can have severe detrimental effects on the survival of B . bassiana (Lingg and
Donaldson 1981; McDowell et al. 1990; Quintelaetal. 1992). In sterile sollBeauveria can
rapidly multipty and increase its population by several levels of magnitude, given optimal
moisture and nutritional levels. On the other hand, fungistasis can occur when soil microflora
rs present.
In nature, there can be a marked increase in the cfu per gram followed by a slow decline
of several orders of magnitude during 4-8 weeks after application of conidia to the top
l0 cm of soil. In some soil types, the initial increase is absent and only a slow steady decline
follows application (S.T. Jaronski, unpublished). Inglis, Duke et al. (1991) discovered that
B. bassiana deposited on soil decreased rapidly in the first 20 days but then stabilized. There
was lessthanoneorderof magnitudelossincfupergramof soil overthe225-212 days of
the study. In another study,.B. bassiana in soil was active against the citrus root weevil up
to 25 weeks (McCoy et al. 1984). At present, the effects of the abiotic and biotic factors in
soil on B. bassiana are difficult to predict.

Virulence and Thrgeting Against Grasshoppers

Schaefer(1936)demonstratedthatB. bassianaisinfectivetolocustsandallofthelocust
adults and nymphs sprayed with conidia in the laboratory died within 5-20 days. Even
untreated locusts kept in separate rooms became infected from contaminating conidia. More
recently, B. bassiana was shown to be infective to grasshoppers that eat wheat leaves
contaminated with conidia (Johnson et at. 1988; Inglis et al. 1993; Delgado et al. 1991).
Careful strain selection is needed when choosing a potential microbial control agent.
Using a lettuce bait inoculation method with third-instar M. sanguinipes, Inglis et"al. (1996a)
compared virulence of the Mycotech strain GHA, which was initially isolated from a
non-orthoperan host and passaged through M. sanguinipes, to isolates collected liom an
alligator and from Canadian and African soils. The GHA isolate was the most virulent with
anLD5{rof5.7x103 conidiaperhopperandanLlroof5daysatadoseof l05conidia.Using
a similar lettuce bait inoculation method, Delgado eI al. (1991) detected no difference in
virulence against Oedaleus senegalensis (Krauss) between the GHA strain and ur isolate
from Burkina Faso soil when the bait was formulated with an emulsifiable oil. Using a topical
applicationofconidiainsunfloweroilagainstO. senegalensis,Ouedraogo(1993)foundthat
the virulence of the GHA strain was similar to that of five isolates obtained from Burkina
Faso soils, but two Burkina Faso soil isolates were much less virulent. The LTrn values for
adult M . sanguinipes inoculated by dipping in an aqueous suspension of conidia of each of
10 isolates of B. bassiana ranged between 4.1 and 7.9 days (Khatchatourians 1992). The two
most virulent isolates were from soil, indicating that the substrate of strain origin may be a
poor predictor of efficacy against a pafticular host.
The long persistence of B. bassiana in soil and the oviposition habits of most orthop-
terans suggest another approach for field application and host targeting (Inglis, Feniuk et al.
1995). Application of '7 x 106 to 4 x 107 cfu per gram of oviposition substrate (sand), in a
controlled environment, caused extensive mortality among addt M. sanguinipes and also
among subsequently emerging nymphs. Application of B. bassiana to oviposition sites may
thus offer an altemative inoculum targeting method, although no field work following this
approach has been conducted.
Moore and Erlandson (1988) found that conidia in an aqueous suspension were more
efficacious when applied topicatly to nymphs of M. sanguinipes than when applied orally.
However, when conidia were formulated in sunflower oil, Ouedraogo (1993) found no

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 231

significant differences in mortality of O. senegalensis inoculated topically or orally. Nymphs

of M. sanguinipes were more susceptible to mycosis if they fed on lettuce or bran treated
with conidia in oil rather than water (Inglis et al. 1996b). This increase in efficacy of conidia
in oil was attributed to increased surface-contamination of nymphs during ingestion;
conspicuously more fluorescent dye was observed on nymphs feeding on oil-based than on
water-based bait formulations. Using a similar bait-inoculation method, Delgado et al.
(1991) showed that emulsifiable oil formulations were more efficacious against
O. senegalensis than were formulations of conidia in flowable oil and clay-based formula-
tions. In contrast, in bioassays against Locusta migratot'ia migratorioides (Reiche and
Fairemaire), formulations of conidia in the flowable oil carrier were more efficacious than
either emulsifiable oil or clay formulations. This discrepancy in efficacy between the two
species remains unexplained, but it could result from differences in insect size.
Incorporation of sublethal amounts of Dimilin (diflubenzuron) in oil formulations of
B. bassiana (GHA) has been demonstrated to increase efficacy of the fungus (Reuter et al.
1996). At an application rate equivalent to 2.5 x 1011 conidia per hectare, mortality of
grasshoppers treated with the fungus/Dimilin combinationwas 600/o as compared with247o
for the fungus only treatment, 10 days post-application. At a higher rate of fungus application
(equivalent to 2.5 x 1013 conidia per hectare), both treatments resulted in >98Vo mortality;
however, the addition of Dimilin increased the speed of kill. The precise role of Dimilin in
infection of grasshoppers by B. bassiana is unknown; however, previous laboratory studies
wing M. anisopliae and Manduca sexta (Joh.) demonstrated that Dimilin facilitated fungal
penetration by altering the structure of the insect's cuticle (Hassan and Chamley 1989).
Strain selection, formulation, and host targeting play an important role in the develop-
ment of a successful microbial control agent (Moore and Caudwell 1991). The difficulty lies
in the development of bioassay methods that provide information that could be used to
predict efficacy under field conditions. Inoculation techniques and environmental conditions
adopted should mimic as much as possible those conditions expected at the host level in the
field situation.

Field Tiials
The first attempts to control Orthoptera with B . bassiana occurred in South Africa. After
observing natural epizootics ofthe fungus among populations ofred locusts, Schaefer (1936)
carried out several experiments evaluating the potential of the fungus to control locust
outbreaks. When locusts in outdoor cages were sprayed with an aqueous suspension of
fungus with an unspecified amount of conidia, TJVo succtrmbed and had signs of infection
by B. bassiana. Schaefer also sprayed locusts in outdoor corrals resulting in l2o/o mycosis;
another 5Jo/o of the locusts died but did not yield sporulating fungus. A swarm in a small
valley was also treated, but with a larger amount of conidia and volume of water, but no
mycosis was detected. Schaefer concluded that control with B. bassiana was not feasible.
Recently, several field trials have evaluated efficacy of B . bassiana against grasshoppers
in Africa and North America. Oil-based formulations of Mycotech strain GH were applied
in 1990 to open field sites in Mali, West Africa (Johnson eI al. 1992). Conidia were applied
at a rate of l.2x 10r3 conidia in 3 L of paraffinic oil per hectare using Micro-Ulva@ spinning
disk sprayers. Although no direct reduction in grasshopper populations was observed,
mortality of grasshoppers held in cages for 14 days after collection from sprayed plots was
J2o/o.
In l99l and 1992, field trials in the United States with Mycotech strain GHA resulted
rn 60-93Vo mortality of six species of Orthoptera held in field cages for 8 days following
ultra low volume (ULV) aerial or ground application of 5 x 1013 conidia per hectare in an
oil-based formulation (Foster et al. 1991, 1992).That same year, a wheat bran formulation
of the fungus, applied at 4 x 70t2 conidia on 2 kg wheat bran per hectare, resulted in greater

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 232 MEMoIRS oF THE ENToMoLocICAL soclBTY oF cANADA No. 171

than 907o mortality among mormon crickets held in the laboratory for 9 days (Mycotech
Corporation 1991). Aparallel trial in Canada using 2 x 1013 conidia on 10 kg wheat bran per
hectare yielded population reductions of 60 and 33o/o by 9 and 15 days after application
(Johnson and Goettel 1993). Mortality of grasshoppers held in cages for up to 30 days was
707o for those collected 2 days afier application, declining to 4lo/a by 13 days and 5a/a by
l9 days.
Various formulations of strain GHA were evaluated in 1991 in arena-type field enclo-
sures of either 20 or 50 m2 in Cape Verde againstO. senegttlerzsis (Delgado etal. 1991).
Wheat bran formulations were more efficacious than were oil or oil-clay formulations. ln
subsequent open-field (2-ha block) tests, using 2.5 x 1013 conidia per hectare applied either
as an emulsifiable suspension at 5 L spray per hectare with Micro-Ulva@ sprayers or as an
emulsifiable suspension amended with clay ar 20 L per hectare with backpack sprayers,
populations were reduced by 45Vo after 1 week. Mortality among grasshoppers collected I h
after fungus application and held in the laboratory fbr 10 days was 98Vo. Additional trials in
Mali indicated an initial population reduction of 887o,96Vo mortality among grasshoppers
in field enclosures and 64-'72a/o mortality among grasshoppers introduced onto sprayed
vegetation I h after fungus application (Montana State University 1993).
Addition of Dimilin at l0o/o of recommended field application rate to an oil formulation
of GHA significantly improved field performance against rangeland grasshoppers in
Nebraska (Foster et al. 1996). GHA was applied aerially at2.5 x 1013 spores per hectare with
or without sublethal doses of either Dimilin, Sevin, Orthene, or Fyfanon to 4-ha plots at a
rate of 9.3 L per hectare. At 14 days after treatment, there was an 88% reduction in
populations of grasshoppers in the GHA + Dimilin treatment whereas the other treatments
resulted in population reductions of 14-3'7o/o as compared with an untreated control plot.
Further investigations are warranted using combinations of sublethal doses of chemical
stressors and B. bassiana combinations.
However, not all field trials have been as successful. For instance, Lobo-Lima et al.
(1992) reported that application of up to 2 x 1013 conidia in 5 L per hectare of an oil
suspension resulted in infection of only 30o/o of field-collected caged grasshoppers held fbr
15 days and no significant reduction in field populations. Foster et al. (1994) reported much
lower than expected mortality in their 1994 field trials which was attributed to a possible
loss of virulence of the conidia. Inglis, Johnson, andGoettel(1991a) found both these conidia
and those of a previous production batch were highly virulent against M. sanguinipes rn
laboratory bioassays. Furthermore, in a field trial, conidia from both production batches were
applied at a rate of 2.5 x 1013 conidia in ll2L per hectare of a 1.57o oil emulsion amended
with 4o/o clay suspension in water. Despite excellent targeting of grasshoppers, neither
treatment reduced total populations or significantly reduced populations of specific grass-
hopper taxa. However, approximately 80o/o of grasshoppers, collected immediately and
2 days after conidial application and maintained in cages in a greenhouse environment for
l0 days, succumbed to mycosis. Mycosis rates decreased to less than 30% for those collected
15 days post-application. The onset of mycosis always occuned 3-4 days after caging
regardless of collection time post-application. A similar phenomenon was observed in a later
trial (Inglis et al. 1997b) and in a previous field trial, but in the latter, rates of mycosis in the
cages corresponded to estimated reductions in field populations of grasshoppers (Johnson
and Goettel 1993). Results of field studies suggest that at times conditions in the field were
not conducive to mycosis, whereas those in the cages held in a greenhouse were conducive
because virulence of conidia was high under laboratory conditions, the field targeting of
conidia was excellent, cage morlality was high, and there was a lag in mortality of caged
field-collected grasshoppers (Inglis, Johnson, and Goettel 1997a, b).
The differences in,B. bassiana virulence between field and cage environments have also
been noted by Mason and Erlandson (1994). These differences have provided an opportunity

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 MICROBIALCONTROLOFGRASSHOPPERS ANDLOCUSTS 233

to elucidate environmental constraints on B. bassiana efficacy (see below). Furlher investi-

gation is needed; in the meantime, however, extreme caution must be exercised when
attempting to evaluate field efficacy based solely on mycosis rates in caged grasshoppers.

Environmental Constraints
Thermoregulation has been implicated in the ability of grasshoppers and flies to
overcome infection by entomophthoralean fungi (Carruthers et al. 1992; Watson et al. 1993).
Grasshoppers thermoregulate by basking in the sun, increasing their temperature up to 1SoC
higher than ambient (Chappell and Whitman 1990; Kemp 1986) and elevation of body
temperature may account for lower rates of mycosis in free-living versus caged grasshoppers.
Inglis et al. (1996c) found that nymphs of M. sanguinipes inoculated with conidia of B.
bassiana and exposed to more than 4 h of 35'C per day had a lower incidence of mycosis
than those held at lower temperatures. The upper constant temperature limit for conidial
germination and mycelial growth of strain GHA is 36"C (S.T. Jaronski, unpublished).
By comparing mycosis in grasshoppers from plots sprayed with B. bassiana and placed
in cages in a greenhouse environment or in the field (exposed to direct sunlight, shaded tiom
sunlight, or protected from UVB radiation), Inglis et al. (1991b) demonstrated the impor-
tance of temperature and quantity and quality of solar radiation; a higher incidence and more
rapid development of disease were obserrred in grasshoppers placed in greenhouse (>90%)
and shaded (>807o) cages than in grasshoppers placed in exposed(l\ok) and UVB-protected
(43Vo) cages.
It may be possible to overcome some of the constraints to consistently high field efficacy
through formulation and/or strain selection (Moore and Caudwell 1997). For instance,
Ouedraogo (1993) demonstrated that several isolates from Burkina Faso soils germinate at
higher temperatures than Mycotech's GHA. Also, one of these strains is more resistant than
GHA to UVB radiation (J. Fargues et al. 1996). Under laboratory conditions, Inglis et al.
(1997c)foundthatapplicationofbothB. bassianaandM.flatovirideconidiasimultaneously
resulted in a final prevalence of disease in M. sanguinipes that was greater than that which
resulted when M..flavoviride was applied in a simulated hot temperature environment, and
equal to that which resulted when B. bassiana was applied in a simulated cool temperature
environment. These results suggest that the use of "cocktails" of isolates and/or species with
specific activities under different environmental conditions may be a way to overcome some
of the constraints of temperature on entomopathogenic Hyphomycetes against grasshoppers
and locusts. Much more effort is needed to identify and evaluate biotic and abiotic factors
that influence the development of fungus inf-ections in grasshoppers and locusts, both in
naturally occurring infestations and as a consequence of artificial augmentation of inoculum.
This will be accomplished only through more studies on epizootiology of the disease in
grasshoppers and other insects.

Conclusions
Field trials show that B. bassiana is a promising microbial control agent of grasshoppers
and locusts. However, much more effort is needed to evaluate this fungus fully as a
commercial microbial control agent. A better understanding of epizootiology and the
environmental constraints is needed. Once these constraints are known, it may be possible
to overcome some of them through novel targeting strategies, strain selection, and/or
formulation. Identification of microclimatic constraints would also allow development of
predictive models which would identity windows of opportunity thereby optimizing effica-
cious use of these mycopesticides. Ultimately, success of B. bassiana as a microbial control
agent will depend on our ability to overcome some constraints and/or to predict its efficacy
under various environmental conditions.

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 234 MEMOIRS OF THE ENTOMOLOGICAL SOCIETY OF CANADA NO, I7I

Acknowledgments
We thank Doug Inglis and Helen McMenamin, Lethbridge Research Centre, for criti-
cally reviewing the manuscript. The SEM micrograph was kindly supplied by Doug Inglis.
This paper is LRC contribution # 3819548.

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