Journal of

INVERTEBRATE
PATHOLOGY
Journal of Invertebrate Pathology 87 (2004) 7783
www.elsevier.com/locate/yjipa

Variations in UV-B tolerance and germination speed of Metarhizium

anisopliae conidia produced on insects and articial substrates
Drauzio E.N. Rangela, Gilberto U.L. Bragaa,b, Stephan D. Flintc,
Anne J. Andersona, Donald W. Robertsa,*
b

a
Department of Biology, Utah State University, Logan, UT 84322-5305, USA
Departamento de Analises Clnicas, Toxicologicas e Bromatologicas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade
de Sao Paulo, Ribeirao Preto, SP, 14040-903, Brazil
c
Department of Forest, Rangeland and Wildlife Sciences and the Ecology Center, Utah State University, Logan, UT 84322-5230, USA

Received 9 March 2004; ; accepted 30 June 2004

Available online 28 September 2004

Abstract
Solar ultraviolet radiation (UV-A and UV-B) is a major factor in failure of programs using the insect pathogenic fungus Metarhizium anisopliae as a biological control agent. Studies were conducted to determine if growth conditions, viz. articial (agar media
or rice grain) or natural (infected insects) substrates for conidial production aect two traits that directly inuence performance of
conidia after eld application: tolerance to UV-B radiation and conidial germination speed. Conidia of two isolates (ARSEF 23 and
ARSEF 2575) of M. anisopliae var. anisopliae produced on potato dextrose agar plus yeast extract (PDAY) or on fungus-killed larvae of two insect species, Galleria mellonella and Zophobas morio, were inactivated by exposure to UV-B radiation. Conidia of both
isolates when produced on insect cadavers were signicantly more sensitive to UV-B radiation than conidia produced on PDAY.
Also, conidia from insect cadavers germinated slower than those from PDAY cultures. A comparison of conidia from articial substrates showed that conidia produced on Czapeks and Emersons YpSs agar media or rice grains had higher tolerance to UV-B
radiation and germinated faster than conidia raised on PDA and PDAY. Accordingly, the growth substrate and nutritional environment in which conidia are produced inuences M. anisopliae conidial UV-B tolerance and speed of germination; and manipulation of these variables could be used to obtain conidia with increased tolerance to UV-B radiation and shorter germination times.

2004 Elsevier Inc. All rights reserved.
Keywords: Metarhizium anisopliae; Galleria mellonella; Zophobas morio; Fungal photobiology; Fungal nutrition; UV-B radiation; Conidial germination; Czapeks medium; Emersons YpSs medium; PDA and PDAY media

1. Introduction
Ecient biocontrol of insects with fungi requires
maintenance of viability and virulence of the fungal
inoculum (usually conidia) after eld application. Solar
radiation is a major environmental factor that negatively aects these traits in the entomopathogenic fungus
Metarhizium anisopliae (Braga et al., 2001a; Fargues
et al., 1996; Moore et al., 1993; Zimmermann, 1982).
*

Corresponding author. Fax: 1 435 797 1575.

E-mail address: dwroberts@biology.usu.edu (D.W. Roberts).

0022-2011/$ - see front matter
2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.jip.2004.06.007

Both the UV-A and UV-B fractions of solar radiation

can damage the conidia of M. anisopliae in a matter of
few hours (Braga et al., 2001a). In addition, conidia
of entomopathogenic fungi exposed to sub-lethal doses
of UV radiation undergo genetic and/or physiological
changes that can impair germination and growth rates;
and thereby reduce the eciency of these microorganisms as biological control agents (Braga et al.,
2001a,b,c,d, 2002a,b; Morley-Davies et al., 1995).
Phenotypic characteristics of a quantitative nature,
as is the case for tolerance to solar radiation, have a
complex genetic base and are usually strongly inuenced

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D.E.N. Rangel et al. / Journal of Invertebrate Pathology 87 (2004) 7783

by the environment. Thus, one interesting possibility

that could be explored to increase tolerance to solar
radiation and to increase conidial germination speed is
the manipulation of the physical and/or chemical
variables of the culture medium during the development
and conidiogenesis of fungi. Prior to the current
study, little was known about the eects of nutritional
environments in which M. anisopliae conidia are produced on germination speed and tolerance to UV radiation. The eects of nutritional environment and
physical factors, i.e., temperature and relative humidity
(RH), on conidial production of M. anisopliae have
been clearly demonstrated (Arthurs and Thomas,
2001; Sun et al., 2002).
Growth conditions also aect the virulence of entomopathogenic fungi. With M. anisopliae, virulence was
increased after a life cycle through insect hosts (Daoust
and Roberts, 1982; Fargues and Robert, 1978, 1983) or
some articial growth media (Daoust and Roberts,
1983; Fargues and Robert, 1983; Kmitowa, 1980; Lane
and Trinci, 1991). Conidia produced on insect cadavers
had on their surface higher concentrations of pathogenicity-related hydrolytic enzymes than conidia produced
on an articial medium (St. Leger et al., 1991), and culture media inuenced the ability of conidia to adhere to
insect cuticle (Ibrahim et al., 2002). Higher virulence and
stress tolerance, therefore, can be expressed phenotypically by physiological manipulation of endogenous reserves stored in or on conidia during conidiation. The
amounts of nutritional reserves apparently depend on
the medium on which the conidia were produced, and
those most frequently associated with germination are
carbohydrates and polyols (Gottlieb, 1976; Magan,
2001). Endogenous reserves accumulated in conidia during the conidiogenesis also may reduce germination
time. Faster germination has been correlated with higher
virulence in M. anisopliae (Al-Aidroos and Seifert, 1980;
Al-Aidroos and Roberts, 1978; Hassan et al., 1989;
Samuels et al., 1989) and Paecilomyces fumosoroseus
(Altre et al., 1999); and, in the eld, germination also reduces exposure of the fungal inoculum to solar UV
radiation.
Since high tolerance to (and/or avoidance of) UV
radiation is very important to successful use of M.
anisopliae conidia for biological control of agricultural
pests, the objectives of this study were to determine if
M. anisopliae conidia produced on dierent articial
growth substrates and on cadavers of insects killed
by the fungus vary in their UV-B tolerance and also
study whether conidia produced on dierent substrates
will cause variability in germination speed. As far as
we know, this is the rst study with M. anisopliae
showing the eects of growth conditions and nutritional environment on germination speed and on
conidial tolerance to environmentally realistic doses
of UV-B.

2. Materials and methods

2.1. Metarhizium anisopliae isolates
Metarhizium anisopliae var. anisopliae isolates ARSEF 23 and ARSEF 2575 were obtained from the
USDA-ARS Collection of Entomopathogenic Fungal
Cultures (US Plant, Soil and Nutrition Laboratory, Ithaca, NY, USA). ARSEF 23 was isolated originally from
Conoderus sp. [Coleoptera: Elateridae] in North Carolina, USA, and ARSEF 2575 from Curculio carryae [Coleoptera: Curculionidae] in South Carolina, USA. Stock
cultures were maintained at 4 C in test tubes on slants
of potato dextrose agar (Difco Laboratories, Sparks,
MD, USA) supplemented with 1 g L 1 yeast extract
(Technical, Difco) (PDAY) adjusted to pH 6.9.
2.2. Insects
Final-instar larvae of Zophobas morio [Coleoptera:
Tenebrionidae] and Galleria mellonella [Lepidoptera:
Pyralidae] were used for production of conidia of the
isolates ARSEF 23 and ARSEF 2575. Both insect species were obtained from Timberline (Marion, IL,
USA). No fungicides or antibiotics were added to the insects diets. Supplementary information on the insects
can be obtained at http://www.timberlinesheries.com.
2.3. Irradiation chambers, lamps, and lters
Irradiation experiments were conducted at 28 1 C
in a Percival growth chamber (Boone, IA, USA). Cellulose diacetate lters (JCS Industries, Le Miranda, CA)
were used to exclude UV-C and short wavelength UVB radiation provided by two UV-B uorescent lamps
(TL 20W/12 RS, Philips, Eindhoven, Holland) as described by Braga et al. (2001d). Lamp output was primarily UV-B (peak at 313 nm) with minimal UV-A
radiation (Braga et al., 2001b,c). Spectral irradiance
was measured as in Braga et al. (2001c). The DNA-damage (cyclobutane pyrimidine dimer formation) action
spectrum developed by Quaite et al. (1992) and normalized to unity at 300 nm was used to calculate weighted
UV irradiances in mW m 2. The reasons for using this
action spectrum and the reasons for selection of this biological spectral weighting function (BSWF) are discussed in Braga et al. (2001c).
2.4. Comparisons of conidia from insect cadavers and
PDAY medium
2.4.1. Production of conidia on PDAY and insects
The conidia used to inoculate articial media and insects were produced on 23 ml of PDAY medium in 95mm polystyrene Petri dishes (Fisher, Pittsburgh, PA,
USA) in the dark at 28 1 C and 100% relative humid-

D.E.N. Rangel et al. / Journal of Invertebrate Pathology 87 (2004) 7783

ity (RH) for 814 days. Suspensions of freshly harvested

conidia in 0.01% Tween 80 were used to inoculate the
agar medium. To produce conidia on insect cadavers,
dry conidia were spread with cotton swabs on dorsal
surfaces of Z. morio and G. mellonella larvae. Thirty insects were used for each fungal isolate. The inoculated
larvae were held without food in Petri dishes with moist
lter paper in dark, high-humidity chambers (100% RH)
at 28 1 C until the conidiation was completed (814
days). PDAY cultures were started at the same time as
the insect inoculations and incubated under the same
conditions as for the insects. A separate batch of conidia
was produced on insects or medium for each
experiment.
2.4.2. UV-B tolerance
To prepare inoculum for UV exposures, a microbiological loop was touched to aerial conidia without
touching the substrate (insect or medium) and suspended in 4 ml of sterile Tween 80 solution (0.01% v/v)
in 15 ml polystyrene tubes (Corning, Corning, NY,
USA). The suspensions were shaken vigorously using a
vortex and ltered through a polycarbonate membrane
(25-mm diameter, 8-lm pore size, Whatman Nucleopore, Clifton, NJ, USA) to remove spore aggregates.
Conidial concentrations were estimated by haemocytometer counts and dilutions made with sterile Tween
80 solution (0.01% v/v) for immediate use in the irradiation and germination studies.
For each of the three trials, conidia obtained from insects and from PDAY (40 ll, 1.5 104 conidia ml 1)
were spread onto PDAY (8 ml) plates (polystyrene
60 15 mm, Fisherbrand Pittsburgh, PA, USA), using
a sterile glass spreader. Conidia were exposed to
768 mW m 2 of weighted UV-B for 1, 2, 3, and 4 h, providing total doses of 2.8, 5.5, 8.3, and 11.1 kJ m 2,
respectively. Four replicate dishes per exposure time
were irradiated in each trial. Control conidia were not
irradiated but were placed in the chamber covered with
an aluminum foil barrier. The eect of UV-B radiation
on conidial relative culturability (%) was estimated as
described by Braga et al. (2001c). Briey, Relative
Culturability = (Mt/Mc) 100, where Mt is the mean
number of colonies of the four replicates at exposure
time t and Mc is the mean number of colonies for all
control-plates. Control-plate data were pooled over all
exposure times because length of time in the chamber
when plates were wrapped with aluminum foil did not
inuence colony numbers. Relative culturability,
where culturability of treated spores is compared to that
of untreated spores from the same lot, was calculated to
allow comparisons of dierent fungal strains or treatments. The basic observations were colony counts.
The eects of culture substrate (PDAY, Z. morio, and
G. mellonella) and exposure time for each isolate (ARSEF 23 and ARSEF 2575) on relative culturability were

79

assessed using analysis of variance of a two-way factorial in a completely randomized design. The use of a
completely randomized design assumes that there was
little or no variability among three trials (trial = a repetition made on a dierent date). Values of relative culturability were transformed using the arcsine square-root
transformation prior to analysis to better meet assumptions of normality and homogeneity of variance. In
addition, the statistical model tested for heterogeneous
(i.e., dierent) variances for conidia produced on dierent substrates (i.e., PDAY, G. mellonella or Z. morio).
2.4.3. Germination speed
To determine whether the substrate on which conidia
were produced inuenced endogenous reserves stored in
conidia during conidiogenesis, the conidial germination
rates were tested. The germination of conidia produced
on Z. morio, G. mellonella, and PDAY were determined
on 8 ml PDAY medium in polystyrene Petri dishes
(60 15 mm). The inoculum (a 20 ll drop, 105 conidia ml 1) was placed on the medium and the dishes kept
in darkness at 28 C. Germination was read at 400
magnication every 2 h until 12 h. Conidia were scored
for germination by moving from one edge of the drop
area to the other on a line that passed through the center
of the drop area. A conidium was considered germinated
when a germ tube projected from it (Milner et al., 1991).
A total of 300 conidia were scored for each treatment in
each of the three trials, and statistical analysis was performed as described above (Section 2.4.2).
2.5. Comparisons of conidia produced on four
mycological media and rice
2.5.1. UV-B tolerance
Studies of UV-B tolerance were repeated using the
procedures described above (Section 2.4.2) with conidia
produced on agar media: Czapek Solution Agar (Difco,
Sparks, MD), Emerson YpSs Agar (Difco), PDA, and
PDAY. Conidia also were produced on commercially
available long grain white rice enriched with ferric phosphate, niacin, thiamine mononitrate, and folic acid
(Smiths Food and Drug Centers, Salt Lake City, UT,
USA,). The rice (100 g) was autoclaved with 100 ml distilled water in 250-ml Erlenmeyer asks for 15 min at
120 C and inoculated after cooling with 5 ml of conidial
suspension (108 conidia ml 1) in 0.1% (v/v) Tween 80
solution. Cultures were incubated in darkness at
28 1 C for 14 days. Conidia were harvested and suspensions prepared as described above (Section 2.4.2).
A separate batch of conidia was produced on each substrate for each experiment, and each UV-B exposure
experiment was repeated three times. Conidia were exposed to a weighted UV-B irradiance at 897 mW m 2
for 2 h, providing a total dose of 6.5 kJ m 2. The eects
of media (Czapek, Emerson, PDA, PDAY, and rice)

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D.E.N. Rangel et al. / Journal of Invertebrate Pathology 87 (2004) 7783

and isolates (ARSEF 23 and ARSEF 2575) on relative

percent of CFU after UV-B exposure were assessed
using analysis of variance as described above. Signicance levels of pairwise comparisons among media were
adjusted for experimentwise Type I error using the TukeyKramer method.
2.5.2. Germination speed
Germination was recorded every 2 h from the time
when conidia were placed on the PDAY medium until
14 h. The eects of isolates (ARSEF 23 and ARSEF
2575), media (Czapek, Emerson, PDA, PDAY, and
rice), and incubation time (2, 4, 6, 8, 10, 12, and 14 h)
on percent germination were assessed using analysis of
variance with a three-way factorial in a completely randomized design as previously described.

3. Results
3.1. Comparisons of conidia from insect cadavers and
PDAY medium
3.1.1. UV-B tolerance
The eects of UV-B exposure on conidia produced on
PDAY and on insects are shown in Figs. 1A and B for
ARSEF 23 and ARSEF 2575, respectively. Relative
culturability decreased as exposure time increased
[(F3, 36 = 267.4, P < 0.001 for ARSEF 23) (F3, 36 =
70.2, P < 0.001 for ARSEF 2575)]. In all exposures,
both isolates showed higher CFU numbers when produced on PDAY as compared to the insect substrates
[(F2, 36 = 33.3, P < 0.001 for ARSEF 23) (F2, 36 =
111.1, P < 0.001 ARSEF for 2575)].
3.1.2. Germination speed
Percent germination increased with time and achieved
similar levels for conidia grown on the insects and on
PDAY (Fig. 2). For ARSEF 23 (Fig. 2A), there was

no dierence in the speed of germination for conidia

produced on Z. morio or G. mellonella, but conidia produced on PDAY germinated faster (F2, 54 = 7.7,
P < 0.001). For ARSEF 2575 (Fig. 2B), percent germination diered among groups (PDAY, Z. morio, and
G. mellonella) (F2, 54 = 10.0, P < 0.001), viz. lower germination of conidia produced on Z. morio than the other
two groups, and no dierence between conidia from
PDAY and G. mellonella.
3.2. Comparisons of conidia produced on four
mycological media and rice
3.2.1. UV-B tolerance
Relative culturability diered among media
(F4, 26 = 12.1, P < 0.001 for both isolates). Conidia of
both isolates produced on Czapek or Emerson media
or on rice had similar tolerance to UV-B radiation,
and this level was higher than that of conidia raised
on PDA or PDAY (Fig. 3). Relative culturability of
conidia exposed for 2 h to UV-B radiation was higher
for ARSEF 2575 than for ARSEF 23 (F1, 26 = 213.2,
P < 0.001 for both isolates) regardless of substrate used
for production.
3.2.2. Germination speed
For both isolates, germination of conidia produced
on Emerson or Czapek media and rice grains was faster
for the rst 8 h than for conidia produced on PDA and
PDAY media (Figs. 4A and B). Germination speed of
ARSEF 23 was highest for conidia produced on Emerson medium (Fig. 4A); whereas conidia of isolate ARSEF 2575 germinated fastest when produced on
Czapek medium (Fig. 4B). With the exception of the
interaction among isolate, media, and time
(F24, 182 = 1.3, P = 0.191), all terms in the model were
statistically signicant (F24, 182 = 13.0, P < 0.001 for
both isolates). These results imply that the proles of
percent germination over time were dierent among

Fig. 1. Mean relative percent culturability of Metarhizium anisopliae conidia produced on three dierent substrates following exposure of 1, 2, 3, and
4 h to irradiance of 768 mW m 2 weighted UV-B. Isolate ARSEF 23 (A) and ARSEF 2575 (B). Relative percent culturability was calculated in
relation to non-irradiated controls. Errors bars are standard errors of three independent experiments.

D.E.N. Rangel et al. / Journal of Invertebrate Pathology 87 (2004) 7783

81

Fig. 2. Germination speed at 28 C of Metarhizium anisopliae conidia produced on PDAY medium and fungus-killed Galleria mellonella and
Zophobas morio larvae. ARSEF 23 (A) and ARSEF 2575 (B). Errors bars are standard errors of three independent experiments.

(F24, 91 = 6.7, P < 0.001 for ARSEF 2575)], implying

that the prole of percent germination over time diered
among media.

4. Discussion

Fig. 3. Mean relative percent culturability of Metarhizium anisopliae

ARSEF 23 and ARSEF 2575 conidia produced on Czapek, Emerson,
PDA and PDAY media and rice grain after 2 h exposure to irradiance
of 897 mW m 2 weighted UV-B. Relative percent culturability was
calculated in relation to non-irradiated controls. Errors bars are
standard errors of three independent experiments.

media and among isolates. Percent germination was

greater for strain ARSEF 2575 than for strain ARSEF
23 (F1, 182 = 277.6, P < 0.001)
For a simple analysis of the eects of media and time
for each isolate, an analysis of variance of a two-way
factorial in a completely randomized design was computed using data for each isolate individually. For both
isolates, a signicant interaction of media and time was
detected [(F24, 91 = 7.9, P < 0.001 for ARSEF 23)

Growth and conidiogenesis of the fungus on dierent

substrates (insect cadavers, articial culture media, and
rice grains) signicantly changed both the tolerance to
UV-B radiation and the germination speed of M. anisopliae conidia. The culture medium also aected colony
morphology and conidial pigmentation (data not
shown). For both isolates, colonies formed on Czapek,
Emerson, PDA, and PDAY were morphologically different. When the isolates were grown on Czapek medium, the sporulation was poor, followed by growth of
a copious overlayer of white mycelium. Isolate ARSEF
23 always produced a mycelial layer (thin or abundant,
depending on the medium) which covered the spores,
but it was easily removed before conidial harvesting.
Abundant growth and sporulation was observed with
Emersons, PDA, and PDAY media and rice grain. On
Emersons medium, the conidia had darker pigmentation than on Czapeks, PDA, or PDAY media. Darker
pigmentation of M. anisopliae conidia by certain culture
media also has been reported by St. Leger et al. (1994)

Fig. 4. Conidial germination speed at 28 C of Metarhizium anisopliae conidia produced on Czapek, Emerson, PDA, and PDAY media and rice
grain. ARSEF 23 (A) and ARSEF 2575 (B). Errors bars are standard errors of three independent experiments.

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D.E.N. Rangel et al. / Journal of Invertebrate Pathology 87 (2004) 7783

and Ibrahim et al. (2002). Altered pigmentation has

been demonstrated in Aspergillus, Ustilago, and Cryptococcus to cause variability in resistance to UV radiation
(Al-Rubeai and El-Hassi, 1986; Wang and Casadevall,
1994; Will III et al., 1987).
Substrate and nutritional environment also aect the
physiology and biochemistry of conidia. Metabolites
present in conidia of M. anisopliae grown on articial
media diered from those present in conidia obtained directly from fungus-killed insects (Magan, 2001). High
amounts of erythritol, mannitol, and glucose with low
amounts of trehalose were found in conidia produced
on insects. In contrast, conidia produced on SDA medium (Sabouraud dextrose agar, containing neopeptone
and dextrose) had high levels of mannitol, glucose, and
trehalose and low levels of erythritol. Hallsworth and
Magan, 1996 showed that culture age and environmental
conditions aect the polyol and trehalose contents of
fungal conidia. Intracellular levels of polyols and trehalose of conidia were manipulated through the alteration
of water activity (aw) of culture medium. Conidia with increased intracellular concentrations of glycerol and
erythritol also germinated faster and at lower aw (Hallsworth and Magan, 1995). Accumulation of trehalose in
the cells cytosol as a carbohydrate reserve is known to
protect the cells against several stresses [e.g., desiccation,
dehydration, low and high temperature, low pH, and
oxidative damage (Benaroudj et al., 2001; Fillinger et
al., 2001; van Laere, 1989)]. The involvement of trehalose
in protection from stress caused by UV-B radiation is not
known; but since one eect of UV radiation, particularly
UV-A, is the formation of reactive oxygen species
(ROS), we may speculate that the intracellular levels of
trehalose may also aect the conidial tolerance to UV
radiation. Therefore, the low accumulation of trehalose
in conidia produced on insects, as evidenced by Magan
(2001), might be one of multiple factors that reduced tolerance to UV-B radiation; and, on the other hand, the
high amount of trehalose found in conidia produced on
articial media may increase tolerance to UV-B radiation and germination speed. Several factors aect germination speed, including age of culture (Hall et al., 1994),
exogenous nutrients (Ibrahim et al., 2002; James, 2001;
St. Leger et al., 1989), temperature (Walstad et al.,
1970), degree of conidial hydration prior to inoculation
(Dillon and Charnley, 1990, 1995), and the genetic background of the isolate, e.g., ARSEF 2575 germinates faster than ARSEF 23. Conidia of M. anisopliae require
signaling factors such as suitable exogenous nutrients
to trigger germination (Braga et al., 1999; Dillon and
Charnley, 1990; St. Leger et al., 1989, 1994).
It is clear that nutritional and physical factors during
conidiation deeply inuence the morphology, physiology, and chemical composition of conidia, as manifested
in various aspects of their biology, such as germination,
pathogenicity, and tolerance to stress-generating factors.

We propose that some enhancement of M. anisopliae

products may be achieved by conidiation under conditions that maximize UV tolerance and rapid germination of conidia. Growth on Czapeks or Emersons
media or rice grain provided the best results in our stress
studies. The rice grain substrate is attractive because of
its simplicity, relatively high yield, and low cost (e.g.,
Alves and Pereira, 1998; Daoust and Roberts, 1983;
Quintela, 1994).

Acknowledgments
We are grateful to Susan Durham for the statistical
analysis and to Dr. Martyn M. Caldwell for providing
use of the spectroradiometer. This project was supported in part by a NRICGP/CSREES #99-353028052 Grant, and a Utah State Mineral Lease (CURI)
grant. We sincerely thank the National Council for Scientic and Technological Development (CNPq) of Brazil for a Ph.D. fellowship for D.E.N.R; as well as the
State of Sao Paulo Research Foundation (FAPESP),
Sao Paulo, Brazil for nancial support to G.U.L.B.

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