E¡ect of nutrition on growth and virulence of the
entomopathogenic fungus Beauveria bassiana
S.A. Safavi1,2, Farooq A. Shah1, Aziz K. Pakdel2, G. Reza Rasoulian2, Ali R. Bandani2 & Tariq M. Butt1
1
Department of Biological Sciences, University of Wales, Swansea, UK; and 2Department of Plant Protection, University of Tehran, Karaj, Iran
Correspondence: S. Ali Safavi, Department
of Plant Protection, University of Tehran,
Karaj, Iran. Tel.: 198 9144048349;
fax: 198 261 2238529;
e-mail: s.a.safavi@gmail.com
Received 8 November 2006; accepted
10 January 2007.
First published online 22 February 2007.
DOI:10.1111/j.1574-6968.2007.00666.x
Editor: Geoffrey Gadd
Keywords
Beauveria bassiana ; Metarhizium anisopliae ;
nutrition; Pr1; CN ratio.
Introduction
Entomopathogenic fungi are attracting attention as biocon-
trol agents for insect pests (Clarkson & Charnley, 1996)
and are being considered as supplements or alternatives to
synthetic insecticides (St. Leger et al., 1996). Beauveria
bassiana (Bals.) Vuil. is a well-known and cosmopolitan
entomopathogen, some isolates of which have been com-
mercialized. Serial passage of entomopathogenic hyphomy-
cetes has been shown to alter virulence (Vandenberg &
Cantone, 2004), but passage through a host may improve
virulence (Aizawa, 1971; Ferron, 1985). Stability of virulence
following in vitro passage is clearly a desirable trait for a
mass-produced biocontrol agent (Vandenberg & Cantone,
2004) to ensure the consistent quality of the marketed
material throughout the mass-production process (Brown-
bridge et al., 2001). Some reports indicate maintenance of
virulence after serial in vitro passage of hyphomycetes
(Ferron et al., 1972; Hall, 1980; Hayden et al., 1992), but
others show that these entomopathogens may be attenuated
c 2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Abstract
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Three isolates of the entomopathogen Beauveria bassiana along with one strain of
Metarhizium anisopliae were cultured on seven media with different carbon/
nitrogen (C/N) ratios. The effect of nutrition on virulence of the isolates was
evaluated via measurement of colony growth, spore yield, germination speed,
conidial C/N ratio and Pr1 (a serine protease) activity. ‘Osmotic stress’ medium
produced the lowest colony growth with low numbers of conidia in all isolates.
However, these conidia showed a high germination rate and virulence. However,
conidial Pr1 activity was low in some isolates. In most but not in all cases conidia
from 1% yeast extract, 2% peptone and low (10 : 1) C/N medium had higher Pr1
activity compared with conidia from other media. However, in some instances we
could not conclude that there was a relationship among germination rate, conidial
Pr1 activity and virulence. C/N ratio of conidia was statistically different among
various media and fungal isolates. Conidia with lower C/N ratio generally
produced lower LT50 (lowest median lethal time) values (more virulent). Insect-
passaged conidia from different media had lower C/N ratio compared with similar
conidia from artificial cultures. Therefore, they should be more virulent than in
vitro produced conidia. As germination rate, conidial Pr1 activity and C/N ratio
are independent of host, it seems that host-related determinants such as insect
cuticle and physiology and environmental conditions may influence host suscept-
ibility and therefore fungal isolate virulence towards host insects.
after consecutive in vitro transfer (Kawakami, 1960; Schaerf-
fenberg, 1964; Aizawa, 1971; Nagaich, 1973; Fargues &
Robert, 1983; Morrow et al., 1989). Spore size, germination
speed and attachment to cuticle have all been considered as
reliable determinants of pathogenicity (Jackson et al., 1985;
Lane et al., 1991; Chandler et al., 1993; Altre et al., 1999). In
addition, bioassays, conidia production in artificial media,
temperature growth optima, tolerance to solar radiation and
production of beauvericin have been considered as good
parameters in strain selection research using Beauveria
bassiana against plant bugs (Leland et al., 2005). The
feasibility of using fungi as biocontrol agents against im-
portant insect pests is dependent on numerous biological
constraints, including the ability to produce high concen-
trations of stable propagules at a reasonable cost (Jaronski,
1986; Latgé et al., 1986). Vega et al. (2003) measured spore
yields in three entomopathogenic fungi in six nutritionally
(C/N ratio) different liquid media. In general, highest spore
yields were obtained in media containing 36 g L 1 carbon
concentrations and a C/N ratio of 10 : 1. Spore production
FEMS Microbiol Lett 270 (2007) 116–123
Nutrition and virulence of Beauveria bassiana
by B. bassiana isolates was variable, with one isolate produ-
cing high spore yields (12.2  108 spores mL 1) after 7 days
of growth.
In addition, these fungi produce an array of hydrolytic
enzymes thought to be utilized in cuticle penetration
(Charnley & St. Leger, 1991). Proteolytic enzymes along
with chitinases and lipases are important factors of entomo-
pathogenic fungi (Samuels & Paterson, 1995). Pr1, a serine
protease, plays a major role in insect penetration and
subsequent pathogenicity (St. Leger et al., 1987, 1988a,
1996; Goettel et al., 1989). Although the factors responsible
for expression of B. bassiana protease are unknown (Urtz &
Rice, 2000), the carbon and/or nitrogen source that results
from growth medium composition may be involved in the
expression of the pr1 gene in conidia.
Studies on Metarhizium anisopliae (Shah et al., 2005)
revealed that the nutrition source of conidia can affect the
levels of transcripts of pathogenicity-related genes. Our aim
here was determine if there the same strategy as in Metarhi-
zium anisopliae occurs in the entomopathogen B. bassiana,
in a disparate systematic situation and with an important
role in host attack. Pathogenicity of entomopathogenic
hyphomycetes towards insects has undoubtedly originated
from disparate pathogenicity factors that interact with each
other. Most studies have focused on inoculum produced
on artificial media and consider its influence on either
colony growth or virulence (Shah et al., 2005). To our
knowledge, this is the first study on a promising entomo-
pathogen, B. bassiana, which concentrates on the interaction
between nutrition and virulence of in vitro and in vivo
produced conidia. Quality control of inoculum with regard
to commercialization of strains of B. bassiana is discussed.
Materials and methods
Fungal strains
Ten strains of B. bassiana from Iran were used against
Tenebrio molitor and Galleria mellonella, and after passaging
and bioassays three strains, one highly virulent, one moder-
ately virulent and one less virulent, were selected for further
nutritional studies. Conidia were recovered from mycosed
insects on Oatmeal dodine agar as a selective medium.
Colonies were made by transferring a single spore to Sabour-
aud dextose yeast agar (SDYA) and these subcultures were
used as the source of subsequent studies. These strains were
DEBI007 from soil, DEBI008 from Acrididae, BEH 1 from
soil and a strain (V275) of M. anisopliae from Finnish soil.
Culture conditions
Culture media representing disparate carbon and nitrogen
sources and ratios were used in this study. They included:
(1) chitin peptone nutrient (CPN) consisting of 1% chitin,
FEMS Microbiol Lett 270 (2007) 116–123
117
0.5% peptone, 0.2% yeast extract; (2) high C/N (75 : 1)
medium consisting of 9.1% glucose and 1% peptone; (3)
low C/N (10 : 1) medium consisting of 0.6% glucose and 1%
peptone; (4) intermediate C/N (35 : 1) medium consisting of
4% glucose and 1% peptone ( = SDA); (5) nutrient-poor
media consisting of either 2% peptone (2P) or 1% yeast
extract (1Y); and (6) ‘osmotic stress’ medium (OSM)
consisting of 8% glucose, 2% peptone, 5.5% KCl. Yeast
extract and peptone have C/N ratios of 3.6 : 1 and 8 : 1,
respectively and represented different carbon and nitrogen
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sources (Wyss et al., 2001). All the media were prepared
using 2% agar except CPN, with 1.8% agar, and OSM, which
required 5.5% agar to solidify. Media were sterilized at
121 1C at 15 psi for 15 min and 15 mL poured into 9-cm-
diameter Petri dishes. Glucose, yeast extract, agar and KCl
were obtained from Sigma, while mycological peptone was
obtained from Oxoid.
Monitoring growth and sporulation
Any probable relationship among growth rate, sporulation
and inoculum virulence was investigated using the above
media. A 2-mm-diameter mycelial plug taken from the
growing plate of a 14-day-old culture grown on SDYA at
27 1C in the dark was used for inoculation of each medium.
Inoculated Petri dishes were sealed with Parafilm and
incubated at 27 1C in the dark. Colony diameter was
measured five times at right angles at 3-day intervals and
radial growth (mm day 1) calculated from the linear por-
tions of the curves plotted from these values. Two samples
including 5-mm plugs from each plate were taken and
conidial yield was determined by suspending the conidia in
0.03% Tween 80. The number of conidia was counted using
an improved Neubauer haemocytometer (Weber Scientific
International Ltd, UK).
Virulence of inoculum from different culture
media
Inoculum from different media was assayed against 4–5th
instar larvae of Tenebrio molitor. Ten larvae were immersed
in 20 mL conidial (1  107 conidia mL 1) suspension or
0.03% v/v aq. Tween 80 carrier (control) for c. 30 s and the
excess moisture removed by filtering over a vacuum in a
Buchner funnel. Batches of 10 larvae were incubated with-
out food in 9-cm-diameter Petri dishes lined with moist
Whatman No.1 filter paper and incubated at 27 1C in the
dark. Each treatment was replicated three times with 10
larvae per replicate. Mortality was recorded daily and dead
insects were transferred to Petri dishes lined with moist filter
paper to encourage external sporulation of the fungus if
present.
c2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
118 S.A. Safavi et al.

Carbon--nitrogen (C/N) analysis of conidia Statistical analysis

produced on different media
Conidial C/N elemental composition was determined using
an Automated Nitrogen Carbon Analysis for Gas Solids and
Liquids (ANCA GSL) elemental analyser (Europa, UK)
interfaced with a PDZ Europa 20/20 mass spectrometer.
Conidia (1 mg) from the disparate media were wrapped in
6  4-mm tin foil discs (Elemental Microanalysis, Ltd, UK),
which were prewashed with acetone, stored in sealed tubes
and kept in airtight desiccators until required. Concentra-
The whole study was repeated twice with each treatment
replicated three times unless stated otherwise. Data were
subjected to one-way ANOVA followed by the Duncan multi-
ple range test. Wherever required, linear regression was also
performed for the calculation of growth rate, LT50 (lowest
median lethal time) and enzyme activities. SAS software
(Version 6.12) was used for all statistical analyses.
Results

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tions between 25 to150 mg of isoleucine (Sigma) were used
as standards. Empty discs were included as negative con-
trols. Each treatment was replicated and the whole proce-
dure repeated twice.
Germination assays
The germination speed of inoculum from the different
media was assessed by inoculating SDA with 10 mL conidial
suspension (1  106conidia mL 1) then counting the num-
ber of germlings following 8 h of incubation at 25 1C.
Conidia with germ tubes equal to or greater than the width
were considered to have germinated. For each treatment,
three separate fields were observed for germination at 40 
and 100 conidia observed randomly in each field.
Effect of nutrition on the activity of conidial Pr1
enzyme
Pr1 bound to conidia harvested from the disparate media
was quantified using the method described by St. Leger et al.
(1996), with some modifications. One milligram of conidia
were incubated in 1 mL of 0.1 M Tris-HCl (pH 7.95)
containing 1 mM succinyl-ala-ala-pro–phe-p-nitroanilide
(Sigma) for 5 min at room temperature. After incubation
the conidia were clarified by centrifugation at 12 000 g (in a
Sanyo, Harrier 18/80 centrifuge) for 5 min. The supernatant
(200 mL) was transferred to wells of a microtitre plate
(Dynatec) and absorbancy measured using a Lab System
spectrophotmeter at 405 nm. Buffered substrate was used as
control.
Radial growth and sporulation of fungal isolates
in different nutritional conditions
Colony growth of three isolates of B. bassiana and one from
M. anisopliae significantly (P o 0.002) varied on the differ-
ent media. As can be seen from Tables 1–4, colony growth
had the broadest (from 2.51 to 5.09 mm day 1) and the
narrowest (from 2.99 to 3.59) range for strains V275 and
DEBI007, respectively. In all isolates studied, high-osmolar-
ity medium had the lowest colony growth per day (Tables
1–4). However, highest vegetative growth in each isolate
varied from high-C/N (75 : 1) medium for strain V275 to
low-C/N (10 : 1) medium and 1% yeast extract in strains
DEBI007 and DEBI008, respectively. Conidial yield of
different isolates of B. bassiana differed significantly
(P o 0.003) among different media, but there was no
significant (P = 0.74) difference in various media for M.
anisopliae. Isolate DEBI008 produced the highest number of
conidia on low-C/N medium, and strain BEH1 the lowest
amount of conidia on this medium (Tables 2 and 3). BEH1
and DEBI007 produced the maximum number of conidia
on medium-C/N (35 : 1) medium (= SDA). Strains DEBI007
and DEBI008 had the minimum conidial yield on OSM and
CPN, respectively.
Virulence of inoculum from different culture
media
There were significant (P o 0.0001) differences among various
nutritional media in virulence towards T. molitor larvae.
According to the data in Tables 1–4, except to conidia from

Table 1. Influence of different nutritional conditions on the growth and virulence of Beauveria bassiana DEBI007
Medium Radial growth (mm day 1) Conidial yield (  107) Conidial C/N Germination (%) Spore-bound Pr1 LT50 (days)
C/N 75 : 1 3.59  0.66bc 1.79  0.91ab 6.49  0.06a 78  1.82ab 0.63  0.11c 3.22d
C/N 35 : 1 3.56  0.54bc 2.85  0.6a 6.14  0.03b 56.4  12.57b 0.88  0.21b 3.05e
C/N 10 : 1 4.32  0.64a 2.5  0.79ab 4.75  0.02f 7  0.81c 0.66  0.06c 3.48c
1% Yeast extract 4.01  0.71ab 0.94  0.31ab 4.94  0.1e 7.5  2.94c 1.12  0.12a 3.77a
2% Peptone 4.24  0.39ab 0.92  0.22ab 5.3  0.07d 0c 1.12  0.08a 2.92e
KCl 2.99  0.16c 0.65  0.13b 5.76  0.07c 98.5  1.29a 0.65  0.09c 2.38f
Chitin peptone nutrient 4.05  0.47ab 0.76  0.25b 4.92  0.01f 11  5.85c 0.77  0.05bc 3.62b

Means within a column labelled with the same letter are not statistically different.

c 2007 Federation of European Microbiological Societies FEMS Microbiol Lett 270 (2007) 116–123
Published by Blackwell Publishing Ltd. All rights reserved
Nutrition and virulence of Beauveria bassiana 119

Table 2. Influence of different nutritional conditions on the growth and virulence of Beauveria bassiana DEBI008
Medium Radial growth (mm day 1) Conidial yield (  107) Conidial C/N Germination (%) Spore-bound Pr1 LT50 (days)
bc
C/N 75 : 1 4.16  0.08 0.92  0.18c 6.56  0.07a 75.5  11.53b 0.71  0.02e 2.88c
C/N 35 : 1 4.16  0.15bc 1.92  0.01ab 6.6  0.01a 82.5  4.71ab 1.41  0.17c 3.12b
C/N 10 : 1 4.53  0.42ab 2.54  0.03a 4.74  0.03d 3.25  2.75c 0.55  0.05f 3.44b
1% Yeast extract 4.59  0.11a 1.69  0.56b 5.2  0.1c 3.25  2.5c 0.99  0.15d 3.64a
2% Peptone 3.98  0.55c 0.58  0.19c 5.24  0.06c 1.33  0.87c 1.56  0.03b 3.21b
KCl 2.84  0.43d 0.69  0.24c 6.1  0.07b 99.5  0.41a 2.26  0.10a 2.48d
Chitin peptone nutrient 4.24  0.22abc 0.65  0.22c 4.51  0.03e 2.33  0.97c 0.69  0.04e 3.31b

Means within a column labelled with the same letter are not statistically different.

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Table 3. Influence of different nutritional conditions on the growth and virulence of Beauveria bassiana BEH1
Medium Radial growth (mm day 1) Conidial yield (  107) Conidial C/N Germination (%) Spore-bound Pr1 LT50 (days)
b
C/N 75 : 1 2.91  0.3 2.53  0.23ab 7.53  0.08a 87  6.92a 1.19  0.11c 11.39a
C/N 35 : 1 2.91  0.48b 3.75  0.32a 5.93  0.01c 75.5  12.42a 1.91  0.04ab 8.86b
C/N 10 : 1 3.88  0.15a 0.59  0.03c 4.83  0.03f 6  3.87b 1.48  0.04bc 6.96c
1% Yeast extract 3.61  0.22a 0.52  0.07c 4.98  0.01e 1.66  0.97b 2.05  0.55a 8.78b
2% Peptone 3.59  0.39a 1.39  0.44bc 5.15  0.03d 2  1.22b 1.91  0.39ab 6.42d
KCl 2.63  0.59b 0.91  0.2c 6.46  0.01b 94.75  1.89a 0.47  0.006d 7.39c
Chitin peptone nutrient 3.92  0.28a 1.05  0.35c 4.75  0.06f 68.2  10.81a 1.85  0.17ab 9.46b

Means within a column labelled with the same letter are not statistically different.

Table 4. Influence of different nutritional condition on the growth and virulence of Metarhizium anisopliae V275
Medium Radial growth (mm day 1) Conidial yield (  107) Conidial C/N Germination (%) Spore-bound Pr1 LT50 (days)
a a a a a
C/N 75 : 1 5.09  0.16 0.08  0.03 7.34  0.03 92.5  5.19 2.83  0.40 2.68bc
C/N 35 : 1 4.64  0.11b 0.26  0.09a 4.95  0.01e 64.7  11.09ab 1.54  0.27c 3.13b
C/N 10 : 1 4.21  0.48c 0.24  0.12a 4.38  0.04g 34.1  18.04bc 0.36  0.004e 4.22a
1% Yeast extract 4.1  0.34c 0.07  0.04a 4.70  0.02f 24  9.16c 0.50  0.01ed 4.26a
2% Peptone 4.22  0.33c 0.14  0.05a 5.37  0.01d 0c 0.29  0.005e 4.03a
KCl 2.51  0.28e 0.18  0.07a 5.77  0.01c 74  9.62a 0.75  0.04d 2.35c
Chitin peptone nutrient 3.78  0.11d 0.06  0.03a 6.26  0.04b 32.5  6.24bc 2.27  0.01b 4.14a

Means within a column labelled with the same letter are not statistically different.

strain BEH1 for which 2% peptone produced the lowest LT50,
in all other isolates conidia from OSM were the most aggressive
inoculum: high mortality with rapid onset of mortality to the
host. Conidia of strain BEH1 were remarkably less virulent
than those of other isolates from the same medium.
Carbon--nitrogen content of conidia from
various media
In each isolate there was a significant (P = 0.0001) variation
in C/N ratio of conidia from different media. As can be
expected, conidia of the high-C/N medium showed the
highest amount among different media (Tables 1–4). Low-
C/N medium exhibited the lowest C/N ratio in both
DEBI007 and V275, while this ratio was the lowest in CPN
in two other isolates. C/N ratio of three isolates of B.
bassiana in chitin nutrient medium was lower than that of
V275 from M. anisopliae.
Germination rate
Germination rate of spores from the seven studied media
was significantly (P o 0.007) different. In all three isolates of
B. bassiana conidia obtained from OSM had the highest
germination ability. Although in M. anisopliae V275 conidia
from the high-C/N medium had the highest germination
rate, there was no statistically significant difference between
this and those for OSM. All conidia from 2% peptone
showed the lowest germination rate.
Effect of nutrition on the activity of conidial
Pr1 enzyme
There was a significant (P = 0.0001) difference among var-
ious media within each isolate in the activity of conidial Pr1
enzyme. However, there was no general profile among
studied isolates in this regard. In strain DEBI007, conidia
from 1% yeast extract and 2% peptone produced the highest

FEMS Microbiol Lett 270 (2007) 116–123

c2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
120
Table 5. C/N ratio in B. bassiana DEBI008 after passage through the
T. molitor larvae
Medium Conidial C/N
C/N 75 : 1 4.27c
C/N 35 : 1 4.71a
C/N 10 : 1 4.20c
1% Yeast extract 4.75a
2% Peptone 4.45b
KCl 4.73a
Chitin peptone nutrient 4.30c
Means within a column labelled with the same letter are not statistically
different.
activity of spore-bound Pr1, 1.12 mmol mL min 1, whereas
conidia of the high-C/N medium had lowest activity,
0.63 mmol mL min 1 (Table 1). Conidia from OSM revealed
the highest activity in strain DEBI008 but the lowest activity
in strain BEH1 (Tables 2 & 3).
C/N ratio in host-passaged conidia
C/N ratio of insect-passed conidia from B. bassiana
DEBI008 (Table 5) showed a decrease in the C/N ratio after
passage through T. molitor larvae in all conidia from
different nutritional conditions. This decrease was consider-
able in some conidia, such as those from high-C/N and
OSM. Conidia from the 1Y medium showed the highest C/N
ratio in comparison with conidia from other media. How-
ever, in conidia from plates, the highest ratio was recorded
for high-C/N medium. The C/N ratios of conidia from low-
C/N, high-C/N and CPN were not statistically different after
passage through host insects.
Discussion
Radial growth of fungal strains changes not only with fungal
species and isolates but also with the C/N ratio of the media
used for production of conidia. Metarhizium anisopliae
V275 showed the highest radial growth in high-C/N med-
ium but with low conidial yield. In B. bassiana, the low-C/N
medium provided high radial growth in three isolates and
high conidial yield in at least in two isolates, DEBI007 and
DEBI008. Strain BEH1 had the lowest radial growth among
B. bassiana isolates, but except for in low-C/N and 1Y media,
the highest conidial yield maintained in comparison with
that of other strains on similar media. Therefore, colony
conidial yield is not definitively correlated with colony
growth. High conidial yield was produced in strains
DEBI007 and BEH1 on 35 : 1 C/N medium. Likewise, high-
est conidial yield of the mycoherbicide Colletotrichum
truncatum was in 30 : 1 C/N medium. Media with higher or
lower C/N ratios had lower conidial yield (Jackson &
Schisler, 1992). In the same way, maximum yields for two
strains of M. anisopliae were achieved in a 35 : 1 CN medium
c 2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
S.A. Safavi et al.
similar to SDA (Shah et al., 2005). Conversely, isolate
DEBI008 produced its maximum spore yield on the low-
(10 : 1) C/N medium. Similar results have been produced in
some isolates of B. bassiana and M. anisopliae using potato
dextrose agar (PDA; C/N ratio 10 : 1) medium (Wyss et al.,
2001; Kamp & Bidochka, 2002). Also, a liquid medium with
C/N ratio of 10 : 1 resulted in the maximum sporulation in
the entomopathogens B. bassiana, M. anisopliae and Paeci-
lomyces fumosoroseus (Vega et al., 2003). Likewise, evalua-
tion of 33 carbon sources on 11 fungal biocontrol agents
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revealed their role in spore germination, hyphal growth and
sporulation (Sun & Liu, 2006). Development of fluffy
colonies in high-C/N medium in two isolates of B. bassiana,
DEBI007 and DEBI008, caused fewer conidia to be pro-
duced in comparison with BEH1, which had smaller radial
growth. Strain BEH1 showed high sporulation in all treat-
ments. In most B. bassiana isolates, colony development in
OSM was fluffy with fewer conidia compared with colonies
from high-C/N medium.
Germination rate is another pathogenicity determinant
that is affected by nutritional conditions. In all three isolates
of B. bassiana, conidia originated from OSM had the highest
germination speed. Correspondingly, the lowest LT50 was
revealed in these conidia (OSM) in strains DEBI007 and
DEBI008. The rapid germination of conidia produced in
OSM may have been due to a high polyol content (Halls-
worth & Magan, 1994, 1995). Our results revealed a correla-
tion between germination rate and virulence. Hall (1984)
reported the germination rate as a trait that is frequently
associated with virulence in Verticillium lecanii towards
aphids and whitefly. Altre et al. (1999) stated that germina-
tion speed is positively correlated with the size of aerial
conidia. Jackson & Schisler (1992) attributed the rapid
germination of conidia to the relatively high protein content
of inoculum. As entomopathogens such as B. bassiana have
a wide range of different insect hosts, most studies have
characterized the host range of different isolates on the basis
of pathogenicity. Therefore, most information is concerned
with the effects of different host components on adhesion
and germination. However, germination on the insect
integument may be slower, owing to limited free moisture
(Ugine et al., 2005). In addition, conidial germination is
inhibited by the presence of phytochemicals found on
potato foliage (Fernandez et al., 2001). Colorado potato
beetle cuticle and extracts repress germination of M. aniso-
pliae var. acridum, although allowing low levels of adhesion.
Hence, there are many stages in the process of infection
during which the ability of an isolate to cause disease may be
influenced, and so a widening of our knowledge in this area
is needed (Wang & St. Leger, 2005).
Virulence of inoculum was dependent upon germination
speed in some isolates (DEBI007 and DEBI008) but not all.
This shows that there are other determinants in
FEMS Microbiol Lett 270 (2007) 116–123
Nutrition and virulence of Beauveria bassiana
pathogenicity. In other words, germination speed cannot be
used individually for virulence prediction. Isolate BEH1 had
the highest LT50 values in all different nutritional conidia.
But there was no a relationship between its germination rate
and virulence. Low-C/N and 2P media revealed a low
germination speed but higher virulence (lower LT50) when
compared to conidia from some other media.
Shah et al. (2005) considered a C/N ratio of 5.2 : 1 as a
virulence indicator in quality control, especially in combi-
nation with other parameters. Although there have been
some data consistent with this idea, we could not find such
an indicator and a general relationship between C/N ratio of
conidia and virulence of inoculum in B. bassiana isolates.
Conidia from OSM were virulent but had a high C/N ratio.
Conversely, conidia from low-CN and 1Y media that had
lower C/N ratios were less virulent (higher LT50 values).
Cuticle-degrading Pr1 enzyme has been determined as a
pathogenicity determinant in most entomopathogenous
hyphomycetes, with an established role in virulence towards
insect hosts (St. Leger et al., 1987, 1988a, 1996; Goettel et al.,
1989). The presence of transcripts of Pr1 in conidia would
increase the production of this enzyme in early stages of
infection and ensure the rapid involvement of the host
insect. It has been suggested that Pr1 enzyme releases
peptides that induce further Pr1 production (Paterson
et al., 1994a). The presence of Pr1 in the conidial cell wall
shows that this enzyme is secreted during conidiation; the
level of activity appeared to be correlated with the amount
of transcripts in the cell (St. Leger et al., 1996). Analysis of
spore-bound Pr1 data with LT50 values to investigate any
relationship between them revealed some interesting results.
Although Pr1 activity in strain BEH1, except in OSM, was
higher than for two other B. bassiana isolates, LT50 values in
this strain were remarkably higher than in DEBI007 and
DEBI008, indicating that cell-wall Pr1 is not the only
pathogenicity determinant in B. bassiana. Similarly, in any
given medium there were contradictory results among
isolates. In OSM, conidia of DEBI008 had the highest
spore-bound Pr1 activity with the most virulence towards
T. molitor larvae. Conversely, the most virulent conidia of
this medium in DEBI007 had low Pr1 activity. In spite of
these discrepancies, if enzyme activity is considered alone
(Tables 1–4), conidial Pr1 activity was higher in media with
low and complex C/N ratio, in some cases at least. Pr1
activity of conidia in two isolates of B. bassiana (DEBI007
and BEH1) was higher in 1Y and 2P media in comparison
with other media, possibly because of carbon and nitrogen
repression suggested for M. anisopliae (St. Leger et al.,
1988b; Paterson et al., 1994a, b; Screen et al., 1997, 1998).
The deleterious effects of synthetic insecticides, environ-
mental problems and resistance development has shifted
attention to biocontrol agents. Hyphomycetous entomo-
pathogenic fungi, especially B. bassiana and M. anisopliae
FEMS Microbiol Lett 270 (2007) 116–123
121
given their wide host range, have been shown to be promis-
ing agents for mass production for insect control and some
isolates of these species have been developed as mycoinsecti-
cides. Although many studies have investigated cuticle-
degrading enzymes and gene structure and expression of
these genes in M. anisopliae, less work has been done on
B. bassiana. Mass production of these fungal agents necessi-
tates having some knowledge of quality control. We quali-
fied conidia based on their germination rate, C/N ratio, LT50
and spore-bound Pr1. Pr1 is a good indicator of pathogen-
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esis that has been introduced for first time in M. anisopliae
(Shah et al., 2005). It seems that host-related determinants
such as insect cuticle and physiology and environmental
conditions may influence host susceptibility and therefore
fungal isolate virulence towards host insects. To our knowl-
edge this is the first work on B. bassiana that had studied
conidial Pr1 as a virulence factor. Quality control not only
culminates in selection of more virulent isolates for devel-
opment as mycoinsecticides but also maintains the effec-
tiveness of inoculum following serial subculturing in mass
production programmes. It seems that further work is
needed to find other virulence determinants with a major
role and prediction ability in entomopathogenic fungi.
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