Advances in Microbiology, 2012, 2, 268-276
http://dx.doi.org/10.4236/aim.2012.23032 Published Online September 2012 (http://www.SciRP.org/journal/aim)
Optimization of the Chitinase Production by Different
Metarhizium anisopliae Strains under Solid-State
Fermentation with Silkworm Chrysalis as Substrate
Using CCRD
Cynthia Barbosa Rustiguel, João Atílio Jorge, Luis Henrique Souza Guimarães*
Department of Biology, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto,
Universidade de São Paulo, São Paulo, Brazil
Received April 20, 2012; revised May 30, 2012; accepted June 7, 2012
ABSTRACT
Entomopathogenic fungi, such as Metarhizium anisopliae, are able to control various insect pests. These fungi attack
the integument of the host using an enzymatic complex. Among the enzymes found in this complex, chitinase is an im-
portant component. However, the relation between the chitinase production and the virulence from different M. ani-
sopliae strains has not been analyzed. In this manuscript it is presented the chitinase production by four M. anisopliae
strains with different potential of virulence in Solid-State Fermentation using silkworm chrysalis as substrate. The
higher chitinase level was obtained with the strain IBCB 360 (7.14 U/g of substrate) with potential virulence of 68% on
Diatrea saccharalis. The enzyme production was optimized for all strains using a factorial planning (CCRD) consider-
ing the cultivation time and medium humidity as independent variables. The maximal production of chitinase was ob-
tained at a range from 8 to 12-days old cultures and from 45% to 62% of moisture according to the surface response
plot, with high R2 value. The enzyme production by the strain IBCB 167 was increased two-folds under optimized con-
ditions, while for the strains IBCB 360 and 425 the chitinase production was increased four-folds and nine-folds for the
strain IBCB 384.
Keywords: Entomopathogenic Fungus; Metarhizium anisopliae; Chitinase; Solid-State Fermentation
1. Introduction
The entomopathogenic fungi are pathogens with a broad-
spectrum of action which are able to attack insects that
live in different ecological niches in different stages of
development. Most of these species are specialized in
penetrating the tegument [1]. The interaction between
pathogen and host is influenced by different factors such
as enzymes production, environment temperature and
humidity, light and ultraviolet radiation, as well as by
nutritional conditions and host susceptibility. Thus, the
complete cycle of infection involves the sequential stages
of adhesion, germination, appressorium formation, for-
mation of staple penetration, penetration, colonization
and reproduction of the pathogen in the insect [1]. En-
zymes play an important role during the penetration of
the fungus in the host, especially during adhesion and
germination which occurs at the germ tube formation,
releasing enzymes that degrade the cuticle of the insect
as, for example, proteases and chitinases, among others
[2]. Some studies have discussed the relation between the
production of enzymes with pathogenicity and virulence
[3-5]. The entomopathogenic fungus Metarhizium ani-
sopliae is a deuteromycete from Monoliaceae family
widely distributed in the nature that can be easily found
in the soil. This fungus has been studied due to its ability
to control insect pests [1,6,7]. Zappelini (2009) [8] analyz-
ed different strains of the entomopathogenic fungi Beau-
veria bassiana and M. anisopliae to verify their potential
of infection and mortality on Diatrea saccharalis and it
was observed that the virulence from each one of the 27
M. anisopliae strains analyzed was variable. The strain
IBCB 167, for example, was able to kill around 55% of
D. saccharalis while the strains IBCB 360, IBCB384 and
IBCB 425 showed a mortality power of 68% - 90%.
These different values can be attributed to the enzymatic
complex associated to the virulence, including chitinase.
Chitinases (EC 3.2.1.14) are enzymes widely spread in
nature that can be found in a great diversity of organisms,
with special attention to the filamentous fungi. Chitinase
catalyzes the hydrolysis of chitin, an insoluble linear
*Corresponding author.
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C. B. RUSTIGUEL ET AL. 269
molecule constituted by N-acetyl glucosamine units
linked by β-1, 4 (GlcNAc) linking [9] found in insect
cuticle. The complete hydrolysis of chitin occurs through
a chitinolytic system which acts synergistically and con-
secutively [10]. Chitinases are divided into two main
classes, the endochitinases and the exochitinases. The
endochitinases cleave chitin at random sites within the
polymer, releasing chito-oligosaccharides (chitotetraose,
chitotriose). The exochitinases cleave chitin from its
non-reducing end, releasing dimers (GlcNAc)2 [11,12].
These enzymes have multiple biological roles as, for
example, in the infection of insects by entomopathogenic
fungi. According to Boldo et al. (2009) [5], M. ani-
sopliae produces different chitinases related to the infec-
tion process on the host. However, the relation between
the level of chitinase production by different strains of M.
anisopliae and their virulence has not been analyzed.
Hence, this manuscript presents the production of chiti-
nase by the strains IBCB 167, IBCB 360, IBCB384 and
IBCB 425 from M. anisopliae with different virulence
potential and the optimization of enzyme production by
factorial design (CCRD).
2. Material and Methods
2.1. Microorganisms
Four different strains (IBCB 167, 360, 384 and 425) from
M. anisopliae classified according to their virulence
potential and deposited in the Collection of Entomo-
pathogenic Microorganisms “Oldemar Cardim Abreu”
from the Laboratory of Biological Control, Biological
Institute of Campinas, São Paulo, Brazil were used. The
strains were maintained on PDA (potato dextrose agar)
slants and stored at 4˚C in refrigerator. Spores from 15
days-old cultures were used to obtain new cultures,
which were initially maintained at 25˚C for 7 days and
then stored at 4˚C.
2.2. Obtainment of Cultures under Solid-State
Fermentation (SSF) and Crude Extract
The cultures under SSF were obtained by inoculating of
1 mL of a spore suspension (105 spores/mL) on 4 g of
crushed dry chrysalis from silkworm (BRATAC S.A.,
Brazil) (Figure 1) as substrate/carbon source moistened
with tap water, yeast extract solution (1% m/V), SR salt
solution [20×] [13] or Khanna salt solution [14], pre-
viously autoclaved at 120˚C, 1.5 atm for 30 minutes. The
cultures were maintained in a stove at 26˚C for different
periods (96 - 312 hours) with relative humidity around
76% monitored by a thermo hygrometer.
After incubation, the cultures were added with 50 mL
of cold distilled water previously autoclaved in the same
conditions cited above, maintained under agitation for 1
(a) (b)
Figure 1. Pictures from intact chrysalis (a) and triturated
chrysalis (b) from silkworm used as substrate for chitinase
production by different strains from M. anisopliae under
SSF.
h and harvested by gauze using Whatman paper No.1.
The free cell filtrate, identified as extracellular crude
extract, was dialyzed against distilled water at 4˚C over-
night and used for chitinase activity determination.
2.3. Determination of the Chitinase Activity
The determination of the chitinase activity was accomp-
lished using 1 mM of the synthetic substrate 4-Nitro-
phenyl N-acetyl-β-D glucosaminide (Sigma®) in 100 mM
sodium acetate buffer, pH 5.0. The reaction was per-
formed using 200 μL of the substrate solution and 200
μL of the enzymatic sample. After incubation at 60˚C,
the reaction was stopped at different time intervals by
adding 1 mL of 1 M NaOH. All experiments were per-
formed in triplicate. The p-nitrophenolate released was
quantified at λ = 405 nm in a spectrophotometer. One
unit of enzyme activity (U) was defined as the amount of
enzyme required to hydrolyze 1 µmol of substrate per
minute under the assay conditions.
2.4. Optimization of the Enzyme Production
The optimization of enzyme production for all M. ani-
sopliae strains under SSF was carried out using a facto-
rial design (22) (CCRD), where the independent variables
were the time of growth (X) and humidity (Y). These
parameters were chosen because the microbial growth
and, consequently, the enzyme production are drastically
influenced by the culture conditions, including water
activity and cultivation time, among others. The tem-
perature was maintained at 26˚C since the influence of
this variable is a well characterized parameter for M.
anisopliae growth. Three repetitions were performed at
the central points (0) and two outer points (–1.41, +1.41)
were considered, totalizing 11 trials (Table 1). The re-
sults were subjected to analysis of variance (ANOVA)
with p values of 0.05 and 0.1 using the software Statis-

C. B. RUSTIGUEL ET AL. 270
tica 8.0 (Stat Soft). Values lower than the p values fixed
were considered statistically significant. The Pareto
charts and surface response graphs were obtained using
the same software. The equations that describe de model
for the influence of each independent variable on the
enzymatic production were obtained using the same
software and validated by the experimental results using
the best conditions of cultivation time (9 days-old cul-
tures) and humidity (48%).
3. Results and Discussion
3.1. Influence of Salt Solution on the Enzyme
Production in SSF
The availability of the nutrients and salts is an important
factor to the growth of microorganisms and, conse-
quently to the enzyme production. In Table 2, the influ-
ence of different salt solutions (tap water, yeast extract
solution, SR and Khanna salt solutions) on chitinase
production under SSF by M. anisopliae strains can be
observed. Higher levels of chitinase were obtained when
the substrate silkworm chrysalis was moistened with
yeast extract (1%) at the ratio 1.3:1 (m/V) for the strains
IBCB 167, 360 and 425, differing than that observed for
the strain IBCB 384 with best enzyme production when
the substrate was moistened with Khanna salt solution
Table 1. Encoded and real values for both independent
variables time of growth and medium humidity used for
factorial design (CCRD) to evaluate the chitinase produc-
tion by four strains of M. anisopliae under SSF using silk-
worm chrysalis as substrate.
Encoded values –1.41 –1.0 0 +1.0+1.41
Time of growth (days) 4 5 9 1213
Real
values
Medium humidity (%) 23 30 48 6572
The encoded axial points were calculated using (2k)1/4, where k is the num-
ber of independent variables in the study.
Table 2. Influence of different salt solutions, tap water and
solution of yeast extract in the production of chitinases by
different isolates of the fungus M. anisopliae in SSF.
Extracellular chitinase activity (U/g substrate)
Solution 167 360 384 425
Tap water 0.73 ± 0.01 0.87 ± 0.01 0.35 ± 0.01 1.09 ± 0.01
Yeast extract
solution 2.42 ± 0.05 1.76 ± 0.03 0.30 ± 0.01 1.33 ± 0.01
SR salt
solution 0.63 ± 0.01 0.71 ± 0.01 0.20 ± 0.01 0.84 ± 0.01
Khanna salt
solution 0.62 ± 0.01 0.72 ± 0.01 0.46 ± 0.01 0.85 ± 0.00
The cultures were kept at 26˚C with relative humidity of around 76% for a
period of 168 hours.
(1.3:1 m/V). The solution of yeast extract has soluble
proteins, amino acids, biotin and it is rich in B complex
vitamins [15]. Amino acids are absorbed and utilized in
cellular metabolism, while the vitamins are important as
growth promoter and as co-enzymes [16]. These com-
pounds present in the yeast extract solution as well as the
composition of Khanna salt solution are able to supply
some nutritional necessity that can not be efficiently sup-
plied by the other solutions analyzed. In addition, the
enzymatic production by IBCB 167 strain in the best
condition was higher than that observed for the other
strains also in the best conditions, regarding 5.3-folds
higher if compared to the IBCB 384 strain.
3.2. Optimization of Enzyme Production by
CCRD
The experimental design is an important tool to analyze
the enzyme production under different culture conditions,
such as SSF, allowing to study the interaction of the in-
dependent variables. Bhanu et al. (2008) [17] used the
factorial design to evaluate the effect of pH, yeast extract
and mixtures of carbon sources on the production of co-
nidia by M. anisopliae. Patel et al. (2007) [18] used a
Plackett-Burman with eight independent variables for
chitinase production by Paenibacillus sabina JD2. The
influence of other parameters can also be evaluated as for
example the cultivation period and the humidity in SSF.
These parameters are determinant for fungal develop-
ment and, consequently, for the enzyme production.
There is an optimal range of water activity for microbial
growth that should be analyzed for each species. Ac-
cording to Table 3, the influence of the independent
variables growth’ time and medium humidity on chiti-
nase production by all four strains of M. anisopliae was
analyzed using a 2nd-order planning. Both variables
showed a positive effect on the production of extracellu-
lar chitinase for all strains (IBCB 167, 360, 384 and 425),
regarding an increase in the enzymatic levels. The Pareto
chart for the enzyme production by the strain IBCB 167
shows that the linear variable time of growth has a posi-
tive effect on the enzyme production, but very long pe-
riods of time already have a negative effect. The linear
variable moisture shows that the addition of tap water is
favorable for chitinase production, but too much water
turns the production into negative. The interaction be-
tween these variables was not significant (Figure 2(a)).
The CCRD performed for each strain was statistically
significant with the F-value calculated higher than the
F-value tabulated. The coefficient of determination (R2),
that explains the proportion of variation related to the
total variation of responses, obtained with the CCRD for
each strain was close to 1. The R2 obtained for the strain
IBCB 167 was 0.87 (Table 4), with p value fixed at 0.1,

C. B. RUSTIGUEL ET AL.
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271
22
.09y 1.88y 
chart (Figure 3(a)) shows that the linear variable time of
growth has a positive effect on the production of chiti-
nase, but a very long time has a negative effect as
showed by the quadratic interaction. In addition, the lin-
ear variable moisture shows that the addition of tap water
is favorable for chitinase production, but the excess of
water becomes unfavorable. The interaction between
both variables had a negative effect. The F-value calcu-
lated was four-times higher than the F-value tabulated,
allowing the obtainment of the response surface plot
(Figure 3(b)) and the equation that describes the model
what allows the obtainment of the response surface plot
(Figure 2(b)) and the equation that describes the model
(Equation 1), where Z is the enzyme activity (U/g of sub-
strate), x is the variable time of growth and y is the vari-
able humidity.
5.36 1.63x1.34x1Z  (1)
Considering the strain IBCB 360, the R2 value was
0.89 with p-value fixed at 0.1, and all variables (linear
and quadratic mode) as well as the interaction between
them were significant (Table 5). The respective Pareto
Table 3. Total extracellular chitinase activity produced by isolates 360, 384 and 425 M. anisopliae cultured in SSF through the
CCRD.
Coded values Real values Chitinase activity (U/g substrate)
Treatments Time growth X Humidity Y Time growth Hum idity % 167 360 384 425
1 –1.00 –1.00 5 30 0.000.00 0.00 0.04
2 1.00 –1.00 12 30 1.277.36 1.63 1.45
3 –1.00 1.00 5 65 1.143.90 0.72 4.26
4 1.00 1.00 12 65 3.494.68 2.81 6.27
5 0.00 0.00 9 48 5.386.18 4.60 6.38
6 0.00 0.00 9 48 5.107.23 3.63 6.02
7 0.00 0.00 9 48 5.596.96 4.51 5.84
8 –1.41 0.00 4 48 0.050.66 0.43 1.50
9 1.41 0.00 13 48 6.706.12 2.18 4.50
10 0.00 –1.41 9 23 0.400.33 0.44 0.55
11 0.00 1.41 9 72 4.176.98 1.29 3.29
p= 0.1
1Lby 2L
(2 )humidity(L)
grow th(Q)
hu midity(Q)
(1)growth(L)
Standardized effect estimate (absolute value)
(a) (b)
Figure 2. Pareto chart (a) and response surface plot (b) of the factorial design for the influence of the independent variable
time of growth and humidity on chitinase production by M. anisopliae IBCB 167 under SSF using silkworm chrysalis as sub-
trate. s

C. B. RUSTIGUEL ET AL. 272
Table 4. Analysis of variance (ANOVA) for chitinase pro-
duction isolated 167 M. anisopliae grown in SSF.
Source for Sum of Degrees of Mean F
Variation Square Freedom Square Calculated
Regression 54.59 3.00 18.19 15.15
Residual 8.39 7.00 1.20
Total 62.99 10.00
Regression coefficient: R = 0.87; Ftab 0.90,3;7 = 3.07.
Table 5. Analysis of variance (ANOVA) for chitinase pro-
duction isolated 360 M. anisopliae grown in SSF.
Source for Sum of Degrees of Mean F
Variation Square Freedom Square Calculated
Regression 76.50 4.00 19.13 12.10
Residual 9.48179 6.00 1.58
Total 85.9845610.00
Regression coefficient: R2 = 0.89; Ftab 0.90,3;7 = 3.18.
-2.39
4.07
p= 0.1
1Lby 2L
humidit y(Q)
(2)humidity ( L)
grow th(Q)
(1)growth(L)
- 2.73
2.72
- 2.50
St andardized effec t es tim at e (absolut e value)
(a) (b)
Figure 3. Pareto chart (a) and response surface plot (b) of the factorial design for the influence of the independent variable
time of growth and humidity on chitinase production by M. anisopliae IBCB 360 under SSF using silkworm chrysalis as sub-
strate.
(Equation (2)), where Z is the enzyme activity (U/g of
substrate), x is the variable growth time and y is the
variable humidity.
22
6.79 1.98x1.59x1.33y 1.45y1.64xyZ 
22
4.250.78x1.42x0.39y 1.65y0.12xyZ  
22
6.070.96x1.40x1.62y 1.95yZ 
(2)
The R2 value obtained for the strain IBCB 384 was
0.97, with p-value fixed at 0.1 (Table 6). The influence
of all variables (linear and quadratic mode) was signifi-
cant. The same can not be observed for the interaction
between both variables. According to the Pareto chart
(Figure 4(a)), the increase of the linear variable time of
growth has a positive effect on the chitinase production,
but a very long period lead to a negative effect. The lin-
ear variable humidity showed that the addition of tap
water is favorable for the enzyme production, but when
the humidity is excessive the production decreases. The
F-value calculated was 47.55 if compared to the F-value
tabulated of 3.62, what allows the obtainment of the re-
sponse surface graph (Figure 4(b)) and the equation that
describes the model (Equation (3)), where Z is the en-
zyme activity (U/g of substrate), x is the variable growth
time and y is the variable humidity.
(3)
According to Table 7, the R2 value was 0.93 for the
planning using the strain IBCB 425. The linear and
quadratic effects for both independent variables were
statistically significant with p-value fixed at 0.05, but not
for the interaction effect (Figure 5(a)). The linear vari-
able time of growth had a positive effect on the produc-
tion of chitinase, but long period has a negative effect as
shown by the quadratic effect. The analysis of the effect
of the moisture showed that the addition of tap water was
favorable for chitinase production, but at high humidity
environment the enzymatic production is reduced (Fig-
ure 5(a)). The F-value calculated was 9 times higher
than the F-tabulated allowing the obtainment of the re-
sponse surface plot (Figure 4(b)) and of the adjusted
equation that describes the model (Equation (4)), where
Z is the enzyme activity (U/g of substrate), x is the vari-
able growth time and y is the variable humidity.
(4)
The higher level of extracellular chitinase was ob-

C. B. RUSTIGUEL ET AL. 273
Table 6. Analysis of variance (ANOVA) for chitinase pro-
duction isolated 384 M. anisopliae grown in SSF.
Source for Sum of Degrees of Mean F
Variation Square Freedom Square Calculated
Regression 26.76 3.00 8.92 47.55
Residual 0.93803 5.00 0.19
Total 27.69867 10.00
Regression coefficient: R2 = 0.97; Ftab 0.90,3;5 = 3.62.
p= 0.1
Standardized effect estim ate (absolute value)
1Lby2L
(2)humidi ty(L)
(1) growth(L)
growth(Q)
humidity(Q)
-9.00
-7.78
5.01
2.54
0.54
(a)
(b)
Figure 4. Pareto chart (a) and response surface plot (b) of
the factorial design for the influence of the independent
variable time of growth and humidity on chitinase produc-
tion by M. anisopliae IBCB 384 under SSF using silkworm
chrysalis as substrate.
tained with the strain IBCB 360 (7.23 U/g of substrate)
with moisture ratio from 45% to 62% and incubation peri-
ods of 8 to 12-days using silkworm chrysalis as substrate
according to the analysis of the surface response plot.
This same ratio for both variables was also observed for
the other strains analyzed. Similarly, the chitinase pro-
duction by Aspergillus sp. S1-13 was increased with 58%
Table 7. Analysis of variance (ANOVA) for chitinase pro-
duction isolated 425 M. anisopliae grown in SSF.
Source for Sum of Degrees of Mean F
Variation Square Freedom Square Calculated
Regression 53.77 3.00 17.92 29.34
Residual 4.27600 7.00 0.61
Total 58.0444510.00
Regression coefficient: R2 = 0.93; Ftab 0.95,3;7 = 3.07.
p= 0.05
-3.63
2.96
0.33
4.99
4.07
1Lby2L
(1)gr owth(L)
growth(Q)
(2)humidity(L)
humidity(Q)
Standardized effect estimate (absolute value)
(a)
(b)
Figure 5. Pareto chart (a) and response surface plot (b) of
the factorial design for the influence of the independent
variable time of growth and humidity on chitinase produc-
tion by M. anisopliae IBCB 425 under SSF using silkworm
chrysalis as substrate.
- 65% of water under SSF using shrimp shellfish as sub-
strate [19]. The optimization process of chitinase produc-
tion using CCRD allowed an increase in chitinase pro-
duction by all isolates if compared to the production
showed in Table 3. The enzyme production by the strain
IBCB 167 was increased two-times under optimized

C. B. RUSTIGUEL ET AL. 274
conditions, while for the strains IBCB 360 and 425 the
chitinase production was increased four-times and nine-
times for the strain IBCB 384. Despite the good enzyme
production with high humidity, M. anisopliae strains
were able to produce chitinases in dried conditions as
well. This flexibility demonstrated by all strains is an
important factor that should be considered, since the
presence of water in the environment is unstable. Chiti-
nase production by the strain IBCB 360 under optimized
conditions was two times higher than that observed for
the production by Trichoderma harzianum (3.14 U/g of
substrate) under SSF using a mixture of chitin and wheat
bran as substrate [20]. In addition, this is the first report
that shows the possibility to use chrysalis, a biological
residue of the textile industry that is discarded into the
environment, as substrate for chitinase production by
different strains of M. anisopliae under SSF.
All equations obtained were validated performing ex-
periments using 9 day-old cultures and humidity adjusted
for 48%. Under these conditions the enzymatic activity
values experimentally obtained were 5.20, 7.07, 4.60 and
6.08 U/ g of substrate for the strains IBCB 167, 360, 384
and 425, respectively, which are in agreement with the
values obtained using the equations (Equations (1)-(4)) of
5.36, 6.80, 4.25 and 6.07 U/g of substrate, respectively.
There are many works which demonstrate that the en-
zymatic production using SSF is higher than those ob-
tained using Submerged Fermentation. The conditions
observed in SSF is much closer to the conditions found
in nature by the fungus. The chitinase production in both
fermentation conditions has been reported for M. ani-
sopliae [17,21]. Usually, the conditions of the SSF are
simple and the substrate used has the necessary nutrients
for the growth of microorganism. The destination of the
biological residues is a preoccupation around the world.
These residues, such as chrysalis can be used as alterna-
tive substrates to produce enzymes, adding aggregate
value to this organic residue and minimizing the negative
impact in the environment. On the other hand, the use of
low cost substrates has a significant advantage under
economic view, reducing the production costs in the in-
dustry. Many works showing chitinase production have
used colloidal chitin or crab and shrimp shell powder
[22-25] as the main substrate/carbon source. Chitinase
production using arthropods cuticle was also demon-
strated by Da Silva et al. (2005) [4].
Chitinase, among other enzymes, from entomophato-
genic fungi is related to pathogenicity and virulence.
Virulence is defined as the capacity degree that the mi-
croorganisms have to cause disease [26]. The virulence
of fungal strains over different arthropods is variable and
it is related to the production of enzymes and other viru-
lence factors [27]. The production of endochitinase CH2
by M. anisopliae as a virulence factor on Dysdercuspe-
ruvianus was demonstrated by Boldo et al. (2009) [5].
Mustafa & Kaur (2009) [28] also evaluated the produc-
tion of extracellular enzymes from 12 strains of M. ani-
sopliae, observing a wide variation in the production of
extracellular enzymes. The extracellular lipase produced
by M. anisopliae also participates in the process of infec-
tion [29]. The cultivation of M. anisopliae under FSbm
using cuticles of Dysdercus peruvianus, Boophilus mi-
croplus and Anticarsia gemmatalis as carbon sources
showed differences in the secretion of total proteins,
chitinases and proteases [4]. These differences in the
enzymes secretion are important as virulence factor di-
rectly related with the potential of the fungus M. ani-
sopliae to recognize the structure of the cuticle of their
host and to secrete the correct enzymatic pool. According
to Freimoser et al. (2003) [30], M. anisopliae can encode
different chitinase isoforms and other proteins as func-
tion of the material used as substrate.
The four M. anisopliae strains (IBCB 167, 360, 384
and 425) studied have been recognized according to their
potential to control the pest Diatraea saccharalis pro-
moting the death of 55%, 68%, 90% and 82%, respect-
tively, of the insect [8]. However, the chitinase produc-
tion by these strains had not been analyzed until this
moment. The chitinases produced by these strains can be
considered an important virulence factor. We found that
the best producer of chitinase is the strain IBCB 360,
what is not coincident with the best percentage of control
found to D. saccharalis. The strain IBCB 425 was the
second best chitinase producer and was able to control
90% of the pest D. saccharalis. The strain IBCB 167 was
the third chitinase producer and did not show a good
percentage of D. saccharalis control. Interestingly, the
strain IBCB 384 was the lowest chitinase producer, but it
was able to control 82% of the pest D. saccharalis. This
is the first work that relates the production of extracellu-
lar chitinase from these strains to understand the poten-
tial capacity to control pests. It is important to highlight
that the potential of virulence of an entomopathogenic
fungus depends on different factors and not only of the
chitinase production, what justifies the differences be-
tween the chitinase production by each strain and its ca-
pacity to control D. saccharalis. These strains are able to
produce other virulence factors that were not quantified
in this study. In addition, the use of chrysalis as substrate
must induce the expression of other genes differently
from that observed for the control of D. saccharalis.
Differences in gene expression of M. anisopliae grown
on cuticle or hemolymph from its host have been ob-
served [31]. The results obtained are interesting and in-
dicate that chitinases produced by the strains IBCB 167,
360, 384 and 425 of the fungus M. anisopliae on chrysa-
lis can have different degrees of participation in the in-
fection process.

C. B. RUSTIGUEL ET AL. 275
4. Conclusion
All strains of the entomophatogenic fungus M. anisopliae
showed flexibility to produce chitinases under SSF using
chrysalis, which was used for the first time as substrate,
indicating their potential to adapt to the environment
conditions, important factor that should be considered for
the infection process. Under optimized condition by
CCRD, the strain IBCB 360 was the best chitinase pro-
ducer although this strain is not the best controller of D.
saccharalis, indicating that other virulence factors are
also involved in the infection.
5. Acknowledgements
This Investigation was supported by Fundação de Apoio
à Pesquisa do Estado de São Paulo (FAPESP) and Coor-
denadoria de Apoio ao Ensino Superior (CAPES). This
manuscript is part of the CBR Doctoral (Comparative
Biology Post-Graduate Program, FFCLRP, USP). We
thank BRATAC S.A. and Mauricio de Oliveira for tech-
nical assistance.
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