ava i lab le at www.sc iencedi rec t . com
journa l homepage : www.e l sev i er . com/ loca te /mycres
m y c o l o g i c a l r e s e a r c h 1 1 0 ( 2 0 0 6 ) 485 – 492
Impact of moisture on in vitro germinationof Metarhizium anisopliae and Beauveria bassianaand their activity on Triatoma infestans
Gustavo Maximiano Junqueira LAZZARINI, Luiz Fernando Nunes ROCHA,Christian LUZ*
DMIPP, Instituto de Patologia Tropical e Saude Publica, Universidade Federal de Goias, CP 131, 74001-970 Goiania (GO), Brasil
a r t i c l e i n f o
Article history:
Received 19 July 2005
Received in revised form
30 October 2005
Accepted 4 December 2005
Published online 20 March 2006
Corresponding Editor:
Richard A. Humber
Keywords:
Biocontrol
Chagas disease
Entomopathogens
Insect pathology
Triatomines
a b s t r a c t
The in vitro germination of 11 Metarhizium anisopliae and 11 Beauveria bassiana isolates orig-
inating from substrates collected in rural peridomestic areas in Central Brazil where triato-
mines are common was tested. Conidia completed germination up to 24 h after exposure to
water activity of >0.99 aw in all isolates tested. At lower 0.93 aw germination was delayed
but conidia of most isolates germinated at high rates (>98 %) within 216 h of incubation.
Activities of 2 M. anisopliae and 2 B. bassiana isolates with different patterns of germination
at 0.93 aw were tested in Triatoma infestans third instar nymphs. There was no relationship
between germination kinetics in vitro at 0.93 aw and their activity in vivo at 98, 75 and 43 %
relative humidity (rh). Isolates with accelerated germination at 0.93 aw were not more vir-
ulent at 75 and 43 % rh compared with isolates with retarded or no germination. Highest
mortalities were observed at 98 % rh, and they did not exceed 25 % after 25 d incubation
at lower 75 and 43 % rh. Isolates that originated from a region with an extensive annual
arid period showed no adaptation to lower humidity in their activity against T. infestans.
ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction
Entomopathogenic fungi are important candidates for biocon-
trol of triatomines (Lecuona et al. 2001; Luz et al. 1998a, b).
These insects transmit Trypanosoma cruzi, the agent of Chagas
disease in Latin America. Recent studies in Central Brazil
showed that Metarhizium anisopliae and Beauveria bassiana oc-
cur naturally in peridomestic habitats and may act as antago-
nists of these vectors (Luz et al. 2004a). Another
entomopathogenic fungus, Evlachovaea sp., was isolated
from a dead Triatoma sordida collected in the same region
(Luz et al. 2003). When tested under laboratory conditions, ac-
tivity of these fungi against T. infestans and other species was
highest at moisture close to saturation, and a reduction and
high variability of mortality was observed at lower relative hu-
midity (rh) of 75 % (Luz et al. 2004a, c). Results obtained re-
cently in field tests with a B. bassiana isolate and T. sordida
underline the high potential of this fungus to control perido-
mestic triatomine populations (Luz et al. 2004b).
Infective conidia adhere to the cuticle, germinate at favour-
able conditions, and the fungus invades the host. After host
death fungi produce new conidia. Extracuticular development
on triatomines depends on the level and exposure time to
favourable moisture as shown for B. bassiana Bb 297 and Rhod-
nius prolixus (Fargues & Luz 1998, 2000; Luz & Fargues 1998,
1999). When tested in vitro the same isolate revealed a clear
* Corresponding author.E-mail address: [email protected]
0953-7562/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.mycres.2005.12.001
486 G. M. J. Lazzarini et al.
threshold for germination at 0.96 aw (Luz & Fargues 1997).
However, other studies showed that T. infestans may be killed
by B. bassiana regardless to ambient humidity (Lecuona et al.
2001). The divergent behaviour of B. bassiana may be related
to genetic variability among strains. Both M. anisopliae and B.
bassiana are found worldwide, and in arid or semi arid regions
strains may occur with a better adaptation to unfavourable
moisture. A better understanding of the impact of moisture
on fungal activity during infection of triatomines is important,
as many species of these vectors are common in regions with
dry climate. We report on the impact of water activity on in
vitro germination of different M. anisopliae and B. bassiana iso-
lates collected in triatomine-infested areas in Central Brazil
and their activity against T. infestans at different humidities
under laboratory conditions.
Materials and methods
Fungus origin and culture
Eleven isolates of Metarhizium anisopliae (IP 220, IP 221, IP 222, IP
225, IP 226, IP 227, IP 230, IP 232, IP 233, IP 234, IP 235) and Beau-
veria bassiana (IP 155, IP 157, IP 161, IP 165, IP 170, IP 171, IP 180,
IP 224, IP 228, IP 229, IP 231) were tested. All isolates originated
from substrates collected in rural peridomestic areas in
Central Brazil where triatomines are common (Luz et al.
2004a). Fungi were obtained from the collection of entomopa-
thogenic fungi at the Institute of Tropical Pathology and Public
Health (IPTSP), Federal University of Goias, Goiania, Brazil.
Fungi were grown on complete medium (CM: 0.001 g FeSO4;
0.5 g KCl; 1.5 g KH2PO4; 0.5 g MgSO4$H2O; 6 g NaNO3; 0.001 g
ZnSO4; 1.5 g hydrolyzed casein; 0.5 g yeast extract; 10 g glu-
cose; 2 g peptone; 20 g agar; 1000 ml water) at 25 �C� 1 �C,
and a 12 �h photophase. All isolates were previously host-pas-
saged on third-instar nymphs of Triatoma infestans at the be-
ginning of the reported experiments in order to stimulate
fungal activity. Conidia were obtained directly from 10 d-old
sporulating cultures by scraping and then suspending in
10 ml of 0.1 % Tween 80. Suspensions were adjusted to defined
concentrations based on haemocytometer counts.
Insect origin and rearing
Triatoma infestans originated from Jacarezinho, Parana, Brazil
and were mass-reared at the IPTSP. Insects were blood-fed
on chickens (Gallus gallus domesticus) at two-weeks intervals,
held at 25� 1 �C, 75� 5 % rh, and 12 h photophase (Silva &
Silva 1988).
In vitro tests
Conidial germination of all isolates was screened in liquid
minimal medium (MM: 2 g glucose; 2 g yeast extract; 1000 ml
H2O) at two different water activities, 0.93 aw and >0.99 aw.
The water activity 0.93 aw was adjusted by equilibration
with sodium chloride (117 g 1000 ml�1 medium) and no salt
was added to MM to obtain>0.99 aw (Luz & Fargues 1997). Con-
idia were inoculated in 125 ml MM at final 1� 106 conidia ml�1
and then incubated at 25� 1 �C and darkness at 120 rev min�1
up to 216 h. Samples of 1 ml were taken 4, 8, 12, 24, 48, 72, 96,
120, 144, and 216 h post inoculation ( p. i.) and stored at�20 �C
until microscopic observation. Water was added daily to MM
in order to provide a permanent volume of 125 ml. Progress
of germination was monitored by scoring conidia as ungermi-
nated, swollen, germination initiated or germinated. Conidia
were considered germinated with an elongating germ tube
longer than the conidial diameter (Luz & Fargues 1997). Germi-
nation rates were determined by examination of 100 conidia
per replicate. Each treatment consisted of four independent
replicates.
In vivo tests
Two Metarhizium anisopliae isolates (IP 225 and IP 230) and two
Beauveria bassiana isolates (IP 170 and IP 229) which showed ac-
celerated (IP 170 and IP 230), retarded (IP 229) or no germination
(IP 225) at 0.93 aw, were assayed on insects. At least five up
to six concentrations of suspended conidia, 1� 106, 3� 106,
1� 107, 3� 107, 1� 108, and 3� 108 conidia ml�1 were tested
corresponding to 2.33� 103, 7� 103, 2.33� 104, 7� 104,
2.33� 105, and 7� 105 CFU (colony forming unit) cm�2 treated
surface, respectively. Ten recently molted and unfed third in-
star nymphs (N3) were treated by directly spraying 5 ml of
each concentration with a Potter spray tower (Burkard, Hert-
fordshire). Final deposit was determined by spraying 5 ml sus-
pended conidia at different concentrations, as mentioned
before, on sterile cover slips (18� 18 mm), transferring in
10 ml sterile 0.1 % Tween 80, and inoculating 100 ml on chlor-
amphenicol added (0.5 g 1000 ml�1) CM. The number of CFU
was checked for 5 d p. i. Control insects were treated with
0.1 % Tween 80 only. After drying for 1 h at ambient tempera-
ture and humidity nymphs were placed on filter paper in plas-
tic Petri dishes (90� 15 mm) and transferred to test chambers
at 43 % rh, 75 % rh and humidity close to saturation. Humidities
of 43, 75 and 98 % inside the chambers were regulated with sat-
urated solutions of potassium carbonate, sodium chloride, and
potassium sulphate, respectively (Winston & Bates 1960). Test
chambers consisted of an airtight plastic box (33� 37� 22 cm)
with the salt solution arranged at the bottom (1000 g salt
500 ml�1 water). Petri dishes were put on a plastic rack that
permitted free air exchange inside the chamber. Nymphs
were incubated at 25� 1 �C and a 12 h photophase and were
not fed during the assays. Four independent replicates of ten
N3 each were used per treatment. Mortality was monitored
daily for 25 d. Dead insects were transferred to a humid cham-
ber and fungal development on cadavers examined for 15 d.
Data analysis
Germination and mortality data were arcsine-square root
transformed and then analysed using either Kruskal–Wallis
test (H) or one- or two-way analysis of variance (ANOVA) (F)
and the Student–Newman–Keuls (SNK) multiple range test
for comparison of means. Means were considered signifi-
cantly different at P< 0.05. Times to obtain 50 and 90 % germi-
nation (GT50 and GT90), lethal times (LT50 and LT90), and
concentrations (LC50 and LC90) to obtain 50 and 90 % mortality
were calculated by non-linear regression analysis or probit
analysis, respectively. The survival curves of nymphs were
Impact of moisture on Beauveria bassiana and Metarhizium anisopliae 487
fixed with the Kaplan–Meier test and analysed using the log
rank test (SAS Institute Inc. 2000).
Results
Germination of conidia at different water activities
At >0.99 aw, the first swollen conidia of all Metarhizium aniso-
pliae and Beauveria bassiana isolates were observed 4 h p. i. For-
mation of germ tubes initiated from then and up to 8 h p. i.
Three M. anisopliae and eight B. bassiana isolates showed ger-
mination rates �10 % at this time (Tables 1 and 2). Except for
IP 235, germination of M. anisopliae isolates was >98 % at
12 h p. i. (Table 1). Germination of B. bassiana isolates varied
from<1 % (IP 180) to 75 % (IP 161) at 12 h p. i. and�98 % germi-
nation of all isolates was observed only at 24 h p. i. (Table 2). A
highly significant effect of the isolate on the germination rate
at 8 h and 12 h p. i. of both fungal species (P< 0.001) was noted.
At 0.93 aw the first swollen conidia of both fungal species
were observed also at 4 h p. i., but germination started only be-
tween 48 h and 72 h p. i. (Tables 1 and 2). Whereas no germina-
tion of M. anisopliae conidia could be detected at 48 h p. i.,
B. bassiana IP 170 and IP 180 had already initiated germination.
At 72 h p. i. germination of M. anisopliae was <10 %, except IP
230 with 47.5 % of conidia germinated. Germination of B. bassi-
ana isolates at 72 h p. i. was distinctly higher compared with
M. anisopliae and reached 83.7 % (IP 170). Nine isolates had
values of germination >10 %. No germination of M. anisopliae
IP 225 incubated at 0.93 aw could be observed at 216 h p. i. Ex-
cept for IP 225 and IP 235 (47.5 %), all other isolates of this spe-
cies showed germination rates >96 % at 216 h p. i. This was
also found for B. bassiana isolates, except for IP 171 and IP
229 where 92 % and 57.5 % of the conidia had germinated
216 h p. i., respectively. A highly significant effect (P< 0.001)
of the isolate on the germination was found for M. anisopliae
and B. bassiana between 72 and 216 h. Regression curves
were fitted for most isolates (F> 120, df¼ 2, P< 0.0001) and
values of GT50 of M. anisopliae isolates varied between 98.2 h
(IP 233) and 137 h (IP 227) and for B. bassiana isolates between
59.3 h (IP 170) and 102 h (IP 155; Table 3). Lowest values of GT90
were found at 104 h for M. anisopliae (IP 233) and 78.8 h for
B. bassiana (IP 170). The number of germinated conidia of
M. anisopliae IP 225 and IP 235 and B. bassiana IP 229 were insuf-
ficient or germination was completed too quickly (IP 230 and IP
234) to calculate GT50/90.
Fungal infection of Triatoma infestans at differenthumidities
The first mortalities of fungus-treated nymphs were found 4–
6 d p. i. at all humidities tested. There was a highly significant
effect of humidity on cumulative mortalities between 10 and
25 d p. i. (P< 0.001; Table 4). At rh close to saturation (98 %
rh) mortalities of both M. anisopliae and B. bassiana-treated
N3 increased with the conidial concentration and time of in-
cubation (Figs 1 and 2). Except B. bassiana IP 170, all isolates
had induced total mortality within a 10 d exposure to this hu-
midity. Only 30.9 % of N3 treated with IP 170 had been killed at
10 d p. i. (Table 4). Values of LC50/90 found for isolates after 10 d
Ta
ble
1–
Cu
mu
lati
ve
germ
ina
tio
n(%
;±
S.E
.)o
fM
eta
rhiz
ium
an
isop
lia
eco
nid
iain
liq
uid
min
ima
lm
ed
ium
ad
just
ed
at
wa
ter
act
ivit
ies>
0.9
9a
wa
nd
0.9
3a
w,
25� C
,1
20
rev
min
L1
aw
Tim
e(h
)Is
ola
tes
test
ed
IP220
IP221
IP222
IP225
IP226
IP227
IP230
IP232
IP233
IP234
IP235
0.9
94
**
**
**
**
**
*
8þ
16.8�
2.6
a18.0�
1.5
a6.3�
1.4
b4.8�
1.5
b4.0�
0.6
b*b
20.5�
3.0
a*
b*
b*
b*b
12þ
**a
**a
**a
**a
**a
**a
**a
**a
**a
**a
61.5�
2.2
b
24
****
****
****
****
****
**
0.9
348
**
**
**
**
**
*
72þ
8.8�
0.9
b*b
*b
*b
1.0�
0.4
b*b
47.5�
2.3
a6.5�
0.6
b4.3�
0.3
b*
b*b
96þ
17.8�
1.5
c9.8�
1.0
d6.0�
0.4
e*f
25.3�
0.9
b10.5�
1.3
d**
a10.2�
0.6
d22.0�
1.1
b*
g4.5�
0.6
ef
120þ
43.3�
2.5
b44.8�
1.9
b44.8�
0.7
b*d
42.3�
0.9
b35.8�
0.6
d**
a12.3�
1.0
c**
a10.5�
0.6
c13.0�
1.3
c
144þ
75.3�
1.0
d86.3�
1.5
b79.8�
1.1
c*g
**a
53.8�
1.3
e**
a**
a**
a**
a38.0�
0.9
f
216þ
**a
96.3�
0.5
b**
a*d
**a
**a
**a
**a
**a
**a
47.5�
1.3
c
*G
erm
ina
tio
n<
1%
.**
Germ
ina
tio
n>
98
%.
þK
rusk
al–
Wa
llis
test
:42.9>
H>
40.6
;P<
0.0
01.
Sig
nifi
can
td
iffe
ren
ces
wit
hin
on
eli
ne
are
ind
ica
ted
by
dif
fere
nt
lett
ers
(Stu
den
t–N
ew
ma
n–K
eu
lste
st,
P<
0.0
5).
488 G. M. J. Lazzarini et al.
Ta
ble
2–
Cu
mu
lati
ve
germ
ina
tio
n(%
;±S
.E.)
ofB
eau
ver
iaba
ssia
na
con
idia
inli
qu
idm
inim
alm
ed
ium
ad
just
ed
at
wa
ter
act
ivit
ies>
0.9
9a
wa
nd
0.9
3a
w,2
5� C
,12
0re
vm
inL
1
aw
Tim
e(h
)Is
ola
tes
test
ed
IP155
IP157
IP161
IP165
IP170
IP171
IP180
IP224
IP228
IP229
IP231
0.9
94
**
**
**
**
**
*
8þ
10.0�
0.8
cd10.2�
0.6
cd16.2�
0.5
b20.5�
0.3
a13.2�
1.2
bc
15.5�
0.3
b*e
9.5�
0.9
d10.8�
0.8
cd10.5�
0.6
cd7.7�
0.8
d
12þ
44.0�
2.5
d13.5�
0.6
e75.0�
1.3
a67.2�
0.6
b71.5�
0.6
a74.2�
0.5
a*f
59.5�
3.5
c57.2�
1.6
c60.0�
1.3
c69.5�
1.8
ab
24
****
****
****
****
****
**
0.9
336
**
**
**
**
**
*
48
**
**
10.0�
0.4
*3.0�
0.4
**
**
72þ
15.5�
2.1
e36.5�
2.3
d40.5�
0.6
d52.7�
0.6
c83.7�
0.5
a5.0�
0.4
f44.0�
0.4
cd36.7�
0.5
d68.5�
0.6
b8.0�
0.4
f74.2�
0.8
b
96þ
57.7�
0.5
e70.7�
0.5
d76.7�
0.5
cd80.5�
0.6
c88.5�
0.6
b48.7�
1.0
f61.2�
1.3
e94.7�
1.7
a88.2�
0.5
b21.0�
0.4
g91.2�
0.5
ab
120þ
63.2�
0.8
bc
**a
80.0�
0.8
ab
86.5�
0.6
ab
**a
79.5�
0.6
ab
**a
**a
**a
26.5�
0.6
c**
a
144þ
67.5�
0.9
b**
a**
a**
a**
a85.2�
3.6
b**
a**
a**
a41.0�
0.4
c**
a
216þ
**a
**a
**a
**a
**a
92.0�
0.9
b**
a**
a**
a57.5�
0.6
c**
a
*G
erm
ina
tio
n<
1%
.**
Germ
ina
tio
n>
98
%.
þK
rusk
al–
Wa
llis
test
;42.9>
H>
42.4
;P<
0.0
01.
Sig
nifi
can
td
iffe
ren
ces
wit
hin
on
eli
ne
are
ind
ica
ted
by
dif
fere
nt
lett
ers
(Stu
den
t–N
ew
ma
n–K
eu
lste
st,
P<
0.0
5).
Table 3 – Germination time (h; ±S.E.) to achieve 50 or 90 %germination (GT50/90) of Metarhizium anisopliae andBeauveria bassiana conidia incubated in liquid minimalmedium adjusted at water activities 0.93 aw, 25 �C and120 rev minL1
Species Isolate GT50 (h) GT90 (h)
M. anisopliae IP 220 123.0� 4.0 f 173.0� 7.5 g
IP 221 120.9� 19.0 ef 157.0� 33.1 e-g
IP 222 122.8� 5.1 f 158.0� 8.7 f
IP 225 * *
IP 226 115.9� 12.1 ef 150.0� 21 e-g
IP 227 137.7� 27.7 ef 191.0� 49.3 fg
IP 230 ** **
IP 232 123.6� 15.4 ef 130.0� 27.1 d-f
IP 233 98.2� 21.8 c-f 104.0� 35.6 a-e
IP 234 ** **
IP 235 * *
B. bassiana IP 155 102.0� 7.9 de 181.2� 13.9 g
IP 157 79.9� 16.0 a-d 112.1� 24.5 b-e
IP 161 78.5� 6.3 c 120.0� 9.9 de
IP 165 72.4� 7.5 a-c 108.1� 11.3 c
IP 170 59.3� 5.1 a 78.8� 7.3 a
IP 171 97.1� 15.8 b-e 150.7� 27.1 de
IP 180 79.6� 11.5 bc 118.1� 18.6 de
IP 224 75.0� 19.4 a-d 90.6� 28.7 a-d
IP 228 66.1� 4.6 ab 87.6� 6.5 a-c
IP 229 * *
IP 231 64.5� 4.9 ab 82.8� 6.9 a
* Number of germinated conidia insufficient to calculate GT50/90.** Number of values insufficient for nonlinear regression analysis.
Significant differences based on confidence intervals (CI¼ 95 %)
within one column are indicated by different letters.
Table 4 – Cumulative mortality (%) (±S.E.) of Triatomainfestans third instar nymphs, 10 and 25 d after treatmentwith Metarhizium anisopliae and Beauveria bassianaisolates (5 ml of 1 3 108 conidia mlL1) and incubation atrh [ 43, 75 and 98 %, 12 h photoperiod, and 25 �C
Day Species Isolate * Relative humidity: ***(rh) 43 %
75 % 98 %
10 * M. anisopliae IP 225 7.5� 4.1 bc 7.5� 4.1 bc 100.0 a
IP 230 0.6� 2.5 c 6.2� 9.5 bc 100.0 a
B. bassiana IP 170 11.2� 6.5 bc 7.5� 4.1 bc 30.9� 11.1 b
IP 229 2.6� 2.9 bc 14.9� 9.1 bc 100.0 a
25 ** M. anisopliae IP 225 7.5� 4.1 b 7.5� 4.1 b 100.0 a
IP 230 0.6� 2.5 b 10.6� 8.7 b 100.0 a
B. bassiana IP 170 15.6� 7.1 b 9.4� 2.5 b 98.7� 7.1 a
IP 229 23.7� 2.9 b 19.2� 8.5 b 100.0 a
* Effect of isolate, df¼ 3;6, F¼ 6.06, P¼ 0.002.** Effect of isolate, df¼ 3;6, F¼ 1.59, P¼ 0.2.*** Effect of relative humidity, df¼ 2;6, F> 146, P< 0.001.
Values followed by different letters within the same day indicate
different mean mortality (two-way analysis of variance and
Student–Newman–Keuls test, P< 0.05).
Impact of moisture on Beauveria bassiana and Metarhizium anisopliae 489
0
20
40
60
80
100
05
1015
2025
CFU cm-2
IP 225
2.323
233 707
Day
s 0
20
40
60
80
100
05
1015
2025
CFU cm-2
IP 230
2.3
2370
7
Day
s
0
20
40
60
80
100
05
1015
2025
CFU cm-2 2.323
233 707
Day
s 0
20
40
60
80
100
05
1015
2025
CFU cm-2 2.3
2370
7
Day
s
0
20
40
60
80
100
05
1015
2025M
orta
lity
(
)
CFU cm-2
Mor
talit
y (
)
Mor
talit
y (
)
Mor
talit
y (
)
Mor
talit
y (
)
Mor
talit
y (
)
2.323
233 707
Day
s 0
20
40
60
80
100
05
1015
2025
CFU cm-2 2.3
2370
7
Day
s
Fig 1 – Cumulative mortality (%) of Triatoma infestans third instar nymphs after treatment with Metarhizium anisopliae iso-
lates [2.33 3 103 up to 7 3 105, corresponding to 3.37 to 5.85 log values of CFU (colony forming unit) cmL2] and
exposure to 43 %, 75 % and 98 % relative humidity, 25 �C and 12 h photophase during 25 d. Number of CFU presented
as linear values (multiplied by 103) on log transformed axis.
incubation at 98 % rh and values of LT50/90 of N3 treated with
1� 108 conidia ml�1 and exposed to the same humidity are
shown in Table 5. The highest LC50/90 data were found for
B. bassiana IP 170, which differed significantly from values
found for the other isolates. Regression curves were fitted
for all isolates (F> 108, df¼ 2, P< 0.0001), and there was
a highly significant effect of the isolate on survival of nymphs
(X2¼ 94.6, df¼ 3, P< 0.0001). LT50 (13.6 d) and LT90 (20.1 d) of IP
170 were highest and differed from the other isolates tested.
Their LT50 values varied between 5.9 d (IP 230) and 6.6 d (IP
225) and LT90 values between 6.7 d (IP 229) and 7.9 d (IP 230; Ta-
ble 5). No distinct relation between conidial concentration and
quantitative mortality could be observed at 43 % or 75 % rh. At
these conditions cumulative mortality within 25 d did not ex-
ceed 40 %, independently of the fungus species or isolates
tested (Figs 1 and 2, Table 4). Mortality of control insects dur-
ing 25 d p. i. did not exceed 10 %, regardless of humidity tested.
Development of inoculated fungi was observed on all
cadavers.
Discussion
Results showed a clear impact of moisture on initiation and
progress of conidial germination in vitro. At optimal water ac-
tivity (>0.99 aw) conidia of both fungi began germinating
490 G. M. J. Lazzarini et al.
0
20
40
60
80
100
05
1015
2025
CFU cm-2
IP 170
23233
70
7
Day
s 0
20
40
60
80
100
05
1015
2025
CFU cm-2
IP 229
23
23370
7
Day
s
0
20
40
60
80
100
05
1015
2025
CFU cm-2
23
23370
7
Day
s 0
20
40
60
80
100
05
1015
2025
CFU cm-2
23233
70
7
Day
s
0
20
40
60
80
100
05
1015
2025
CFU cm-2
23
23370
7
Day
s 0
20
40
60
80
100
0
510
1520
25
CFU cm-2
232.3
23370
7
Day
s
Mor
talit
y (
)
Mor
talit
y (
)
Mor
talit
y (
)
Mor
talit
y (
)
Mor
talit
y (
)
Mor
talit
y (
)
Fig 2 – Cumulative mortality (%) of Triatoma infestans third instar nymphs after treatment with Beauveria bassiana iso-
lates [2.33 3 103 up to 7 3 105 CFU (colony forming unit) cmL2] and exposure to 43 %, 75 % and 98 % relative humidity, 25 �C
and 12 h photophase during 25 d. Number of CFU presented as linear values (multiplied by 103) on log transformed axis.
between 4 and 8 h of incubation. Most Metarhizium anisopliae
isolates completed germination up to 12 h and B. bassiana up
to 24 h p. i. Water activity below 0.93 had a considerable neg-
ative effect on the germination kinetics. Conidia initiated
spherical growth within 4 h of incubation but germinated
only between 48 and 72 h p. i. Interestingly, Beauveria bassiana
conidia germinated faster at 0.93 aw than did M. anisopliae con-
idia. Even at the unfavourable 0.93 aw, most isolates of both
fungal species completed germination within 216 h, except
M. anisopliae IP 225 where no germination was detected. Differ-
ences of quantitative germination between isolates of the
same species were more apparent at unfavourable 0.93 aw,
than at >0.99 aw.
An increasing delay of germination and growth of M. aniso-
pliae, B. bassiana and other entomopathogenic fungi at de-
creasing water activities has been reported by other authors
(Florido et al. 2002; Hallsworth & Magan 1999; Humphreys
et al. 1989; Inch & Trinci 1987; Luz & Fargues 1997; Milner
et al. 1997). A distinct species- and strain-related variability
of germination was reported by Hywel-Jones and Gillespie
(1990) for B. bassiana and M. anisopliae conidia on Sabouraud
dextrose agar (SDA) at 100 % rh. These authors found, as did
we in this study, that conidia of M. anisopliae strains germi-
nated generally more quickly than those of B. bassiana strains.
Various other studies showed that water activities of 0.90–0.96
aw are necessary for in vitro development of these fungi. In
Impact of moisture on Beauveria bassiana and Metarhizium anisopliae 491
Table 5 – Lethal concentration [LC50/90: CFU (colony forming unit) cmL2] of Metarhizium anisopliae and Beauveria bassiana, 10and 25 d after treatment, and lethal time (LT50/90: days) to kill 50 or 90 % of Triatoma infestans third instar nymphs atrh [ 98 % and 25 �C
Days aftertreatment
Species Isolate LC50/90 LT50/90 (1� 108 conidia ml�1)
LC50 (CI) LC90 (CI) LT50 (CI) LT90 (CI)
10 M. anisopliae IP 225 2.8� 103 a (5.8� 102–4.9� 103) 1.8� 104 a (9.3� 103–1.8� 105) 6.6� 1.1 a 7.5� 1.1 a
IP 230 2.8� 103 a (2.2� 102–6.3� 103) 2.1� 104 a (9.1� 103–3.3� 105) 5.9� 1.2 a 7.9� 1.2 a
B. bassiana IP 170 1.2� 106 b (4.4� 105–1.4� 107) * 13.6� 1.8 b 20.1� 1.8 b
IP 229 2.8� 103 a (9.1� 100–5.1� 103) 8.6� 103 a (4.9� 103–1.9� 1010) 6.0� 1.5 a 6.7� 1.5 a
25 M. anisopliae IP 225 ** ** d d
IP 230 ** ** d d
B. bassiana IP 170 5.6� 102 (3.3� 101–2� 103) 7.7� 104 (4.4� 104–1.8� 105) d d
IP 229 ** ** d d
* Number of dead nymphs insufficient to calculate LC50/90.** Number of dead nymphs too high to calculate LC50/90.
Significant differences within one column and different days (LC50/90) based on confidence intervals (CI 95 %, probit analysis) and within one
column (LT50/90) based on nonlinear regression and T test are indicated by different letters.
addition to inter- and intraspecific variability, techniques of
submerged and solid cultivation, the solute used to adjust wa-
ter activity and nutrients added to the medium interfered with
test conditions and determined fungal development at re-
duced water availability. B. bassiana conidia germinated either
on nutrient agar adjusted to 0.90 aw and exposed to a corre-
sponding relative humidity or in liquid medium at 0.92 aw
(Luz & Fargues 1997). Hallsworth and Magan (1999) found
that M. anisopliae and B. bassiana inoculated on SDA medium
developed at 0.93 aw. However, germination of M. anisopliae
conidia in liquid medium was reported to be completely
inhibited in another study at <0.96 aw by Milner et al. (1997)
and was not observed, even after as much as 240 h of incuba-
tion on SDA, for either M. anisopliae and B. bassiana at �0.94 aw
(Hallsworth & Magan 1995). The same authors showed that
manipulation of the polyol content in conidia extends the
range of water availability as conidia of B. bassiana and M. ani-
sopliae with higher intracellular concentrations of glycerol and
erythritol germinated at water activities as low as 0.89 aw
(Hallsworth & Magan 1995).
In this study, daily compensated liquid medium with
added minimal nutrients and salt provided constant and pre-
cise conditions of water activity. However, in vitro conditions
did not reflect conditions in the microhabitats on the insect
and complex interactions of conidia and cuticle. Results
obtained in vitro should be extrapolated carefully. Previous
studies showed that B. bassiana, M. anisopliae and other fungi
were able to infect insects at lower humidities (Ferron 1977;
Hsiao et al. 1992; Lecuona et al. 2001; Marcandier & Khacha-
tourians 1987; Milner et al. 1997; Ramoska 1984).
Fungal activity is directly related to the dose. Quantitative
contamination of the cuticle with conidia depends on applica-
tion techniques in standard laboratory assays. James et al.
(1998) found five times greater doses per insect after immer-
sion than after spray application. Moreover, a boundary layer
of elevated moisture on the cuticle may permit germination of
a higher number of conidia even at lower ambient humidities.
Obviously there was no relationship between germination
kinetics at the unfavourable 0.93 aw and their activity against
T. infestans nymphs at different humidities. Isolates such as
M. anisopliae IP 230 and B. bassiana IP 170 that showed a faster
germination and higher number of germinated conidia at 0.93
aw, did not induce higher mortalities at the unfavourable rel-
ative humidities of 75 % or 43 %. On the contrary, IP 170 was
the least virulent isolate at all humidities and among isolates
tested. In addition, isolates with reduced rates of germination,
(B. bassiana IP 229) or without germination (M. anisopliae IP 225)
at 0.93 aw were not less virulent against nymphs, regardless of
the humidity tested. In vivo tests showed no difference in ac-
tivity between both fungal species in comparison with the in
vitro tests. The reduced virulence at lower 43 % and 75 % rh
may be related to a low number of germinating conidia and in-
vading propagules or to a loss of conidial adherence after pro-
longed exposure on the cuticle. In the present study mortality
did not increase at 43 % rh as observed by other authors
(Romana 1992; Luz 1990).
There are only few studies correlating in vitro and in vivo be-
haviour of entomopathogenic fungi. Drummond et al. (1987)
found no relationship between speed of germination in vitro
of Verticillium lecanii isolates and their activity against white-
flies. In contrast, Matawele et al. (1994) observed an elevated
virulence against green leafhopper (Nephotettix virescens)
among mutants of M. anisopliae and Paecilomyces farinosus
that germinated and grew better in vitro at reduced water ac-
tivity or humidity than the parental strains.
It is important to mention that our results regarding the
correlation between in vitro and in vivo behaviour of B. bassiana
and M. anisopliae are based on only few fungal isolates and
may be different for other strains or insect models. Isolates
of M. anisopliae and B. bassiana collected in an area with an ex-
tremely dry climate during several months of the year germi-
nated under unfavourable conditions of water activity after
prolonged incubation but were not more virulent against
T. infestans at unfavourable humidities. No relation between
behaviour in vitro and activity in vivo was found for the tested
isolates. More studies on the impact of moisture and other fac-
tors during host invasion will be useful to develop a bioinsec-
ticide based on these fungi.
492 G. M. J. Lazzarini et al.
Acknowledgements
The authors thank Ionizete G. da Silva for providing Triatoma
infestans, Alexandre S. G. Coelho for advice on statistical anal-
ysis, Mark S. Goettel for the constructive review of the manu-
script and the National Council of Scientific and Technological
Development (CNPq) for financial support.
r e f e r e n c e s
Drummond J, Heale JB, Gillespie AT, 1987. Germination and effectof reduced humidity on expression of pathogenicity in Verti-cillium lecanii against the glasshouse whitefly Trialeurodesvaporariorum. Annals of Applied Biology 111: 193–201.
Fargues J, Luz C, 1998. Effects of fluctuating moisture and temper-ature regimes on sporulation of Beauveria bassiana on cadaversof Rhodnius prolixus. Biocontrol Science and Technology 8: 323–334.
Fargues J, Luz C, 2000. Effects of fluctuating moisture and tem-perature regimes on the infection potential of Beauveriabassiana for Rhodnius prolixus. Journal of Invertebrate Pathology75: 202–211.
Ferron P, 1977. Influence of relative humidity on the developmentof fungal infection caused by Beauveria bassiana (Fungi Imper-fecti, Moniliales) in imagines of Acanthoscelides obtectus (Col.:Bruchidae). Entomophaga 22: 393–396.
Florido JEB, Rosas RA, Rojas MG, Gonzalez GV, Castaneda GS,2002. Criteria for the selection of strains of entomopathogenicfungi Verticillium lecanii for solid state cultivation. Enzyme andMicrobial Technology 30: 910–915.
Hallsworth JE, Magan N, 1995. Manipulation of intracellularglycerol and erythritol enhances germination of conidia at lowwater availability. Microbiology 141: 1109–1115.
Hallsworth JE, Magan N, 1999. Water and temperature relations ofgrowth of the entomogenous fungi Beauveria bassiana, Meta-rhizium anisopliae, and Paecilomyces farinosus. Journal of AppliedEntomology 74: 261–266.
Hsiao WF, Bidochka MJ, Khachatourians GG, 1992. Effect of tem-perature and relative humidity on the virulence of the ento-mopathogenic fungus, Verticillium lecanii, toward the oat-berryaphid, Rhopalosiphum padi (Hom., Aphidae). Journal of AppliedEntomology 114: 484–490.
Humphreys AM, Matawele P, Trinci APJ, Gillespie AT, 1989. Effectsof water activity on morphology, growth and blastosporeproduction of Metarhizium anisopliae, Beauveria bassiana andPaecilomyces farinosus in batch and fed-batch culture. Mycolog-ical Research 92: 257–264.
Hywel-Jones NL, Gillespie AT, 1990. Effect of temperature onspore germination in Metarhizium anisopliae and Beauveriabassiana. Mycological Research 94: 389–392.
Inch JMM, Trinci APJ, 1987. Effects of water activity on growth andsporulation of Paecilomyces farinosus in liquid and solid media.Journal of General Microbiology 133: 247–252.
James RR, Croft BA, Shaffer BT, Lighthart B, 1998. Impact oftemperature and humidity on host–pathogen interactionbetween Beauveria bassiana and a coccinelid. EnvironmentalEntomology 27: 1506–1513.
Lecuona RE, Edelstein JD, Berretta MF, Rossa FR, Argas JA, 2001.Evaluation of Beauveria bassiana (Hyphomycetes) strains aspotential agents for control of Triatoma infestans (Hemiptera:Reduviidae). Journal of Medical Entomology 38: 172–179.
Luz C, 1990. Zur Pathogenitat von Beauveria bassiana (Fungi, Im-perfecti) gegenuber mehreren Raubwanzenarten (Reduviidae,Triatominae) und Einfluss der relativen Luftfeuchtigkeit auf dieInfektion von Rhodnius prolixus. Mitteilungen der DeutschenGesellschaft fur Allgemeine und Angewandte Entomologie 7: 510–511.
Luz C, Fargues J, 1997. Temperature and moisture requirementsfor conidial germination of an isolate of Beauveria bassiana,pathogenic to Rhodnius prolixus. Mycopathologia 138: 117–125.
Luz C, Fargues J, 1998. Factors affecting conidial production ofBeauveria bassiana from fungus-killed cadavers of Rhodniusprolixus. Journal of Invertebrate Pathology 72: 97–103.
Luz C, Fargues J, 1999. Dependence of the entomopathogenicfungus, Beauveria bassiana, on high humidity for infection ofRhodnius prolixus. Mycopathologia 146: 33–41.
Luz C, Rocha LFN, Humber RA, 2003. Record of Evlachovaea sp.(Hyphomycetes) on Triatoma sordida in the State of Goias, Braziland its activity against Triatoma infestans (Reduviidae, Triato-minae). Journal of Medical Entomology 40: 451–454.
Luz C, Rocha LFN, Nery GV, 2004a. Detection of entomopatho-genic fungi in peridomestic triatomine-infested areas in Cen-tral Brazil and fungal activity against Triatoma infestans (Klug)(Hemiptera: Reduviidae). Neotropical Entomology 33: 783–791.
Luz C, Rocha LFN, Nery GV, Magalhaes BP, Tigano MS, 2004b.Activity of oil-formulated Beauveria bassiana against Triatomasordida in peridomestic areas in Central Brazil. Memorias doInstituto Oswaldo Cruz 99: 211–218.
Luz C, Rocha LFN, Silva IG, 2004c. Pathogenicity of Evlachovaea sp.(Hyphomycetes), a new species isolated from Triatoma sordida,in Chagas disease vectors under laboratory conditions. Revistada Sociedade Brasileira de Medicina Tropical 37: 189–191.
Luz C, Silva IG, Cordeiro CMT, Tigano MS, 1998a. Beauveria bassi-ana (Hyphomycetes) as a possible control agent for the vectorsof Chagas disease. Journal of Medical Entomology 35: 977–979.
Luz C, Tigano MS, Silva IG, Cordeiro CMT, Aljanabi SM, 1998b.Selection of Beauveria bassiana and Metarhizium anisopliae tocontrol Triatoma infestans. Memorias do Instituto Oswaldo Cruz93: 839–846.
Marcandier S, Khachatourians GG, 1987. Susceptibility of themigratory grasshopper, Melanoplus sanguinipes (Fab.) (Orthop-tera: Acrididae), to Beauveria bassiana (Bals.) Vuillemin (Hypho-mycetes): influence of relative humdity. Canadian Entomologist119: 901–907.
Matawele P, Trinci APJ, Gillespie AT, 1994. Mutants of entomo-pathogenic fungi that germinate and grow at reduced wateractivities and reduced relative humidities are more virulent toNeophotellix virescens (green leafhopper) than the parentalstrains. Mycological Research 98: 1329–1333.
Milner RJ, Staples JA, Lutton GG, 1997. The effect of humidity ongermination and infection of termites by the hyphomycete,Metarhizium anisopliae. Journal of Invertebrate Pathology 69: 64–69.
Ramoska WA, 1984. The influence of relative humidity on Beau-veria bassiana infectivity and replication in the chinch bug,Blissus leucopterus. Journal of Invertebrate Pathology 43: 389–394.
Romana CA, 1992. Recherches sur les potentialites des hypho-mycetes entomopathogenes (Fungi Imperfecti) dans la luttecontre les Triatominae (Heteroptera). These de Doctorat,Montpellier, 129 pp.
SAS Institute Inc., 2000. What’s New in SAS� Software for Release8.1. SAS Institute Inc., Cary, N.C.
Silva IG, Silva HHG, 1988. Influencia da temperatura na biologia detriatomıneos. IV. Triatoma infestans Klug, 1834 (Hemiptera, Re-duviidae). Anais da Sociedade Entomologica do Brasil 17: 443–454.
Winston PW, Bates DH, 1960. Saturated solutions for the controlof humidity in biological research. Ecology 41: 232–237.
Top Related