Biocontrol Science and Technology

ISSN: 0958-3157 (Print) 1360-0478 (Online) Journal homepage: https://www.tandfonline.com/loi/cbst20

Microcycle conidiation and the conidial properties

in the entomopathogenic fungus Metarhizium
acridum on agar medium

Shizhu Zhang , Guoxiong Peng & Yuxian Xia

To cite this article: Shizhu Zhang , Guoxiong Peng & Yuxian Xia (2010) Microcycle
conidiation and the conidial properties in the entomopathogenic fungus Metarhizium
acridum on agar medium, Biocontrol Science and Technology, 20:8, 809-819, DOI:
10.1080/09583157.2010.482201

To link to this article: https://doi.org/10.1080/09583157.2010.482201

Published online: 08 Apr 2010.

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Biocontrol Science and Technology,
Vol. 20, No. 8, 2010, 809
819

RESEARCH ARTICLE
Microcycle conidiation and the conidial properties in the
entomopathogenic fungus Metarhizium acridum on agar medium
Shizhu Zhang, Guoxiong Peng and Yuxian Xia*

Genetic Engineering Research Center, College of Bioengineering, Chongqing University,

Chongqing Engineering Research Center for Fungal Insecticides, and Lab of Functional Gene
and Regulation Technologies under Chongqing Municipal Education Commission
Chongqing, China
(Received 10 January 2010; returned 5 February 2010; accepted 29 March 2010)

Conidiation of the entomopathogenic fungus Metarhizium acridum on agar

media was investigated. M. acridum CQMa102 exhibits two different conidiation
patterns on agar media: normal conidiation in which conidia are formed on
extended hyphae and microcycle conidiation in which conidiation occurs directly
after conidia germination. Microcycle conidiation resulted in a mass of conidia
produced via budding by accelerated development at the inoculation site. The
mean total conidial yield (conidiation at day 10) was 4
5-fold greater after
microcycle conidiation than during normal conidiation. Insect pathology assays
indicated that microcycle conidia produced on SYA agar were as effective as
normal aerial conidia against the locust. Ultraviolet (UV)-resistance tests showed
no significant differences between the two types of cell propagules. However,
microcycle conidia were more heat resistant than normal aerial conidia, and
accumulated higher levels of trehalose in response to heat induction compared to
normal aerial conidia.
Keywords: Metarhizium acridum; microcycle conidiation; conidial production;
conidial tolerance; UV; heat tolerant; endogenous trehalose

Introduction
Microcycle conidiation occurs when conidia germinate and develop secondary
conidia on conidiophores produced from germ tubes or conidial cells (Anderson and
Smith 1971; Hanlin 1994; Ahearn et al. 2007). It is a method of asexual spore
formation in which the normal life cycle of the fungus is bypassed. To date,
microcycle conidiation has been described in more than 100 fungal species (Hanlin
1994; Lapaire and Dunkle 2003; Ahearn et al. 2007). In Cercospora beticola,
secondary conidia penetrated host stomata as efficiently as the original primary
conidia (Rathaiah 1977). In the entomopathogenic fungus Beauveria bassiana,
microcycle conidia with distinct morphological, biochemical and pathological
characteristics have been examined from liquid cultures and attempts have been
made to exploit these properties for biological control of insects (Thomas,
Khachatourians, and Ingledew 1987; Bosch and Yantorno 1999; Holder and
Keyhani 2005; Holder, Kirkland, Lewis, and Keyhani 2007). However, there is
very little information available regarding microcycle conidiation in M. acridum.

*Corresponding author: Email: yuxianxia@cqu.edu.cn

ISSN 0958-3157 print/ISSN 1360-0478 online
# 2010 Taylor & Francis
DOI: 10.1080/09583157.2010.482201
http://www.informaworld.com
810 S. Zhang et al.

Aerial conidia produced on agar media are considered easily dispersible,

relatively resistant to varying environmental conditions, and represent the most
commonly used cell form in biological control applications (Jones et al. 2004; Ye,
Ying, Chen, and Feng 2006). Our studies on the conidiation process in M. acridum
isolates reveal that M. acridum CQMa102 exhibits two different conidiation patterns
depending upon the formulation of the agar media: normal conidiation in which
conidia are formed on extended hyphae and microcycle conidiation in which
conidiation occurs directly after conidial germination. An analysis of genes up-
regulated during microcycle conidiation indicated robust and stage-specific gene
expression profiles as compared to cells undergoing normal conidiation (Zhang and
Xia 2008).
In the present study, we examined the process of the microcycle conidiation in M.
acridum on agar medium, as well as conidial yield, virulence to locusts, ultraviolet
(UV) and heat-resistance. The conidial accumulation of trehalose in response to heat
in microcycle conidia was compared with normal aerial conidia. The potential
advantages in mass-production are also discussed.

Materials and methods

Fungal strains and culture conditions
Metarhizium acridum isolate CQMa102 from the Genetic Engineering Research
Center of Chongqing University was used in this study. This isolate was obtained
from Ceracris Kiangsu (Orthoptera: Acrididae) and has been used in the microbial
control of locusts in China (Peng, Wang, Yin, Zeng, and Xia 2008). The strain was
cultured on 1/4 strength Sabouraud’s dextrose agar medium supplemented with 0.5%
(w/v) yeast extract (1/4 SDAY) at 288C for 10
14 days for production of conidia.
Hyphae were removed by filtration through sterile cheesecloth and the conidia were
washed three times using sterile 0.05% (v/v) Tween 80. The conidial concentrations
were determined by a standard hematocytometer.

Morphogenesis of conidia development

Conidial suspensions (100 mL, 1105 conidia mL 1) were evenly spread onto agar
media plates (d  6 cm). Cultures were incubated at 288C either on microcycle
conidiation medium SYA (3% sucrose, 0.5% yeast, extract, 0.3% NaNO3, 0.05%
MgSO4, 0.05% KCl, 0.1% KH2PO4, 0.001% FeSO4, 0.001% MnSO4, and 2% agar) or
on 1/4 SDAY medium for normal conidiation. Conidial development was monitored
using a digital light microscope (MOTIC, China). Small fragments of the fungal
colonies were prepared on microscope slides with glass coverslips for examination.
Colony morphologies were recorded using a digital camera.

Conidial production
Conidial production was assessed daily over a 10-day period. Conidial suspensions
(100 mL, 1 105 conidia mL 1) were spread evenly over the surface of SYA and 1/4
SDAY media plates (d  6 cm) and were incubated at 288C. Conidia were harvested
by flooding the plates with 2 mL sterile 0.05% (v/v) Tween 80 followed by scraping of
 Biocontrol Science and Technology 811

the colony using sterile forceps. Conidial concentrations, determined by hematocyt-

ometer counts, were used to calculate the number of conidia per cm 2 agar surface.
Four replicates were counted per sample and the whole trial was performed in
triplicate using three different batches of cultures.
For assay of the properties of the cells, M. acridum was cultured on 1/4 SDAY and
SYA medium at 288C for 10 days for production of normal aerial conidia and
microcycle conidia, respectively.

Insect bioassays
Locusta migratoria was reared at 308C and 75% relative humidity with a 12 h L:12 h
D photoperiod as previously described (He et al. 2006). Male and female insects were
separated after adult emergence. Male adult locusts (2
3 days after eclosion) were
used for the bioassays. Locusts were treated with 5 mL solution of 2 107 conidia
mL 1 of either microcycle conidia or normal aerial conidia in cottonseed oil (Sigma,
USA) on the head
thorax junction. Control locusts were treated with cottonseed oil.
Mortality was recorded daily and the estimated lethal time value for 50% mortality
(LT50) was used to compare speed of kill using the t-test statistical analysis. For each
experiment, 25 locusts were used and all experiments were repeated three times.

Effect of UV-B radiation on conidial relative germination

Conidial resistance to UV-B radiation was tested according to Braga, Flint, Messias,
Anderson, and Roberts (2001), with some modifications. In brief, UV-B irradiance
was provided by a 40-W fluorescent lamp (Zhongruida Light Corporation, China).
Lamp output was primarily UV-B (peak at 308 nm). Spectral irradiance was
measured using a spectroradiometer (Taina, China). A 20-mL aliquot of either
microcycle conidia or normal aerial conidia in suspension (5 106 conidia mL 1)
was spread evenly onto potato dextrose agar (PDA) plates. The plates were
immediately exposed to irradiances of 1350 mW m 2 for 30, 60, 90 and 120 min
which provided a total dose of 2.43, 4.86, 7.29, 9.72 kJ m 2, respectively. Control
plates were covered with aluminum foil to block radiation. After irradiation, the
plates were incubated in darkness at 288C for 24 h. The relative percent germination
was calculated by comparing germination of treated conidia in relation to conidia
not exposed to UV-B radiation (Braga et al. 2001).

Effect of heat on conidial relative germination

A 100-mL aliquot of microcycle conidia or normal aerial conidia suspension at a
concentration of 5106 conidia mL 1 was transferred to sterile eppendorf tubes
and immediately placed in a water bath at 40 or 458C for 1, 2, 3, 4 and 5 h. Control
conidial suspensions were maintained at 288C. After exposure, a 20-mL aliquot was
spread evenly on PDA agar. Plates were incubated at 288C for 24 h before
microscopic observation of the germlings at 400 magnification. The relative
percent germination was calculated by comparing germination of heat treated with
untreated conidia.
812 S. Zhang et al.

Conidial accumulation of trehalose in response to heat

Samples of microcycle conidia and normal aerial conidia were heated at 45 8C for 1,
2, 3, 4 and 5 h as indicated as above. Trehalose levels of conidia were determined as
described by Foster, Jenkinson, and Talbot (2003). In brief, 1 mL conidial
suspensions (1 108 conidia mL 1) were sonicated for 4 min and boiled for 10
min to inactivate enzyme activities and to release soluble sugars. After centrifugation
at 13,000g for 5 min, the supernatant was collected and a 60-mL aliquot was added
to 60 mL of 0.1 M sodium citrate buffer. Duplicate samples were incubated in either
the presence or absence of 1 mL porcine kidney acidic trehalase (Sigma, USA) at
378C overnight. The concentration of glucose in each sample was assayed using a
glucose assay kit (Bioscience, China).

Statistical analysis
The whole study was repeated three times with each treatment replicated three times
unless stated otherwise. For UV-B resistance and heat-resistance tests, at least 300
conidia per plate and three plates per exposure period were evaluated. Data were
subjected to Independent-samples t-test and one-way ANOVA. SPSS 13.0 for
Windows was used for all statistical analyses.

Results
Morphogenesis of microcycle conidiation on agar media
In order to study the conidiation process of M. acridum, a series of trials were
designed to optimize the cultivation conditions for conidiation. M. acridum
CQMa102 exhibits two different conidiation patterns depending upon agar media
growth substrate: microcycle conidiation in which conidia are produced directly from
the germinating conidia during growth on SYA (Figure 1a, b) and normal

Figure 1. Photomicroscopy of development stages of conidiation patterns of M. acridum

CQMa102 on agar media. (a
c) Development stages of microcycle conidiation: (a) at 28 h; (b)
at day 5; (c) at day 10. (d
f) Development stages of normal conidiation: (d) at 28 h; (e) at day
5; (f) at day 10. Scale bar  10 mm in a, c, d, f and 40 mm in b and e.
 Biocontrol Science and Technology 813

conidiation in which conidia are formed at the tip or sides of extended hyphae
(Figure 1d, e) during growth on 1/4 SDAY plates. Microcycle conidiation developed
from germinating conidia was observed after 20 h incubation on SYA medium,
whereas no conidia were evident after growth on 1/4 SDAY plates during a similar
time course. In addition, microcycle conidia were smaller and more uniform in size
(Figure 1c, d 3.2290.12 mm) than the normal aerial conidia (Figure 1f,
d 4.2191.22 mm).
In normal conidiation, the mycelia spread over the agar surface thus the colonies
took on a rough appearance (Figure 2c, d). Compared to normal conidiation, the
colonies undergoing microcycle conidiation were smoother with a mucoid appear-
ance (Figure 2a, b).

Conidial production
Conidial production by M. acridum growth on either SYA or 1/4 SDAYover a 10-day
time period was determined (Figure 3). Microcycle conidial yield at day 10 was
significantly greater by 4
5-fold compared to normal conidial production (t22.9;
P B0.0001).

Insect bioassays
No significant differences in LT50 values were noted between the normal aerial
conidia (5.1490.06 days) and the microcycle conidia (5.3490.18 days) (t1.04;
P 0.36) (Figure 4). This indicates that microcycle conidia appear to be as efficient
as normal aerial conidia in targeting insects.

Figure 2. Colony morphology of M. acridum CQMa102 grown on SYA and 1/4 SDAY agar
media. (a) Colony undergoing microcycle conidiation at day 5; (b) colony undergoing
microcycle conidiation at day 10. (c) colony undergoing normal conidiation at day 5; (d)
colony undergoing normal conidiation at day 10. Scale bar  5 mm.
814 S. Zhang et al.

1.00E+08
Microcycle conidiation
1.00E+07 Normal conidiation

Conidial count (±SE) cm–2

1.00E+06

1.00E+05

1.00E+04

1.00E+03

1.00E+02

1.00E+01
0 1 2 3 4 5 6 7 8 9 10
Day after inoculation

Figure 3. Conidial production of M. acridum CQMa102 grown on SYA and 1/4 SDAY agar
over a 10-day period. Error bars are standard errors of four trials.
Effect of UV-B radiation on conidial germination
Percent germination of both conidial types declined with increasing exposure time to
1350 mW m 2 UV-B irradiance. Exposure to UV-B radiation for 90 min (7.29 kJ m 2)
resulted in 39.2093.51% germination of microcycle conidia and 42.4092.62% for
normal aerial conidia after 24 h growth. After exposure for 120 min (9.72 kJ m 2),
the irradiated conidia displayed only 21.0891.36% (microcycle conidia) and
18.2491.26% (normal aerial conidia) germination (Figure 5). There were no
significant differences in UV-B resistance between the microcycle and normal aerial
conidia (t3.25, P 0.89).

Effect of heat on conidial germination

Both normal aerial and microcycle conidia can tolerate exposure to 408C for 5 h (data
not show). At 458C, after 3
5 h exposure, highly significant differences were observed
100

80
% Survival (±SE)

Normal conidia
60 Microcycle conidia
Control
40

20

0
0 1 2 3 4 5 6 7
Days after infection

Figure 4. Survival of Locusta migratoria following topical application of 5 mL suspensions of

2 107 conidia mL 1 of normal aerial conidia or microcycle conidia (control insects were
inoculated with 5 mL cottonseed oil). LT50 values were 5.190.06 days for the normal aerial
conidia, 5.390.18 days for microcycle conidia.
 Biocontrol Science and Technology 815

100
Microcycle conidiation

Relative germination (%)

80 Normal aerial conidia

60

40

20

0
30 60 90 120
Exposure time (h)

Figure 5. Relative germination of normal aerial and microcycle conidia after exposure for 30,
60, 90 and 120 min to UV-B irradiance of 1350 mWm 2. Germination was evaluated at 24 h
after exposure. Relative germination was calculated in relation to non-irradiated controls.
Error bars are standard errors of three trials.

between normal aerial and microcycle conidia (t5.07; P 0.007) (Figure 6). After
exposure to 458C for 3 h, microcycle conidia were more thermo tolerant (8095.38%
germination) than normal aerial conidia (3594.31% germination); after 5 h at 458C,
microcycle conidia displayed a viability greater than 65.195.82%, compared to only
4.290.67% for the normal aerial conidia (Figure 6).

Conidial accumulation of trehalose in response to heat

There were no significant differences in trehalose content between non-treated
microcycle and normal aerial conidia (t0.648, P 0.541). The trehalose content of
the cells was determined following microcycle and normal aerial conidia exposure to
458C over time. The mean trehalose accumulation in microcycle conidia was
significantly higher than in normal aerial conidia after exposure to 458C for 5 h
(t2.32, P0.04). For normal aerial conidia, the highest trehalose content was

100
Microcycle conidia
Normal aerial conidia
Relative germination (%)

80

60

40

20

0
1 2 3 4 5
Exposure time (h)

Figure 6. Effect of heat on conidial germination. Relative germination of normal aerial and
microcycle conidia was determined after exposure to 458C for 1, 2, 3, 4 and 5 h. Relative
germination was calculated in relation to non-heat controls. Error bars are standard errors of
three trials.
816 S. Zhang et al.

observed when the conidia were exposed to 458C for 4 h, at which time the trehalose
content increased to about 1.290.15-fold as compared to the control. For microcycle
conidia, the highest trehalose content also appeared when the conidia were exposed
to 458C for 4 h. However, trehalose accumulation was 290.3-fold greater as
compared to the control (Figure 7).

Discussion
Microcycle conidiation is a process which the hyphal life cycle of a fungus is
bypassed, with conidia germinating to directly form secondary spores. Microcycle
conidiation can be induced through manipulation of environmental conditions,
especially culture conditions that are stressful to the organism. However, it also has
been observed in several fungi from field samples or in cultures that have not been
subjected to such stress conditions (Hanlin 1994; Alves, Rossi, Lopes, Tamai, and
Pereira 2002; Lapaire and Dunkle 2003). In most fungi, microcycle conidiation does
not occur in response to a unique set of environmental conditions and there are no
universal factors known for eliciting the microcycle conidiation phase (Bosch and
Yantorno 1999). In Aspergillus, microcycle conidiation can be effectively induced by
heat shock in germinating conidia (Anderson and Smith 1971, 1972). However, there
was no similar effect regarding microcycle conidiation in B. bassiana (Bosch and
Yantorno 1999). A previously reported MacConkey medium that resulted in
microcycle conidiation/yeast-like growth for B. bassiana did not induce microcycle
conidiation in Metarhizium isolates (Alves et al. 2002). In this study, a well defined
agar medium (SYM) that induces microcycle conidiation in M. acridum CQMa102
is described.
The ability of M. acridum to grow and produce conidia in large quantities on
artificial media is one of the main advantages in its commercial development. M.
acridum CQMa102 exhibited two different conidiation patterns, normal and
microcycle conidiation, depending on the agar media. Our results indicate that
high conidial yield may present valuable practical characteristics for microcycle
conidiation (Figure 3). The mean total conidial yield was 4.7-fold greater in

4
Microcycle conidia
Normal aerial conidia
pg trechalose / conidium

0
0 1 2 3 4 5
Exposure time (h)

Figure 7. Trehalose levels in microcycle and normal aerial conidia of M. acridum CQMa102
after exposure of conidia to 458C for 0, 1, 2, 3, 4 and 5 h. Error bars are standard errors of
three trials.
 Biocontrol Science and Technology 817

microcycle conidiation than in normal conidiation. Equally important, bioassays

against the locust showed that microcycle conidia were equally effective as normal
aerial conidia in causing mortality.
Low resistance to stresses such as high temperature and solar irradiation are
principal factors limiting the practical applications of entomopathogenic fungi
particularly in sun-exposed agricultural environments (Braga et al. 2001; Rangel,
Braga, Anderson, and Roberts 2005). These stresses delay germination or reduce
survival of conidia greatly decreasing their applicability in the field. Several thermo/
UV tolerant fungal candidates have been selected which were of greater potential for
formulation (Ying and Feng 2004; Rangel, Anderson, and Roberts 2008). We found
no significant differences between microcycle conidia and normal aerial conidia in
UV-B tolerance. However, microcycle conidia appear to be more heat resistant than
normal aerial conidia. After exposure to 458C for 5 h, only 4.290.67% of the normal
aerial conidia germinated compared to greater than 65.195.82% for microcycle
conidia (Figure 6). The heat resistance of microcycle conidia offers great potential in
formulation.
Microorganisms that live in extreme environments usually have several mechan-
isms for protecting themselves from stresses. One important mechanism is the
endogenous accumulation of trehalose (Fillinger et al. 2001; Doehlemann, Berndt,
and Hahn 2006). In entomopathogenic fungi B. bassiana trehalose accumulation
contributes to thermal stress and recovery (Liu, Ying, Feng, and Jiang 2009). In our
study, microcycle conidia and normal aerial conidia were exposed to 458C for
different times, and their trehalose content was subsequently determined. As
expected, microcycle conidia which were more heat resistant, accumulated more
trehalose after heat treatment than normal aerial conidia. This result was consistent
with previous reports. Besides trehalose accumulation, several heat-shock proteins
(HSP) are involved in thermal resistance by folding the newly synthesized proteins or
repairing the misfolded and/or aggregated proteins (Iwahashi, Nwaka, Obuchi, and
Komatsu 1998). The mechanism of microcycle conidia in thermal tolerance need to
be further explored.
In summary, we report on the properties of microcycle conidia generated by M.
acridum on agar medium. Microcycle conidia displayed significant potential in
regards to conidial production and heat resistance. These findings may provide
insights into a more efficient use of M. acridum with implications in mass-production
and studies of nutrition, pathogenesis, and genetic manipulation of this fungus.

Acknowledgements
The research was supported by grants from the National Natural Science Foundation of
China (No. 30170630), the Key Natural Science Foundation of Chongqing, China (No. 8564)
and the National High Technology Research and Development Program ‘863’ of China (No.
2006AA10A212).

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