A Novel Two-Stage Cultivation Method to Optimize Carbon ...

Folia Microbiol. 54 (2), 142–146 (2009)
http://www.biomed.cas.cz/mbu/folia/
A Novel Two-Stage Cultivation Method to Optimize
Carbon Concentration and Carbon-to-Nitrogen Ratio
for Sporulation of Biocontrol Fungi
L. GAO a,b , X.Z. LIU a *
a Key Laboratory of Systematic Mycology and Lichenology, Institute of Microbiology, Chinese Academy of Sciences,
Chaoyang District, Beijing 100 010, P.R. China
fax +86 10 6480 7505
e-mail liuxz@im.ac.cn
b State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection,
Chinese Academy of Agricultural Sciences, Beijing 100 193, P.R. China
Received 2 July 2008
Revised version 1 December 2008
ABSTRACT. A two-stage solid cultivation method was used to determine the precise requirements of carbon
concentration (1–16 g/L) and C : N ratio (0.625 : 1 to 80 : 1) for the sporulation of six biocontrol fungi.
The C concentration and C : N ratio producing the highest conidia yield were 1 g/L and 5 : 1 for Paecilomyces
lilacinus IPC-P; 2 g/L and 10 : 1 for P. lilacinus M-14; 16 g/L and 80 : 1 for Metarhizium anisopliae
SQZ-1-21; 4 g/L and 5 : 1 for M. anisopliae RS-4-1 and Lecanicillium lecanii CA-1-G; and 2 g/L and 10 : 1
for Trichoderma viride TV-1. Sporulation was more affected by C : N ratio than by C and N concentration
per se. More spores per colony were produced by the two-stage method than by a conventional, single-stage
cultivation method. These results should be useful for improving the mass production of these biocontrol
agents.
Abbreviations
CC continuous cultivation TC two-stage (solid) cultivation
LSD least significant difference PDA potato dextrose agar
Lecanicillium lecanii, Metarhizium anisopliae, Paecilomyces lilacinus, and Trichoderma viride have
been extensively studied as promising biocontrol agents (Ekbom 1979; Papavizas 1985; Mittal et al. 1995;
Arthurs and Thomas 2001; Azaizeh et al. 2002; Sun and Liu 2006), and some have been commercialized
(Liu and Li 2004). Their mass-production at present is largely dependent on a two-phase system (Feng et al.
1994), i.e., mycelium growth in liquid medium and sporulation on solid substrate. Solid incubation for sporulation,
however, generally requires a long fermentation period and a large space relative to the quantity of
spores produced (Jenkins et al. 1998). In view of these problems, many studies have been carried out to increase
production efficiency by optimizing nutritional requirements and other cultivation conditions for sporulation
(Walker and Riley 1982; Mortensen 1988; Weidemann 1988; Jackson and Slininger 1993).
The C concentration is an important factor affecting fungal sporulation (Elson et al. 1998; Gao et
al. 2007); our previous studies have measured the effects of numerous C sources (and N sources, vitamins
and minerals) on mycelial growth and sporulation of biocontrol fungi (Sun and Liu 2006; our unpublished
data). A lower C concentration usually increases sporulation of filamentous fungi, and several researches
have demonstrated that sporulation could be enhanced by the exhaustion of the saccharide source for Aspergillus
niger (Galbraith and Smith 1969) or by the depletion of the N source for Aspergillus nidulans (Carter
and Bull 1969). Pycnidia also formed more frequently when nutrient concentration was low (Nebane and
Ekpo 1992; Xiao and Sitton 2004). The phosphate, C, and N levels in liquid media significantly influenced
blastospore production of Beauveria bassiana (Thomas et al. 1987; Hegedus et al. 1990; Bosch and Yantorno
1999). The finding that media with high C concentration are unfavorable for conidia production may
explain why some fungi produce relatively few conidia on PDA, which provides ≈10 g of C per L (Jackson
and Bothast 1990). In some cases, however, media with a high C and N concentration support good sporulation
of fungi, e.g., a medium with 80 g of glucose per L and 13.2 g of casamino acids per L resulted in a high
blastospore yield of Paecilomyces fumosoroseus (Jackson and Bothast 1990; Jackson and Schisler 1992).
*Corresponding author.

2009 NOVEL TWO-STAGE CULTIVATION METHOD FOR OPTIMIZING SPORULATION OF FUNGI 143
The C : N ratio is another important factor affecting fungal sporulation (Elson et al. 1998; Gao et al.
2007). Jackson and Bothast (1990) found that Colletotrichum truncatum produced more conidia in media
with a C : N ratio of 15 : 1 than 40 : 1 or 5 : 1, and the biological control agent Talaromyces flavus produced
more ascospores as the C : N ratio increased from 5 : 1 to 30 : 1 (Engelkes et al. 1997). However, higher
C : N ratio or higher C concentration reduced sporulation of isolate HSND07 of Helminthosporium solani
(Elson et al. 1998). However, Jackson et al. (1997) found that the blastospore yields of P. fumosoroseus
were insensitive to C : N ratio at 10 : 1, 30 : 1 or 40 : 1 at C concentration of 4 g/L. The optimal C : N ratio
for sporulation depends on the fungal species and/or isolates.
Traditionally, researchers used CC (continuous cultivation) on agar plates to investigate nutritional
and other cultural requirements. With continuous growth on agar, however, the vegetative growth may
deplete nutrients in the medium before sporulation takes place. Thus, the exact nutritional requirements for
sporulation are unknown. To better understand how nutrition affects sporulation, we applied here a TC (twostage
cultivation) method (Sun et al. 2009). In the first stage, fungi are grown for 4 d on an agar medium
with a defined nutrient content. To induce sporulation, the colonies are then transferred to a second agar medium
containing a wider range of known C concentration and C : N ratio.
MATERIALS AND METHODS
Fungi. Six isolates included three nematophages (P. lilacinus M-14, P. lilacinus IPC-P, M. anisopliae
SQZ-1-21), two entomopathogens (M. anisopliae RS-4-1, L. lecanii CA-1-G), and a mycoparasite (Trichoderma
viride TV-1). All fungi were deposited in the Center of General Microorganisms Culture Collection
in the Institute of Microbiology, Chinese Academy of Sciences (Table I).
Table I. Details of fungal isolates used
Species
Isolate
Deposition
no. CGMCC
Host Location Isolated by
Paecilomyces lilacinus IPC-P 3.10031 Meloidogyne incognita Lima, Peru P. Jatala
P. lilacinus M-14 3.10032 Heterodera glycines Huanan County, Heilongjiang, China X.Z. Liu
Metarhizium anisopliae SQZ-1-21 3.10033 Meloidogyne arenaria Qingzhou, Shandong, China M.H. Sun
M. anisopliae RS-4-1 3.10035 soil a Jiangsu, China Z.A. Chen
Lecaticillium lecanii CA-1-G 3.10036 Myzus persicae Fujian, China M. Xie
Trichoderma viride TV-1 3.10038 Alternaria alternata Yunnan, China G. Wang
a With Galleria mellonella baiting.
For preparing homogeneous spore suspensions, each fungus was cultured on PDA (Oxoid, UK) plates
at room temperature for 2 weeks. Two plugs ( 5.0 mm) of each isolate growing on PDA plates were
transferred to 10 mL of sterile 500 ppm Tween 80 in a 50-mL centrifuge tube, and conidia were then suspended
with a WH-861 vortex shaker (Koled, China). Conidia number was determined with a hemocytometer
and adjusted to 5 × 10 4 conidia per mL (Elson et al. 1998).
Medium. One L of the basal medium contained 19 g of sucrose (equal to 8 g of C), 17 g of Bacto
(Difco) agar, 4 g of soy peptone (equal to 330 mg of N), 1 g of K 2 HPO 4 , 500 mg of KCl, 500 mg of MgSO 4 ,
and 10 mg of FeSO 4 . C concentrations were adjusted with sucrose (42 % C) to 1, 2, 4, 8, and 16 g/L; N concentrations
were adjusted with soy peptone (8 % N) to 0.2, 0.4, 0.8, and 1.6 g/L. The combinations of C and
N concentrations resulted in C : N ratio ranging from 0.625 : 1 to 80 : 1.
Determination of optimal carbon concentration and C : N ratio for sporulation. Sterile basal medium
(15 mL) was poured into plastic plates ( 90 mm; Miniplast, Israel). After agar solidified and 2 d before
inoculation, a sterile cellophane membrane disc was placed on the surface of the agar in each plate; the disks
90 mm for plates to be inoculated with T. viride and 35 mm for plates with the other fungi. A conidia
suspension (5 L at ≈5 × 10 4 conidia per mL) was transferred onto the center of the sterile cellophane disk,
and the plate was then sealed with double Parafilm (Pechiney Plastic Packaging, USA). After 4 d at 25 °C,
the cellophane disks and associated colonies were transferred to fresh media with different C concentrations
and C : N ratios, and cultured for further 4 d for sporulation. CC for 8 d on the basal medium with the cellophane
disk was used as control. To quantify sporulation, both the colony and the disk were transferred into
a 50-mL centrifuge tube containing 10 mL of 500 ppm Tween 80; spores were counted with a hemocytometer.
Triplication for each medium and each isolate were used in both TC and CC method.

144 L. GAO and X.Z. LIU Vol. 54
Statistical analysis. Sporulation data (number of conidia per colony) were transformed logarithmically
before statistical analysis with Statistical Analysis System software (Version 8.2, SAS Institute, USA).
Data were subjected to one-way analysis of variances (ANOVA), and means were separated using Fisher’s
protected LSD or Duncan’s multiple-range test.
RESULTS
C concentration significantly affected the spore production of five of the six fungi, while N concentration
significantly affected the spore production of three fungi (Table II). The effect of C : N ratio was extremely
significant on the sporulation of all fungi, which indicated that we cannot predict the sporulation
effect of C independently of N and vice versa. Generally, most sporulation occurred at lower C concentration
and lower C : N ratio, but abundant sporulation sometimes occurred with higher C concentration and
higher C : N ratio (Table III).
Table II. Effects of carbon (C) and nitrogen (N) concentrations, and their combination (C × N) on sporulation of biocontrol
fungi*
Concentration
g/L
P. lilacinus M. anisopliae L. lecanii T. viride
IPC-P M-14 SQZ-1-21 RS-4-1 CA-1-G TV-1
C a a b a b ns
N ns a ns a ns c
C × N c c c c c c
*a, b, c represent p < 0.05, p < 0.01, and p < 0.001, respectively; ns – no significant difference at p ≥ 0.05.
Two isolates of the nematophage P. lilacinus required different C concentration and C : N ratio for
good sporulation. For IPC-P, C at 1 and 2 g/L with higher C : N ratio, and at 4 and 8 g/L with lower C : N
ratio, supported abundant sporulation (Table III). IPC-P sporulation was significantly inhibited at the highest
C concentration (16 g/L) with all C : N ratios, and the CC on the basal medium (control plates) led to low
spore production. For M-14, C at 2 g/L with all C : N ratios supported abundant sporulation, and the highest
conidial yield was obtained with C at 2 g/L with a C : N of 10 : 1. The highest C concentration (16 g/L) with
the highest C : N ratio (80 : 1) also supported abundant sporulation (Table III). All treatments with the TC
(except for C at 8 g/L with a C : N ratio of 20 : 1) resulted in significantly higher spore yield than the control
(CC on the basal medium).
For M. anisopliae SQZ-1-21, lower C concentrations at 1 and 4 g/L with lower C : N ratios supported
abundant sporulation but spore production was highest at C of 16 g/L with a C : N ratio of 80 : 1
(Table III). All C : N ratios at C of 8 g/L inhibited sporulation. For M. anisopliae RS-4-1, higher C concentrations
at 8 and 16 g/L with all C : N ratios (except 8 g/L with a C : N of 20 : 1) inhibited sporulation, and
lower C concentrations with lower C : N ratios increased sporulation. Spore yield for M. anisopliae RS-4-1
was highest at 4 g/L of C with a C : N of 5 : 1, followed by 2 g/L with a C : N of 1.25 : 1, and 1 g/L with
a C : N of 0.625 : 1. All other treatments (except C at 16 g/L with a C : N of 10 : 1) supported higher M. anisopliae
RS-4-1 spore yields than did the control.
The responses of the entomopathogen L. lecanii CA-1-G to C concentration and C : N ratio were
similar to those of the other fungi, but the differences between treatments were less (Table III). More spores
were produced at lower C concentrations with lower C : N ratios and at higher C ones with higher C : N
ones. Sporulation of L. lecanii CA-1-G was poor with 8 g/L of C regardless of C : N ratio.
For the mycoparasite T. viride TV-1, the spore yield was highest with 2 g/L of C and a C : N of
10 : 1, followed by 1 g/L and a C : N of 5 : 1 and 8 g/L with a C : N of 20 : 1. All the C : N ratios at 16 g/L
of C greatly inhibited sporulation (Table III).
DISCUSSION
TC provides a new method to determine the nutritional requirements for fungal sporulation. Our results
indicated that TC resulted in much greater spore yields than CC. Relative to CC, TC increased maxi-

2009 NOVEL TWO-STAGE CULTIVATION METHOD FOR OPTIMIZING SPORULATION OF FUNGI 145
mum spore yields 3× for P. lilacinus IPC-P, M. anisopliae SQZ-1-21, L. lecanii CA-1-G, and T. viride TV-1;
19× for P. lilacinus M-14, and 27× for M. anisopliae RS-4-1.
Table III. Effect of carbon concentration (g/L) and C : N ratio on the sporulation of biocontrol agents by two-stage cultivation method
(10 6 conidia per colony)*
Carbon
C : N
P. lilacinus M. anisopliae L. lecanii T. viride
IPC-P M-14 SQZ-1-21 RS-4-1 CA-1-G TV-1
1 5 28.9 a 167. j 55.5 fg 291. eghi 337. j 286.0 b
1 2.5 22.9 b 107. n 194. c 359. def 269. k 108.9 f
1 1.25 6.8 fgh 117. m 195. c 386. de 225. l 210.0 c
1 0.625 8.5 ef 185. h 126. e 642. bc 438. ef 59.9 g
2 10 24.7 b 630. a 189. c 186. fghijkl 438. ef 451.7 a
2 5 24.3 b 443. c 147. de 252. efghi 612. c 17.0 h
2 2.5 5.2 hi 466. b 32.9 gh 344. defgh 860. b 167.7 de
2 1.25 3.4 ij 201. g 159. d 712. b 34.8 o 6.7 h
4 20 3.0 jk 173. i 65.3 f 204. efghijkl 415. fg 185.7 cd
4 10 3.2 j 215. f 131. e 358. defg 122. n 56.5 g
4 5 16.7 c 163. j 194. c 1540. a 983. a 2.8 h
4 3 24.7 b 231. e 247. b 246. efghij 419. efg 14.5 h
8 40 9.3 e 111. n 43.7 fg 161. hijkl 170. m 5.9 h
8 20 8.5 ef 35.8 p 67.3 f 512. cd 172. m 270.7 b
8 10 17.2 c 123. l 53.2 fg 64.2. jkl 161. m 5.3 h
8 5 11.5 d 118. m 56.3 fg 164. hijkl 343. j 2.0 h
16 80 1.2 k 315. d 801. a 228. efghijk 439. e 6.5 h
16 40 8.3 efg 154. k 47.3 fg 171. hijkl 582. d 1.9 h
16 20 6.5 gh 84.0 o 18.3 h 109. ijkl 402. gh 0.8 h
16 10 6.7 fgh 116. m 39.9 gh 20.8 l 386. hi 1.2 h
8** 24 8.5 ef 34.0 p 242. b 56.4 kl 377. i 134.6 e
LSD 2.0 5.1 24.0 187. 23.9 29.0
*Means of triplicates; values in the same column followed by the same letter are not significantly different (LSD; p < 0.05).
**Control.
Although sporulation varied here in response to the C and N concentrations, it was always very sensitive
to C : N ratio (Table II). The concentration of C had more effect on sporulation than that of N, in that
five of six tested fungi were sensitive to C concentration, while only three were sensitive to N concentration.
Although higher spore yields sometimes occurred with higher C concentration along with higher C : N ratios,
the highest sporulation generally occurred at lower C concentration with lower C : N ratios.
We used a wider range of C concentration and lower one (1–16 g/L) than previous papers (Evans and
Black 1981; Jackson and Bothast 1990; Elson et al. 1998). For the maximum spore production of fungi with
lower costs, C concentration was set up as low as 1 g/L which is lower than any previous related researches,
such as 6 g/L in Evans and Black (1981) medium, 2 g/L in Jackson and Bothast (1990) medium and 1.25 g/L
in Elson et al. (1998) medium. A wider range of C concentration resulted in a wider range of C : N ratio
(from 0.625 : 1 to 80 : 1), which allowed us to obtain more details about the nutritional requirements for sporulation.
We were interested in reducing costs for spore production and therefore in finding the minimal C requirement.
Substrates that support the highest production of spores do not necessarily result in their highest
quality. For example, although a medium with a C : N of 30 : 1 produced the greatest number of conidia of
C. truncatum (a biocontrol fungus against weeds), a medium with a C : N of 10 : 1 produced conidia that
were most effective against the weed Sesbania exaltata (Schisler et al. 1991); the C : N ratio influenced the
lipid content of C. truncatum spores, and lipid content regulates appressorium formation (Jackson and Schisler
1992). C concentration and C : N ratio can also affect shelf-life and survival after application, e.g., conidia
of C. truncatum produced in media with excess C vs. N would often accumulate and store trehalose (Jackson
and Bothast 1990; Jackson and Schisler 1995), which has been associated with desiccation tolerance (Costa
et al. 2000). Zhang et al. (2005) found that Cryptococcus nodaensis OH 182.9 produced more freeze-tolerant
cells on media with higher C : N ratio.

146 L. GAO and X.Z. LIU Vol. 54
This project was jointly supported by the National Hi-Tech Program of China (no. 2006–AA10 A211), the Knowledge
Innovation Program of the Chinese Academy of Sciences (KSCX2-YW-G-037) and National Natural Scientific Foundation of China
(no. 3077 0072 and 3062 5001). The authors also thank Prof. B. Jaffee (University of California at Davis) for serving as pre-submission
reviewers and for his valuable comments and suggestions.
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