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nutritional requirements for fungal growth and sporulation
Effects of carbon concentration and carbon to nitrogen ratio
on the growth and sporulation of several biocontrol fungi
Li GAOa,b, Man H. SUNa, Xing Z. LIUa,*, Yong S. CHEa
aKey Laboratory of Systematic Mycology and Lichenology, Institute of Microbiology,
Chinese Academy of Sciences, PO Box 2714, Beijing 100080, P. R. China
bGraduate School of the Chinese Academy of Sciences, Beijing 100080, P. R. China
a r t i c l e i n f o
Received 11 May 2005
Received in revised form
10 June 2006
Accepted 18 July 2006
Published online 8 December 2006
Richard A. Humber
a b s t r a c t
Effects of carbon concentration and carbon to nitrogen (C:N) ratio on six biocontrol fungal
strains are reported in this paper. All fungal strains had extensive growth on the media
supplemented with 6–12 gl?1carbon and C:N ratios from 10:1 to 80:1, and differed in nutri-
ent requirements for sporulation. Except for the two strains of Paecilomyces lilacinus, all
selected fungi attained the highest spore yields at a C:N ratio of 160:1 when the carbon con-
centration was 12 gl?1for Metarhizium anisopliae SQZ-1-21, 6 gl?1for M. anisopliae RS-4-1
and Trichoderma viride TV-1, and 8 gl?1for Lecanicillium lecanii CA-1-G. The optimal condi-
tions for P. lilacinus sporulation were 8 gl?1carbon with a C:N ratio of 10:1 for M-14 and
12 gl?1carbon with a C:N ratio of 20:1 for IPC-P, respectively. The results indicated that
the influence of carbon concentration and C:N ratio on fungal growth and sporulation is
strain dependent; therefore, consideration for the complexity of nutrient requirements is
essential for improving yields of fungal biocontrol agents.
ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Biocontrol offers an attractive alternative or supplement to
the use of chemical pesticides in agricultural crop protection.
Fungal biocontrol agents play a more important role in agri-
cultural and horticultural pest management (Lacey & Goettel
1995). Currently, 66 products representing at least 38 taxo-
nomically diverse species or varieties of fungi have been de-
veloped or are being developed (Liu & Li 2004). However,
developing some fungi as potential biopesticides is difficult
because their mycelial growth and sporulation on artificial
media usually depends on the fungal species and on the com-
ponents used in the culture media (Latge ´ & Sanglier 1985). The
nematophagous fungi are greatly influenced by nutrients
and culture conditions (Coscarelli & Pramer 1962; Faust &
Pramer 1964; Blackburn & Hayes 1966; Hayes & Blackburn
1966; Olthof & Estey 1966; Satchuthananthavale & Cooke
1967a,b,c; Bricklebank & Cooke 1969; Saxena et al. 1989; Li &
Holdom 1995). Although fungi are able to utilize a wide range
of carbohydrates and nitrogen compounds (Liu & Chen 2002),
their mass-production and commercialization (Bowers 1986).
Various experiments have been conducted to investigate
the influence of basal medium nutrients on fungal growth
and sporulation. Leite et al. (2003) found that the nitrogen
components had more impact than carbohydrates on the
growth of three genera of Entomophthorales: Batkoa, Furia and
Neozygites. Liu & Chen (2002, 2003) reported that the best
* Corresponding author.
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0953-7562/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
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mycological research 111 (2007) 87–92
Author's personal copy
carbon sources are glycogen for Hirsutella rhossiliensis growth
on agar Plates and casein for Arkansas Fungus 18 (ARF18)
growth in liquid cultures. The nutritional requirements for
sporulation ofH. rhossiliensis, Pochoniachlamydosporia(syn.Ver-
ticillium chlamydosporium) and ARF18 were varied. Elson et al.
(1998)studiedthe effectsof amino acid composition, C:N ratio,
as well as carbon concentration for fungal growth and sporu-
lation of Helminthosporium solani which caused silver scurf on
potatoes in storage. They concluded that higher C:N ratios or
higher carbon concentrations reduced sporulation. The com-
position of the culture medium also greatly influenced the
sporulation of the weed biocontrol agent Colletotrichum trunca-
tum NRRL 13737, the carbon concentration and C:N ratio
significantly affected the number of conidia produced and co-
nidial attributes of C. truncatum in liquid culture (Jackson &
Bothast 1990; Schisler et al. 1991; Jackson & Schisler 1992; Jack-
son & Slininger 1993). Similar work has been conducted on the
biological control agent Talaromyces flavus (Engelkes et al.
1997). All of these studies have demonstrated that the carbon
sources, nitrogen sources, and C:N ratios play an important
role on fungal growth and sporulation.
The nutrient requirements of fungi are the key to the effec-
rected toward the development of cost-effective methods for
tion of carbon concentration and the C:N ratio for the growth
and conidiation of six fungal biocontrol agents: Paecilomyces
lium lecanii), and Trichoderma viride. By evaluating the effects of
and conidiation, we can optimize these conditions to maxi-
mize mycelial or spore production of these biocontrol fungi.
Materials and methods
Fungi and inocula
Three isolates of nematophagous fungi, Paecilomyces lilacinus
M-14 and IPC-P, and Metarhizium anisopliae SQZ-1-21, two iso-
Trichoderma viride TV-1 were used in this study (Table 1). All
fungi were deposited in the Center of General Microorganisms
Culture Collection as single-conidium isolates and cultured on
slants of potato dextrose agar (PDA; Oxoid, Basingstoke)
at room temperature, and on PDA plates at 25?C. Conidial inoc-
ula were prepared by transferring three plugs (5 mm diam) of
each isolate from the colony margin after 2 wk incubation to
10 ml sterile 0.05 % Tween 80 solution in a 50-ml centrifuge
tube. Tubes were vigorously shaken using a vortex shaker
(WH-861, KOLED, Beijing) for 3–5 min to dislodge and suspend
the conidia. The conidial concentration was determined using
pension was used for inoculation.
A defined basal salts medium composed of 1 g K2HPO4, 0.50 g
KCl, 0.50 g MgSO4, 0.01 g FeSO4and 17 g Bacto-agar (Difco)
per liter of distilled water was used in all medium prepara-
tions. Sucrose and soy peptone were used as the carbon and
nitrogen sources, respectively. The medium was autoclaved
for 30 min at 121?C. Stock solutions of sucrose (100 g l?1) and
soy peptone (10 g l?1) were autoclaved separately and added
to the basal salts medium as required.
Carbon concentration and C:N ratio studies were con-
ducted with the basal salts medium supplemented with vari-
ous amounts of sucrose and soy peptone. The carbon
concentration and C:N ratio were calculated as carbon and ni-
trogen presented in the sucrose (40 % carbon) and soy peptone
(8 %nitrogen).DuringC:Nratio studies,carbonconcentrations
were maintained at a certain level and the C:N ratio was ad-
justed by varying the amount of soy peptone concentrations.
Three concentrations of carbon at 6 gl?1, 8 gl?1and 12 g l?1
and corresponding soy peptone at the C:N ratio of 10:1, 20:1,
40:1, 80:1 and 160:1 were supplemented with basal salts
The mediumwas adjusted to pH 7 by adding1 N sodiumhy-
droxide or 1 N hydrochloric acid. Fifteen millilitres of each ex-
perimental medium was poured into (9 cm diam) sterile
plastic plates. A piece of sterile cellophane (3.5 cm diam)
was overlaid on each plate 2 d before inoculation. Because
Trichoderma viride grows faster, larger pieces of cellophane
(9 cm diam) were used. Each treatment was carried out in
Assays of fungal growth and sporulation
Five microlitres of homogeneous conidial suspension (about
5 ?104conidia) prepared as above were transferred onto the
centre of the plate and cultured at 25?C for 2 wk. Colonies
on each plate were collected with a sterile scalpel, weighed
and transferred to a 50 ml centrifuge tube containing 10 ml
sterile 0.05 % Tween 80 solution. Spores were dislodged, and
the resulting suspension was vigorously mixed in a tube
with a vortexshakerfor3–5 min,the number ofsporespercol-
ony was determined using a haemocytometer.
Table 1 – Fungal isolates used in this study
soil with Galleria mellonella baiting
88L. Gao et al.
Author's personal copy
10:1 1.230.46 0.700.220.34
20:11.52 0.560.73 0.270.56
40:1 1.42 0.460.80 0.290.54
80:1 1.04 0.53 0.71 0.22 0.42
160:1 0.700.290.43 0.21 0.25
Statistical analysis of all data for fungal growth and sporula-
tion were subjected to one-way analysis of variances (ANOVA)
and the means of various treatments were separated using
Fisher’s protected least significant difference (LSD) at P¼
0.05 with SAS software (Version 8.2, SAS Institute, Cary, NC).
A logarithmic transformation was applied to the sporulation
data before statistical analysis.
Effects of carbon concentration and C:N ratio
on fungal growth
Both the carbon concentration and C:N ratio had a significant
effect on fungal growth (Table 2). In general, the C:N ratio had
more influence on fungal growth than the carbon concentra-
tion. The optimal C:N ratio varied among fungal isolates
even within the same species. A C:N ratio of 160:1 produced
the poorest fungal growth for all isolates, although each iso-
late had different C:N ratio preferences. Paecilomyces lilacinus
M-14 had better biomass yields in the media with C:N ratios
from 10:1 to 40:1 than 80:1 and 160:1. P. lilacinus IPC-P and
Metarhizium anisopliae SQZ-1-21 favoured a relatively wide
range of C:N ratios from 10:1 to 80:1. The C:N ratios of 20:1
and 40:1 were good for fungal growth of Lecanicillium lecanii
CA-1-G and M. anisopliae RS-4-1. The optimal C:N ratio of Tri-
choderma viride TV-1 growth ranged from 10:1 to 40:1. The
carbon concentration had no effect (P >0.05) on the growth
of any isolates except for P. lilacinus M-14, M. anisopliae SQZ-
1-21 and L. lecanii CA-1-G. Media supplemented with high car-
bon concentrations (12 gl?1) enabled P. lilacinus M-14 to grow
quickly and to produce a large biomass but were unfavourable
for the biomass production of M. anisopliae SQZ-1-21, and the
biomass yields of L. lecanii CA-1-G were better on media con-
taining 6 and 12 of carbon than 8 gl?1.
There were significant interactive effects of the carbon
concentration and C:N ratios on fungal growth (Table 3).
Table 2 – Effectsof carbonconcentration and carbonto nitrogen (C:N) ratioon fungalgrowthand sporulation on agarculture
(g per colony)
Sporulation (105conidia per colony)
M-14 IPC-P SQZ-1-21 RS-4-1CA-1-GTV-1 M-14 IPC-P SQZ-1-21RS-4-1 CA-1-GTV-1
Carbon (g l-1)6
Values are means of three replicates at the same level of carbon concentration or C:N ratio. LSD stands for standard bias of different carbon
concentrations or C:N ratios.
Table 3 – Effects of carbon concentration and carbon to
nitrogen (C:N) ratio on fungal growth on agar culture
(g per colony)
C:N ratioLSD1 LSD3
10:1 20:1 40:1 80:1 160:1
Values are means of three replicates. LSD1stands for standard bias
of different C:N ratios at the same carbon concentration; LSD2
stands for standard bias of different carbon concentrations at the
same C:N ratio; LSD3stands for standard bias of interactions of dif-
ferent carbon concentrations and C:N ratios within the same
Effects of nutrients on biocontrol fungi89
Author's personal copy
8 51.8 91.0
Fungal growth varied among different carbon concentrations at
the C:N ratiostested and amongdifferentC:N ratios of the same
lilacinus M-14 and L. lecanii CA-1-G; 6 gl?1and 20:1 or 8 gl?1and
80:1 for P. lilacinus IPC-P; 8 gl?1and 40:1 for M. anisopliae SQZ-1-
Effects of carbon concentration and C:N ratio
The effects of carbon concentration and C:N ratio on fungal
sporulation were similar to those on fungal growth in that
the C:N ratio was more influential for fungal sporulation than
the carbon concentration (Table 2). However, fungal nutri-
tional requirements for sporulation were different from those
M-14 sporulation. P. lilacinus IPC-P and Metarhizium anisopliae
SQZ-1-21 produced more spores in the media with a C:N ratio
derma viride TV-1 sporulation was 160:1. Lecanicillium lecanii
CA-1-G produced more conidia in the media with a C:N ratios
of 10:1 and 160:1 than other C:N ratios. The carbon concentra-
tion had no effect (P >0.05) on the sporulation of any isolates
except for T. viride TV-1 in whicha lowercarbon concentration
(6 gl?1) was beneficial for its sporulation.
tration and C:N ratio on fungal sporulation (Table 4). The opti-
mal nutrition for sporulation was distinctive from that for
growth. The favourable interaction of carbon concentration
and C:N ratio for fungal sporulation was 8 g l?1and 10:1 for
P. lilacinus M-14, 12 gl?1and 20:1 for P. lilacinus IPC-P. At the
C:N ratio of 160:1, the highest conidiation was achieved when
the carbon concentration was 12 gl?1for M. anisopliae SQZ-1-
21, 6 g l?1for M. anisopliae RS-4-1 and T. viride TV-1, and 8 g l?1
for L. lecanii CA-1-G, respectively. The least favourable interac-
tion varied without any obvious pattern for each isolate.
We evaluated the suitability of various carbon concentrations
control fungi able to infect nematodes, insects, or fungi. Al-
though we assumed that fungal requirements for carbon and
nitrogen were related to the host or fungal species, there were
no correlations between them (P>0.05). For example, nema-
tophagous fungi, such as M-14, IPC-P, and SQZ-1-21, had differ-
ing preferences for carbon concentration and C:N ratio, even
thoughboth M-14and IPC-P are isolatesofPaecilomyceslilacinus.
Ithas been reportedthatthe influenceofamino acids onfungal
sporulation is species dependent (Tarr & Kafi 1968; Evans &
Table4 –Effectsof carbonconcentrationand carbontonitrogen(C:N)ratioonfungalsporulationonagarculture(105conidia
C:N ratio LSD1
Values are means of three replicates. LSD1stands for standard bias of different C:N ratios at the same carbon concentration; LSD2stands for
standard bias of different carbon concentrations at the same C:N ratio; LSD3stands for standard bias of interactions of different carbon concen-
trations and C:N ratios within the same isolates.
90 L. Gao et al.
Author's personal copy
Black 1981; Latge ´ & Sanglier 1985) or strain dependent (Elson
tio varied among fungal species and isolates indicating that the
carbon and nitrogen sources required by these fungi for growth
and sporulation may be strain dependent.
The advantage of using the defined basal medium as
a starting point for further medium optimization studies is
that its basic recipe permits further directed selection of car-
bon and nitrogen sources to optimize conidiation. In this
study, the C:N ratio and carbon concentration studies were
conducted with the basal salts medium supplemented with
various amounts of sucrose/soy peptone as the carbon source
and nitrogen source. Sucrose and soy peptone are easily
obtained and inexpensive and have been demonstrated to
support good growth and sporulation of those fungi used in
present study. Three carbon concentrations and five C:N ra-
tios were supplemented to the basal medium and directed to
define optimal growth conditions for sporulation of biocontrol
fungal agents.This study provideda great deal of basic and es-
sential information for mass production of these fungi as bio-
control agents. We have confirmed that the influences of
carbon concentrations and C:N ratios on conidial production
of P. lilacinus, Metarhizium anisopliae, Lecanicillium lecanii, and
Trichoderma viride. Optimal sporulation occurred when cul-
tures weresuppliedwith sucroseandsoypeptonewithcarbon
concentration of 8–12 gl?1at a C:N ratio between 10:1 and 20:1
for P. lilacinus, and 6–12 g l?1at a C:N ratio of 160:1 for M. ani-
sopliae, L. lecanii, and T. viride.
been reported that the C:N ratios of media had significant ef-
fects on the spore yield of the bioherbicidal fungus Colletotri-
chum truncatum (Jackson & Bothast 1990; Schisler et al. 1991;
and casamino acid with a C:N ratio of 15:1 supported more co-
dition, the C:N ratio not only affects the mycelial and conidial
production but also affects the biocontrol efficacy (Jackson &
Schisler 1992). Conidia of C. truncatum produced in a medium
duced in 30:1 or 80:1 media, germinated more rapidly, and
formed appressoria more frequently than did conidia pro-
duced in media with C:N ratios of 30:1 or 80:1. In view of these
differences, it is prudent to evaluate a wide range of media in
order to optimize nutritional conditions for mass production
of fungal biocontrol agents (Jackson et al. 1997). Continuous
studies to increase the quality and quantity of conidiation by
varying the carbon and nitrogen sources and C:N ratios will
aid future research by providing a means of efficient produc-
tion. Future studies should be undertaken to determine the
nutritional conditions maximize sporulation optimize the po-
tential of the spores to germinate and virulence.
This project was jointly supported by the National 863 plan of
China (No. 2001AA246011) and a foundation of Institute of
Microbiology, Chinese Academy of Sciences.
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