Antifungal

Metabolite
from Trichoderma harzianum Mutant Strain
PHILIPP AGRIC
SCIENTIST

Vol. 92 No. 4, 392-397

December 2009

Intana
et al.
ISSNWarin
0031-7454

Bioactive Compound of Antifungal Metabolite from

Trichoderma harzianum Mutant Strain for the Control of
Anthracnose of Chili (Capsicum annuum L.)
Intana Warin1,*, Suwanno Chaiyawat1, Chamswarng Chiradej2, Issarakraisila Montree1,
Koysomboon Sorwaporn3 and Chantrapromma Kan3
1

The School of Agricultural Technology, Walailak University, Nakhon Si Thammarat, 80161, Thailand
Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Kamphaeng Sean Campus,
Nakhon Pathom 73140, Thailand
3
The School of Science, Walailak University, Nakhon Si Thammarat, 80161, Thailand
*
Author for correspondence: iwarin@wu.ac.th; Tel.: + 66 75 672373; Fax: + 66 75 672302
2

The antifungal metabolite was purified from a mutant strain of Trichoderma harzianum T-156co5 by a chromatography procedure. The chemical constituent of the antifungal metabolite
was determined by 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy. The
NMR spectroscopic data showed that the antifungal compound was closely related to
isoharziandione. The purified antifungal metabolite at concentrations of 50 and 100 mg L-1
was tested for the inhibition of spore germination of Colletotrichum capsici, a causal agent
of anthracnose on chili (Capsicum annuum L. var. annuum). The result showed that both
concentrations gave high percentages of inhibition, with 100 mg L-1 providing the highest
efficacy of 83.4% compared with control 2 (sterilized water). Purified antifungal metabolite
at 100 mg L-1 was also comparable with 100 mg L-1 benomyl in controlling anthracnose on
detached chili fruits. The treatment with 100 mg L-1 gave the highest disease control efficacy
of 89.9% compared with control 2, whereas 88.5% disease inhibition was observed in chili
fruits treated with 100 mg L-1 benomyl. Control 1 (2% methanol) treatment showed only 6.4%
disease inhibition.
Key Words: biological control, chemical constituent analysis, Colletotrichum capsici, tetracyclic diterpene
Abbreviations: NMR nuclear magnetic resonance, PDA potato dextrose agar, PDB potato dextrose broth

INTRODUCTION
Anthracnose caused by Colletotrichum capsici is one
of the major diseases of chili (Capsicum annuum L.
var. annuum) fruits. Economic losses as a result of this
disease are very high each year. C. capsici is a pathogen
of chili in both the pre- and postharvest stages. The
diseased chili is withered with brown to black necrosis
patches which expand very quickly under high moisture
conditions of tropical climate. There are various methods

392

of controlling the disease, but the two most common

are use of chemical fungicides such as benomyl and
biocontrol agents. Nowadays, consumers are more aware
of food safety, especially harmful pesticide residue in
agricultural products. Biocontrol and biological products
are thus promising and provide a safe means for disease
management.
Trichoderma harzianum has been studied and
used to control plant disease in many countries (Intana
et al. 2003; Altintas and Bal 2007). A mutant (induced

The Philippine Agricultural Scientist Vol. 92 No. 4 (December 2009)

Antifungal Metabolite from Trichoderma harzianum Mutant Strain

by ultraviolet irradiation) strain of T. harzianum has

also been reported to give higher biocontrol properties
compared with a wild type strain. The mutant T.
harzianum strain has been used to control dampingoff of cucumber caused by Pythium aphanidermatum
(Intana et al. 2003) and black rot of rambutan caused
by Lasiodiplodia theobromae (Intana et al. 2005). The
mutant strain was also found to produce both cell wall
lytic enzyme and chemicals that inhibited the growth of
pathogenic fungus (Intana 2003; Coskuntuna and Ozer
2008; Chandra et al. 2009). The aims of this research
were (1) to extract and purify the antifungal metabolite
from the mutant strain of T. harzianum, (2) to study the
effect of antifungal metabolite on spore germination of C.
capsici and (3) to determine the efficacy of this purified
antifungal metabolite for controlling disease on detached
chili fruit in the laboratory.

MATERIALS AND METHODS

Strain of Microorganisms
The ultraviolet derived mutant strain of Trichoderma
harzianum T-156-co5 used in this research was provided
by the Plant Disease Biocontrol Laboratory, Department
of Plant Pathology, Faculty of Agriculture at Kamphaeng
Sean, Kasetsart University, Thailand. This mutant
strain is able to grow on potato dextrose agar (PDA)
supplemented with 100 mg L-1 of benomyl, whereas the
wild type strain T-156-wt does not grow on this medium.
Moreover, the mutant strain has been found to give higher
biocontrol efficacy on damping-off of cucumber caused
by Pythium aphanidermatum and black rot of rambutan
caused by Lasiodiplodia theobromae compared with the
wild type (Intana 2003).
For the pathogen, Colletotrichum capsici strain
Cc-NST-07 causing anthracnose of chili was provided by
The Tropical Fruits Research Unit at Walailak University,
Thailand. This strain provided the highest pathogenicity
on chili fruits (Capsicum annuum L. var. annuum)
(Suwanno 2007).
Extraction of Culture Filtrate of Trichoderma
harzianum
The T. harzianum T-156-co5 strain was cultured on PDA
(HIMEDIA, HiMedia Laboratories Pvt. Ltd., India) for
2 d at room temperature (27 2C). The margin of the
colony was cut by using a 7 mm sterilized cork borer.
Twenty-five plugs of T. harzianum strain were inoculated
into a 3-L flask containing 1 L of 1/5 strength potato
dextrose broth (PDB), and incubated at room temperature
and with stay-put condition. Twenty-eight days after
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Intana Warin et al.

incubation, spores and mycelia of T. harzianum strain

were removed from the broth culture by filtration using
filter paper (Whatman No. 1) (Worasatit et al. 1994).
The culture filtrate was used for extraction of
antifungal metabolite (Oda et al. 2009). The antifungal
metabolite was extracted three times in sequence with
350 mL of ethyl acetate (EtOAc). Separation of the two
phases was facilitated by the addition of 5 g of sodium
chloride to the first extraction. The extracted solutions
were combined and evaporated at 40 C in a rotary
evaporator and the dry weight of the crude extract was
recorded (Suwanno 2007).
Efficacy of Crude Extract to Inhibit Spore
Germination
Spore suspension of C. capsici was prepared from 7-d-old
culture growing on PDA using 1/5 strength PDB as
suspension agent. The concentration of spore suspension
was adjusted with a haemacytometer to 1 x 105 spores
per mL. Then, 0.1 mL of spore suspension was mixed
with 0.1 mL of crude extract to make a concentration
of 1,000 mg L-1 in a microplate and kept for 24 h at
room temperature before the sample was stained with
lactophenol cotton blue (Chu et al. 2006). Germinating
spores in a microplate were observed under a compound
light microscope (objective lens working distance: 0.65
mm 40X) and compared with an untreated control 2
treatment where the spore suspension was mixed with
0.1 mL of sterilized water. A treatment with 2% methanol
was used as control 1 (Yenjit et al. 2004).
Percent inhibition was calculated in relation to
the germination of the control by using the formula:

[(Sc-St)/Sc] x 100

where Sc is the mean of total numbers of germinating

spore of C. capsici in the control 2 treatment (spore
suspension mixed with 0.1 mL of sterilized water), and
St is the mean of total numbers of germinating spore of
C. capsici in the presence of the crude extract. Each
treatment was performed in four replicates, with 100
spores observed in each replicate.
Isolation and Purification of Antifungal Metabolite
The antifungal metabolites from T. harzianum T-156-co5
crude extract were isolated by column chromatography
containing silica gel (SiO2) before they were eluted with
hexane (C6H14) then followed by high polarity solutions,
dichloromethane (CH2Cl2), EtOAc and methanol (MeOH)
resulting in four fractions. All fractions were separated on
thin layer chromatography (TLC) plates, and were eluted
with mixed solution of 5% MeOH and chloroform (CHCI3)
(v v-1) before visualization by UV light and iodine vapors.
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Antifungal Metabolite from Trichoderma harzianum Mutant Strain

The extract from fraction 3 was separated by column

chromatography containing Sephadex LH-20 before it was
eluted with methanol. The extract was further purified by
preparative TLC which was eluted by mixed solution of
10% EtAOc and n-Hexane (v v-1) to afford pure compound.
The band was sprayed with vanillin and scraped from the
plates before it was extracted with CH2Cl2 and then dried
under vacuum (Evidente et al. 2003). The dry weight of
the pure compound was recorded.
The chemical structure of the compound was
elucidated by analysis of 1H and 13C Nuclear Magnetic
Resonance (NMR) spectra recorded at 300 MHz and at
75 MHz, respectively, in deutero-chloroform (CDCL3), on
Bruker (Kalsruhe, Germany) spectrometers (Evidente et
al. 2003) and compared with the published data.
Efficacy of Purified Antifungal Metabolite to
Inhibit Spore Germination
An aliquot of 0.1 mL of spore suspension of C. capsici
was adjusted to 1 x 105 spores per mL and mixed with
0.1 mL of purified antifungal metabolite at concentrations
of 50 and 100 mg L-1 in a microplate and kept for 24 h at
room temperature. Germinating spores were observed
and recorded under compound light microscope. The
germination was then compared with the control 2 treatment
(sterilized water) and the treatment with 100 mg L-1 of
commercial fungicide, benomyl (Yenjit et al. 2004).
Analysis of inhibition level was calculated in
relation to germination of spores in the control by using
the formula described above.
Control of Anthracnose on Detached Chili Fruits
The fruits of chili (Capsicum annuum L. var. annuum)
were surface disinfected by 0.5% sodium hypochlorite.
One wound, 1.0 mm in diameter, was made on each
chili fruit with a sterilized needle. Each fruit was treated
with 0.1 mL of 100 mg L-1 selected purified antifungal
metabolite and kept for an hour at room temperature.
Then, 1.0 mL of spore suspension of C. capsici (1 x 105
spores per mL) was sprayed on the wounded chili fruit.
The treated fruits were kept in plastic boxes and incubated
for 4 d in a plant growth cabinet (25 2 C, 12 h light and
80% humidity). For each treatment, four replicates with
ten chili fruits per replicate were made. The percentage
of disease inhibition was then observed and recorded
compared with the control 1 treatment on which the
wound was treated with 2% (v v-1) methanol. Treatment
with sterilized water was used as control 2. The percentage
of disease inhibition was calculated by using the formula:

[(RcRt)/Rc] x 100

where Rc is the mean of disease radius on chili fruit in

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Intana Warin et al.

the control 2 treatment; Rt is the mean of disease radius

in the treatment with purified antifungal metabolite or
benomyl chemical fungicide.

RESULTS AND DISCUSSION

Extraction of Culture Filtrate of Trichoderma
harzianum
The culture T. harzianum strain T-156-co5 in 15 L of
medium provided 45.482 g of crude extract. The crude
extracted was white solid and smelled like coconut milk
[similar to the reports of Ghisalberti and Sivasithamparam
(1991) and Intana (2003)]. They found that after 28 d of
culture incubation, T. harzianum produced antifungal
metabolites with a distinct smell like coconut milk in
1/5 strength of normal PDB concentration under room
temperature.
Efficacy of Crude Extract to Inhibit Spore Germination
The crude extract of T. harzianum strain T-156-co5 at
1,000 mg L-1 inhibited spore germination of C. capsici
by 76.3% compared with the control 2 (sterilized water).
This result suggested that the crude extract might
contain some antifungal metabolites which inhibited
germination of the spore of C. capsici. With an in vivo
bioassay, many researches also indicated that the crude
culture filtrates from Trichoderma spp. could inhibit
both spore germination and germtube elongation of some
postharvest plant pathogens such as Alternaria alternata
of ber fruits (Ziziphus mauritana Lamk) (Nallathambi
et al. 2009), Penicillium expansum of pear and Botrytis
cinerea of strawberry (Batta 2007).
Isolation and Purification of Antifungal Metabolite
The antifungal metabolite isolated by chromatography
techniques from EtOAc extract fraction was a whiteyellow solid residue, 576.4 mg in weight. The pure
compound was an amorphous colorless solid. It was
determined to be a tetracyclic diterpene and closely
related to isoharziandione, according to the NMR
spectroscopic data (Table 1), in comparison with
published data (Mannina et al. 1997). This pure compound
had a molecular formula of C20H28O2.
Efficacy of Purified Antifungal Metabolite to Inhibit Spore
Germination
The results showed that the antifungal metabolite at
concentrations of 50 and 100 mg L-1 and 100 mg L-1
benomyl inhibited spore germination of C. capsici by
76.1, 83.4 and 78.5%, respectively, compared with
control 2 (sterilized water). Control 1 with 2% methanol

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Antifungal Metabolite from Trichoderma harzianum Mutant Strain

Intana Warin et al.

Table 1. The NMR spectroscopic data of the purified antifungal metabolite in comparison with published data of Mannina et
al. (1997).
H (ppm)
Position

C (ppm)

Antifungal
Metabolite

Isoharziandione

Antifungal
Metabolite

Isoharziandione

Type of Carbon

213.53

214.49

CO

2.096

2.08

42.619

42.816

CH2

2.890

2.89

2.934

2.92

29.930

30.02

CH

51.136

51.80

1.416

1.41

29.814

29.89

CH2

1.901

1.91

2.024

2.03

29.512

29.71

2.438

2.43

145.699

146.57

148.976

149.62

2.466

2.44

59.975

60.18

CH2

2.548

2.57

197.119

198.02

10

CH2

CO

11

39.444

40.14

12

2.489

2.47

53.036

53.23

CH

13

1.515

1.52

26.652

26.85

CH2

2.053

2.03

14

2.261

2.27

59.289

59.49

CH

15

49.013

49.74

16

1.111

1.11

20.923

20.89

CH3

17

2.127

2.13

22.503

22.71

CH3

18

1.537

1.52

20.685

20.89

CH3

19

0.982

0.98

25.020

25.22

CH3

20

1.001

0.99

23.226

23.43

CH3

provided only 5.2% inhibition (Table 2). Similar to the

report of Mannina et al. (1997), T. viride could produce
a new tetracyclic diterpene, named isoharziandione, and
this antifungal compound was able to inhibit mycelial
growth of fungal pathogens such as Sclerotium rolfsii.
Control of Anthracnose on Detached Chili Fruits
Both purified antifungal metabolite and benomyl at a
concentration of 100 mg L-1 showed significant (P<0.05)
inhibition of anthracnose disease by 89.9% and 88.5%,
respectively, compared with control 1 (2% methanol), after
spraying with spore suspension of C. capsici for 4 d. Control
1 showed only 6.4% disease inhibition (Table 3). This result
was supported by the report previously described by Cabras
et al. (2003) that isoharziandione antifungal metabolites of T.
viride could control crown and stem rot of artichoke caused
The Philippine Agricultural Scientist Vol. 92 No. 4 (December 2009)

Table 2. Percentage of germination inhibition of Colletotrichum

capsici spores by purified antifungal metabolite from
Trichoderma harzianum mutant strain T-156-co5 after
incubation at room temperature for 24 h.
Treatment

Inhibition (%)

50 mg L-1 of purified antifungal

metabolite

76.1 b1

100 mg L-1 of purified antifungal

metabolite

83.4 a

100 mg L-1 of benomyl

78.5 b

Control 1 (2% methanol)

5.2 c

Control 2 (sterilized water)

0.0 d

Means followed by the same letter are not significantly different

according to DMRT (P < 0.05).

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Antifungal Metabolite from Trichoderma harzianum Mutant Strain

Table 3. Percentage of anthracnose disease suppression

on chili fruits treated with 100 mg L-1 of purified antifungal
metabolite from Trichoderma harzianum mutant strain T-156co5 and incubated at room temperature for 4 d.
Treatment
-1

useful for both pre- and postharvest treatments on chili

fruits and other ergot as well. The methods for mass
production of antifungal metabolites and their application
in the field require further studies.

Disease Suppression (%)

100 mg L of purified
antifungal metabolite

89.9 a1

100 mg L-1 of benomyl

88.5 a

Control 1 (2% methanol)

6.4 b

Control 2 (sterilized water)

0.0 c

Means followed by the same letter are not significantly different

according to DMRT (P < 0.05).

by S. rolfsii. Moreover, Trichoderma spp. could produce a

lot of antifungal metabolites that inhibited spore germination
and germtube growth of plant pathogens, and these were
identified as glioviridin (Ghisalberti and Sivasithamparam
1991), pentyl pyrone (Intana 2003), gliotoxin (Wilcox et al.
1992), trichorzianines (El Hajji et al. 1987), trichodermin B
(Zafari et al. 2008) and oxazole (Lee et al. 1995).
Various publications have reported the successful
use of antifungal metabolites extracted from Trichoderma
spp. to control fungal pathogens. These include Fusarium
oxysporum, which causes wilt of melon and tomato
(Ashrafizadeh et al. 2005; Mohamed and Haggag 2005),
S. rolfsii, which causes disease on vegetables (Maiti et al.
1991), P. aphanidermatum, which causes wilt of cotton
and water melon (Ordentlich et al. 1992), damping-off of
cucumber (Intana 2003), Phytophthora sp., which causes
various plant diseases (Wilcox et al. 1992) and A. porri,
which causes blotch disease of onion (Imtiaj and Lee
2008). This research has shown an additional successful
use of antifungal metabolite from T. harzianum mutant
strain in controlling anthracnose disease in chili caused by
C. capsici.

CONCLUSION
Trichoderma species have been shown to be an effective
antagonistic fungus used for plant disease biocontrol.
The biocontrol mechanisms of Trichoderma spp.
include competing with the growth of plant pathogens,
mycoparasitism, antibiosis, promoting plant growth
and inducing resistance in plant (Harman 2000; Howell
2003). The efficacy of purified antifungal metabolite,
closely related to isoharziandione, obtained from mutant
strain of T. harzianum T-156-co5, was comparable to the
use of benomyl chemical fungicide to control anthracnose
on chili fruits. It indicates the ability of this strain to
produce effective antifungal metabolite which will be
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Intana Warin et al.

ACKNOWLEDGMENTS
This work was partially supported by the Thailand
Research Fund and the National Center for Genetic
Engineering and Bioresources Utilization Program Grant
(Grant No. BUP 014 G-48) and the Thailand Research
Fund and the Higher Education Commission (Grant No.
MRG 5080084).

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