Full text of DOI:10.1093/chromsci/38.10.421

Journal of Chromatographic Science, Vol. 38, October 2000
Separation and Identification of Volatile
Components in the Fermentation Broth of
Trichoderma atroviride by Solid-Phase Extraction
and Gas Chromatography–Mass Spectrometry
Ágnes Keszler*, Esther Forgács, and László Kótai
Institute of Chemistry, Chemical Research Center, Hungarian Academy of Sciences, H-1525 Budapest, P.O. Box 17, Hungary
Juan Antonio Vizcaíno, Enrique Monte, and Isabel García-Acha
Department of Microbiology and Genetics, CSIC/University of Salamanca, Salamanca, Spain
a strain aggregate grouped on the basis of morphological
features. Recent studies (6) have served to neotypify and
redistribute T. harzianum (after molecular examination) into
two major groups: T. harzianum and T. viride–T. atroviride
complex. According to this new classification, the studied
strain 11 has been replaced with the T. atroviride species (7).
The described molecular diversity can justify the following
great array of antimicrobial compounds with respect to struc-
ture and function produced by the T. harzianum aggregate:
pyrones (6-n-pentyl-2H-pyran-2-one) (8), anthraquinone,
butenolide (9), cyclopentyl isocyanide, isonitrine-type com-
pounds, short-chain-length hydrophobic peptides containing a
high proportion of amino acid residues, an alkyl N-terminus,
and a hydroxy amino C-terminus (generic name, peptaibols)
(8,10,11). Cyclonerodiol and four octaketide derivatives were
found in liquid culture (12). An antibiotic harzianic acid was
isolated from a culture filtrate of the SY-307 strain (13) and tri-
chothecene (C23H18O6) on spores of ATCC 90237 (14). Some
nonantibiotic-producing strains yield isonitrile-type antibi-
otics after ultraviolet (UV) mutagenesis (15,16). The pyrone for-
mation can also be stimulated by UV irradiation (16). T. species
grown on solid media (in the best case ground corn) leads to an
excess of pyrone accumulation. The addition of liquid supple-
ments and cultivation time affect the yield (17).
Production of some hydrolytic enzymes and their syner-
gism with the peptaibol antibiotics has been observed in a
strain of T. harzianum aggregate (18,19).
The Gas Chromatographic (GC)–Mass Spectrometric (MS)
method is a modern and widely used technique for the analysis
of volatile compounds (e.g., drugs, antibiotics, and toxins) in
biological systems (20–24).
In this study, we have analyzed volatile components found in
T. atroviride strain 11 by mass-spectral detection after GC
separation with the aim of establishing in further studies a
valid correlation between the production of antibiotic metabo-
lites by this isolate and their effectiveness as BCAs.
Abstract
A preseparated fermentation broth of Trichoderma atroviride
strain 11 is analyzed by gas chromatography followed by mass-
spectral detection using a Finnigan MAT GCQ apparatus. After
preseparation in a C18 and a silica gel column, nineteen pyrone
and dioxolane derivatives and two aliphatic esters are obtained,
respectively. Among these, the four dioxolane derivatives have not
been identified previously. The main component is found to be
5,5'-dimethyl-2H-pyran-2-on. The relative standard deviation for
the determination of the retention time and the peak area
(measured in ion counts) is 0.1% and 4.5%, respectively.
Introduction
Trichoderma (T.) species are ubiquitous fungi that can be
widely found in soil. They produce volatile (e.g., ethylene,
hydrogen cyanide, alcohols, aldehydes, and ketones up to C4
chain-length) and nonvolatile (e.g., peptides) compounds that
are able to inhibit the mycelial growth of fungi. This ability
gives the T. species an ecological advantage in soil and the
rhizosphere of cultivated plants and trees. Antibiosis is one of
the main antagonistic interactions between micro-organisms
and T. species, and this species with adequate antibiotic pro-
duction can be used as biological control agents (BCAs) for
several economically important plant-pathogenic fungi (4).
However, the role of antibiosis in biocontrol needs to be deeply
explored, because a large number of T. species and strains (i.e.,
homokaryotic mycelium) can yield antibiotics not only in
nature, but also in selected laboratory cultures. Metabolite
production also depends on whether sporulating or mycelial
cultures were examined (5).
The most important BCAs belong to T. harzianum, which is
* Author to whom correspondence should be addressed: e-mail agi@cric.chemres.hu.
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421

Journal of Chromatographic Science, Vol. 38, October 2000
tives. After elution on the silica gel column, two aliphatic
esters were obtained. The major components were denoted
number 11 (31.8%), 9 (17.0%), 2 and 6 (in the same quantity),
and 15 (13.6%), and the minor components were 20 (7.0%), 12
(6.0%), and 18 (4.4%). The relative quantities of all the other
compounds were below 3%. The reproducibility of the deter-
mination of retention time and of the peak area was as follows:
average relative standard deviation 0.1% and 4.5%, respec-
tively.
The pyrones are well-known volatile constituents of the T.
species (12), and dioxolane derivatives were the first identified
compounds in this fungus. The major part of the latter com-
ponents were present in very low quantity, and the 2-ethyl-2-
acetyl-1,3-dioxolane was observed at the highest level (7%).
Dioxolides were found in another micro-organism (on the
culture filtrate of the bacterium Streptomyces tendae Tu
4042), but no biological activity was found against other bac-
teria, yeast, and fungi (25). In general, dioxolanes have var-
ious influences in biological systems. 1,3-Dioxolane is known
as an antimicrobial agent (26) and 2-(2,4-dichlorophenyl)-1,3-
dioxolane has antifungal activity against pathogenic fungi
Experimental
Sample preparation
Strain 11 of T. atroviride (International Mycological Insti-
tute, Egham, U.K., strain IMI 352941) was cultured in a syn-
thetic medium (10) for 14 days at 25°C without shaking. The
culture broth was as follows: 0.5% glucose, 0.08% KH2PO4,
•
0.07% KNO3, 0.02% Ca(H2PO4)2, 0.05% MgSO4 7H2O, 0.001%
•
•
•
MnSO4 5H2O, 0.0005% CuSO4 5H2O, and 0.0001% FeSO4
7H2O at pH 6.
In vitro antimicrobial susceptibility tests were performed
using a panel that included both clinical pathogens and labo-
ratory control strains.
The solid-phase extraction (SPE) was carried out in the
following way: 100 mg mycelium of T. atroviride (strain 11) was
eluted both in 2-g Hypersyl C18 5-µm (Phenomex, Torrance,
CA) and Hypersyl silica gel 5-µm (Phenomex, Torrance, CA)
columns with 5 mL of methanol. The eluates were concen-
trated to 1 mL by evaporation in a nitrogen flow.
Chromatographic instrumentation
GC analysis was performed on a Finnigan MAT GCQ type
GC–MS. A 30-m long apolar DB-5MS (J&W Scientific, Folsom,
CA) fused methyl silicone column (0.25-mm i.d., 0.25-µm film
thickness) was used. The column temperature setting was pro-
grammed to begin at 60°C for 5 min, then increase at a rate of
20°C/min to 250°C and hold for 10 min with a final injection
temperature of 250°C. The linear velocity of the He carrier
gas was 35 cm/s. Samples were injected by splitless mode with
0.5 min and 2 min for the close and open time, respectively, of
the split valve before and after injection, also respectively.
The ionization in MS detection was performed using the
electron impact (EI+) mode at 70 eV ionization energy in full-
scan mode (10–650 amu mass range) at a 0.5-s/scan velocity
and an acquisition threshold equal to zero. The temperatures
of the ion source and the transfer line were 160°C and 220°C,
respectively. The starting time for the acquisition was 5 min
after the injection. The detected components were identified by
matching the EI+ spectra against the NIST library (Finnigan
Corp. San Jose, CA), which contains approximately 100,000
compounds.
Time (min)
Figure 1. Total ion chromatogram of volatile compounds detected and
identified by GC–MS after preconcentration in a C18 column for Tricho-
derma atroviride strain 11. The names of the components (denoted by the
peak numbers) are found in Table I.
Results and Discussion
The names, retention times, and relative quantities (com-
paring the peak areas expressed in total ion counts) of the
volatile compounds that were analyzed are displayed in Table I.
The matching of mass spectra with the representatives of the
spectral library was also indicated. For cases in which the
matching was less than 50%, only the type of compounds was
determined. The total ion chromatograms are shown in Fig-
ures 1 and 2. An explanation for the peaks’s denoted numbers
is found in Table I.
Time (min)
Figure 2. Total ion chromatogram of volatile compounds detected and
identified by GC–MS after preconcentration in a silica gel column for Tri-
choderma atroviride strain 11. The names of the components (denoted by
the peak numbers) are found in Table I.
Among the 21 components found, 19 were detected after the
preseparation of T. atroviride strain 11 in the C18 column.
These compounds were mainly pyrone and dioxolane deriva-
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Journal of Chromatographic Science, Vol. 38, October 2000
Table I. Volatile Compounds in Trichoderma atroviride Strain 11 Detected and Identified by GC–MS After
Preconcentration on C18 and Silica Gel Columns
Retention time
(min, n = 3) and
average (% RSD*)
Relative intensity
(%, n = 3) and
average (% RSD)
Matching of
spectra (%)
Compound number
Name of identified compound
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
10:36 (0.01)
10:57 (0.02)
11:28 (0.13)
11:48 (0.11)
11:56 (0.14)
12:02 (0.04)
12:10 (0.10)
12:18 (0.02)
12:46 (0.21)
13:21 (0.07)
13:59 (0.01)
14:29 (0.11)
14:37 (0.11)
14:49 (0.13)
15:07 (0.07)
15:56 (0.21)
16:10 (0.10)
16:34 (0.03)
18:44 (0.13)
20:06 (0.21)
21:11 (0.20)
1.0 (2.0)
**
1.0 (2.3)
1.0 (4.1)
1.2 (4.1)
**
2-methoxy-1,3-dioxolane
methylacetate
dioxolane derivative
1,3-dioxolane-2-(1-hydroxyethyl)-methylate
dioxolane derivative
methylisopropyonate
pyranone derivative
pyranone derivative
pyranone derivative
pyranone derivative
5,5-dimethyl-2H-pyran-2-one
pyranone derivative
6-n-butanal-2H-pyran-2-one
pyranone derivative
2-n-heptyl-8-hydroxy-2H-1-benzopyran-5-one
3-methyl-6-(1,3-dioxo-butan)-2H-pyrane-2,4-dione
pyranone derivative
3,3',4,4',5,5'-hexamethyl,6-(5-hydroxy)pentyl-2H-pyran-2-one
6-n-pent-1,2-enyl-2H-pyran-2-one
2-ethyl-2-acetyl-1,3-dioxolane
dioxolane derivative
86.1
84.6
< 50
75.7
< 50
64.5
< 50
< 50
< 50
< 50
85.2
< 50
71.8
< 50
82.5
75.4
< 50
79.5
62.0
72.0
< 50
1.0 (5.0)
1.8 (2.8)
17.1 (0.8)
2.2 (11)
31.8 (0.6)
6.0 (1.7)
1.9 (0.6)
1.2 (2.3)
13.6 (0.7)
1.7 (12)
2.0 (10)
4.4 (0.8)
1.7 (11)
7.0 (7.1)
2.7 (7.4)
* Relative standard deviation.
** Preseparated on silica gel column. The intensity is identical with the value of compound 9.
lite production in that novel species had been cited as part of
T. harzianum. However, their biological activity as BCAs has
demonstrated the difference between these species (7), and
the presence of the new metabolites produced by T. atroviride
is in agreement with the redistribution of both species in a
molecular scheme.
Table II. Results of the Antibiotical Test of T. atroviride
Strain 11 on Synthetic Media
Type of isolates
Quantity (%)
Total active
Antibacterial
Antifungal
8.7
25.0
0
Antiyeast
0
Antiphytopathogenic
0
Conclusion
In the fermentation broth of T. atroviride, nineteen
pyrone and dioxolane derivatives, and two aliphatic esters
were detected after preseparation in a C18 and a silica gel
column, respectively. The major part of them was identified
by greater than 50% probability matching with a spectral
library.
To the best of our knowledge, four compounds (the diox-
olane derivatives) have never been previously found in the fer-
mentation broth of any T. species when identified by the
GC–MS method.
for humans and animals as a result of the oxime group com-
bined with chlorine atoms (27). The halogen (especially
iodine-substituated 1,3-dioxolanyluracils) has antiviral activity
(28).
Antibiotical tests (made in the University of Salamanca,
Salamanca, Spain) have shown that T. atroviride strain 11
has slight antibiotical activity. Only an antibacterial effect
was observed (Table II).
A good agreement was observed between the low antibiotical
activity and the volatile pattern of T. atroviride strain 11. Diox-
olanes have no presumable biological activity (25) and their
toxicity (29) appears to be very low. 6-n-pentyl-Pyranone, the
main volatile antibiotically active constituent of T. harzianum
(8), was completely missing, and 6-n-pentenyl-2H-pyran-2-
one was not found in large quantity either.
Acknowledgments
Because T. atroviride had previously been taxonomically
included within T. harzianum, the description of the metabo-
This work was supported by Scientific-Technological Co-opera-
tion Hungary–Spain.
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Journal of Chromatographic Science, Vol. 38, October 2000
Effect of solid substrate, liquid supplement, and harvest time on
6-n-pentyl-2H-pyran-2-one (6PAP) production by Trichoderma
spp. J. Agric. Food Chem. 45: 531–34 (1997).
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