Vol.2, No.3, 301-307 (2011) Agricultural Sciences
doi:10.4236/as.2011.23040
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
Fungicide tolerance of Trichoderma asperelloides and T.
harzianum strains
Adriana Paola Chaparro1, Lilliana Hoyos Carvajal2*, Sergio Orduz3
1Research Associate Microbiology Department, San Antonio, USA;
2Facultad de Agronomía, Universidad Nacional de Colombia, Sede Bogotá, Colombia, USA;
*Corresponding Aut hor:
3Facultad de Ciencias, Universidad Nacional de Colombia, Sede Medellín, Colombia, USA.
Received 15 June 2011; revised 23 July 2011; accepted 31 July 2011.
ABSTRACT
Tolerance in isolations of Trichoderma was de-
veloped by exposing t wo strains of T. harzianum
and three of T. asperelloides to increasing con-
centrations of chemical fungicides. This isola-
tion of Trichoderma was exposed to three fun-
gicides: Captan, Thiabendazol and the mixture
Captan-Carboxin. Some selected lines of these
strains reached tolerance to Captan and partial
tolerance to the mixture Captan-Carboxin. The
biological and genetic changes in these tolerant
lines were monitored by determining the relative
growth rate of the fungus, inhibition of Fusa-
rium and by analyzing the genomic changes
through UP-PCR. The results show that the tol-
erance to fungicides can be developed without
affecting the parameters of biological activity in
these lines of Trichoderma (growth and para-
sitism against Fusarium). Chemical tolerance to
the fungicide was verified by means of changes
at the DNA level (UP-PCR), mainly in the lines
tolerant to Captan. This suggests that Tric h o-
derma survives in environments with remnants
of fungicide molecules.
Keywords: Trichoderma; Mutation; Chemical
Fungicide; Biological Co ntrol; Tolerance
1. INTRODUCTION
A strategy of biological control of plant diseases
caused by soil-borne plant pathogen fungi is the use of
species of Trichoderma, these includes species of eco-
nomic importance on industrial purposes for production
of antibiotics and enzymes. In agriculture, these fungi,
improves plant growth and development, has biological
control activity against other fungi and nematodes [1-4].
It has been found that the persistent use of fungicides
could weak the natural antagonistic activity [5]. How-
ever, Trichoderma has the capability of degradading
xenobiotic compounds [6-8]. There are Trichoderma
tolerant strains that can survive field concentrations of
chemical fungicides. We now have several approaches
that can be used to obtain Trichoderma strains resistant
to chemical fungicides. Goldman et al. [9] and Mukher-
jee et al. [10] have sccesfully obtained T. viride and T.
pseudokoningii strains tolerant to chemical fungicides.
The resistance mechanism of some fungi to chemical
fungicides is due to genetic mutations , which reduces th e
susceptibility to the fungicides and decreases their effi-
cacy [9,11-13].
In order to study the consequences of fungicide resis-
tance, were obtained selected fungicide tolerant lines of
the strains of three T. asperelloides strains and two of T.
harzianum by exposure to increasing concentrations of
the fungicides Captan ((3aR,7aS)-2-[(trichloromethyl)
sulfanyl]-3a,4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-dione),
a mix of Captan/Carboxin ((3aR,7aS)-2-[(trichloromethyl)
sulfanyl]-3a,4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-dione)
/5,6-dihydro-2-methyl-1,4-oxathiine-3-carboxanilide) and
Thiabendazol (4-(1H-benzimidazol-2-yl)-1,3-thiazole). Tak-
ing into account of a possible mutation caused by induc-
tion of fungicide resistance can also cause alterations in
the fungal adaptation an d fitness, antag onistic assays an d
growth evaluation were carried out in the selected toler-
ant lines and compared to the parental strains.
2. MATERIALS AND METHODS
2.1. Fungal Strains
All fungal strains used in these experiments were iso-
lated in Colombian soils and are identified as T. har-
zianum strains T-7, T-53, T. asperelloides strains T-19, T-
4, T-109 [14]. All strains demonstrated antagonist activ-
ity under in vitro conditions against Fusarium ox-
ysporum, Botrytis cinerea, Colletotrichum sp., Rhizocto-

A. P. Chaparro et al. / Agricultural Science 2 (2011) 301-307
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
302
nia solani, and Sclerotium rolfsii.
2.2. Strategy for the Selection of Tolerant
Trichoderma Lines to Chemical
Fungicides
Before the selection experiments were started, the
Trichoderma strains were grown in potato dextrose agar
(PDA) supplemented with the chemical fungicides at
increasing concentrations. The final concentrations used
in the field are: Captan 1132.5 ppm, a mix 1:1 Captan-
Carboxim 2000 ppm, and Thiabendazole 450 ppm. The
objective was to determine the natural fungicide toler-
ance of the five Trichoderma strains. The selection of
tolerant lines to chemical fungicides was performed by
successive cultures of the Trichoderma strains in PDA
supplemented with the correspondent fungicide at in-
creasing concentrations. Five mm diameter disks from
Trichoderma 10 days old cultures were placed on PDA
with the chemical fungicides, and mycelial growth was
measured on days 1, 2, 3, 4, 5, 7 and 11. Trichoderma
lines displaying more than 20 mm of growth were se-
lected to be grown under the following chemical fungi-
cide concentration in subsequent rounds of selection.
Strains that did grow 20 or more mm in diameter after
10 days of incubation, continue in the selection media;
on the contrary, strains that grew poorly (less than 20
mm of diameter) or did not sporulate, were discarded.
Tolerant strains were subjected to further selection ex-
periments with increasing fungicide concentrations until
the Trichoderma lines were able to sporulate.
To evaluate the tolerance of the selected Trichoderma
lines to the chemical fungicides, they were grown in 30
ml of liquid medium (yeast extract 2.5%, glucose 2.5%,
NaNO3 0.2%) in 125 ml flasks erlenmeyer supplemented
with the fungicides, for four days at 28˚C, at 125 rpm.
This experiment was performed twice, and in each one 2
replicates were set for each Trichoderma line.
2.3. Evaluation of the Antagonistic Activity
and Growth of the Tolerant
Trichoderma Selected Lines
The antagonistic activity of the selected tolerant
Trichoderma strains was compared to the wild type
strains by placing a 3 mm diameter disk from a Fusa-
rium oxysporum 5 to 8 day old culture on PDA. After 24
h, a 3 mm diameter disk of the Trichoderma strain was
placed 3 mm apart from the plant pathogen. Each treat-
ment was done by triplicate, and incubated at 25  1˚C
under lighT. The antagonistic activity of the Tric ho-
derma strains was estimated according to two criteria,:
the plant pathogen growth inhibition radius (IR) and the
antagonism class system described by Bell et al. [15].
Means of growth rate and IR was analyzed by ANOVA
and Fisher’s least significant difference (LSD) test to
determine statistically significant differences.
2.4. Identification of Molecular Characteristics
of Trichoderma Fungicide Tolerant Selected
Lines
The DNA of the Trichoderma fungicides tolerant
strains was analyzed through universal primer PCR
marker (UP-PCR), a multi-site amplification technique
[16,17]. The amplifications pattern s of these strains were
compared to the wild type strain. DNA extraction was
performed from 200 mg of lyophilized fungal mycelia
according to the method described by [18]. PCR ampli-
fication mixture was compose of PCR buffer 1X, MgCl2
3 mM, dNTPs 0.2 mM, primer 1.6 M, Taq DNA poly-
merase 1 U, 25 ng of DNA distilled water to a final
volume of 25 l. The following amplification program
was used: initial denaturation at 94˚C during 2.5 min,
followed by 30 cycles of 92˚C during 50 s, 53˚C during
90 s and 72˚C during 30 s, with a final extension at 72˚C
during 3 min. UP primers used were L-45 (5’ GTAAAA
CGACGGCCAGT 3’) and L-15 (5’ GAGGGTGGCGG
CTAG 3’). All amplification reactions were performed at
least by duplicate. Amplification products were sepa-
rated in 2% agarose, stained with ethidium bromide and
visualized on a UV transilluminator. Additionally, a spe-
cific DNA fragment of the β-tubulin gene was amplified
and used as target to diagnosed resistance to the fungi-
cide Thiabe ndazole [19].
3. RESULTS
3.1. Selection of Tolerant Trichoderma
Lines to Chemical Fungicides
After five rounds of selection, it was noticed that al-
though 10 out of 15 Trichoderma lines used in the ex-
periments accomplished the mycelial growth selection
parameter, of at least 20 mm of colony radius in 10 days,
the speed of growth in all cases was lower than the wild
type strains (Table 1). Natural tolerance to the field dose
of the chemical fungicide Captan (1132, 5 ppm) was
achieved in all the T. asperelloides and T. harzianum
strains evaluated in this study. In general, isolates of T.
harzianum were less tolerant to the chemical fungicides
than isolates of the T. asperelloides species.
At the end of the 9 rounds of selection with the chemical
fungicides, tolerance to Captan varied between 176%
and 207% of the dose recommended for field application.
Isolates of T. harzianum could not develop tolerance to
the fungicide Thiabendazole and the mixture Captan-
Carboxim.

A. P. Chaparro et al. / Agricultural Science 2 (2011) 301-307
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
303303
Table 1. Mean growth value of selected Trichoderma asperelloides and T. harzianum isolates exposed to several concentrations of
the chemical fungicides Thiabendazole, Captan-Carboxin, and Captan compared to the wild type strains after 5 rounds of selection.
Radius of the Trichoderma colony after (hr)
Strain Treatment
Active ingredient
concentration (ppm)24 48 72 96 120 168 240
Wild type 0 21.2 46.5 46.5 46.5 46.5 46.5 46.5
Thiabendazole 5 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Captan-Carboxim 750 0.0 0.0 0.0 0.0 0.0 0.0 0.0
T. harzianum T-7
Captan 1750 0.85 5.9 12.6 18.5 26.6 37.8 40.5
Wild type 0 24.8 46.5 46.5 46.5 46.5 46.5 46.5
Thiabendazole 20 2.6 7.6 10.1 13.0 14.2 18.8 26.3
Captan-Carboxim 1500 0.5 3.1 5.9 9.0 12.7 20.1 28.6
T. asperelloides
T-19 Captan 2000 3.1 8.7 13.9 20.1 21.0 24.8 25.1
Wild type 0 17.9 43.3 43.3 46.5 46.5 46.5 46.5
Thiabendazole 5 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Captan-Carboxim 750 0.0 1.2 1.3 1.9 4.0 6.3 9.7
T. harzianum T-53
Captan 1750 1.4 6.5 14.4 23.4 30.1 37.7 38.3
Wild type 0 28.7 46.5 46.5 46.5 46.5 46.5 46.5
Thiabendazole 20 3.4 6.8 8.9 11.5 15.5 20.8 24.4
Captan-Carboxim 1500 0.7 1.4 5.9 8.5 11.2 13.9 20.1
T. asperelloides
T-84 Captan 2000 2.6 14.8 24.2 29.8 37.2 43.1 46.5
Wild type 0 24.7 46.5 46.5 46.5 46.5 46.5 46.5
Thiabendazole 5 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Captan-Carboxim 750 0.0 5.2 13.7 20.4 24.6 31.3 32.4
T. asperelloides
T-109 Captan 2000 2.9 6.4 13.8 25.1 32.1 39.3 45.8
Growth mean value in bold correspond to the Trichoderma isolates selected to continue in the fungicide tolerance selection experiments.
Table 2. Maximum concentrations tolerated by Trichoderma
asperelloides and T. harzianum strains after multiple increasing
exposures to the chemical fungicides Thiabendazole, Captan-
Carboxin, and Captan, under laboratory conditions.
Strain Active ingrediente
Maximum concentration
tolerated (ppm)
Thiabendazole 0
T. harzianum T-7 Captan-Carboxin 0
Captan 2350
Thiabendazole 20
T. asperelloides
T-19 Captan-Carboxin 1500
Captan 2350
Thiabendazole 0
T. harzianum T-53 Captan-Carboxin 0
Captan 2000
Thiabendazole 20
T. asperelloides
T-84 Captan-Carboxin 1500
Captan 2350
Thiabendazole 0
T. asperelloides
T-109 Captan-Carboxin 1500
Captan 2000
T. asperelloides isolates T-19, T-84, and T-109 were
able to grow and to sporulate in the culture medium
containing 75% of the dose recommended for field ap-
plication (2000 ppm). In contrast, none of the evaluated
strains were able to develop tolerance to the fungicide
Thiabendazole at a concentration below 20 ppm. Se-
lected tolerant strains cultured in liquid medium sup-
plemented with chemical fungicides Captan and Captan-
Carboxin (Table 2) do not shown differences from the
wild type strains grown without fungicides, after four-
days of culture (data not shown).
Analysis of the growth rate, (mm/hr) of the chemical
fungicide tolerant Trichoderma lines compared to the
wild type strains, show that this parameter was affected
in 8 of the 10 tolerant selected lines. Statistical analysis
indicate that the growth rate of six tolerant lines was
lower than that of the wild type strains (T. harzianum T-7
Captan, T. asperelloides T-19 Thiabendazole, T. asperel-
loides T-84 Thiabendazol, T. asperelloides T-84 Captan,
T. asperelloides T-84 Carboxin-Captan, and T. asperel-
loides T-109 Captan),. Also in two tolerant lines, growth
rate was higher than the wild type strains (T. harzianum
T-53 Captan and T. asperelloides T-109 Captan-Car-
boxin) (Table 3).
3.2. Antagonism Tests of Tolerant
Trichoderma Lines to Chemical
Fungicides
The antagonism test was performed with the plant
pathogen Fusarium oxysporum and measured as the IR.
It was observed that all tolerant Trichoderma strains kept
their antagonism class 2 similar to th e Trichoderma wild
type, but strain T. asperelloides T-19 Thiabendazole
shifted to class 3 of antagonism (Ta b le 3). Comparison
of the IR mean values displayed by the Trichoderma
fungicide tolerant lines in dicated that so me lines have IR
values that are significantly higher than the wild type
strain, as in the case of T. harzianum T-7 selected with

A. P. Chaparro et al. / Agricultural Science 2 (2011) 301-307
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
304
Table 3. Mean growth ra te of Trichoderma strains and antago-
nism against to Fusarium oxysporum caused by wild-type and
selected fungicide tolerant lines of Trichoderma asperelloides
and T. harzianum.
Trichoderma strain Antagonism
class1
Mean growth
rate
(mm/hour)2
Mean inhi-
bition radius
(mm)2
T. harzianum T-7 Wild type 2 0 .98a 25.17b
T. harzianum T-7 Captan 2 0.77b 32.65a
T. asperelloides T-19 Wild
type 2 0.74a 33.11b
T. asperelloides T-19
Thiabendazole 3 0.06b 6.38c
T. asperelloides T-19
Captan-Carboxin 2 0.76a 46.70a
T. asperelloides T-19
Captan 2 0.73a 39.45ab
T. harzianum T-53 Wild
type 2 0.67b 36.51a
T. harzianum T-53 Captan 2 0.99a 31.31a
T. asperelloides T-84 W ild
type 2 0.81a 42.45a
T. asperelloides T-84 Thia-
bendazole 2 0.73b 25.45c
T. asperelloides T-84
Captan-Carboxin 2 0.60c 32.91b
T. asperelloides T-84
Captan 2 0.72b 30.26bc
T. asperelloides T-109
Wild type 2 0.67b 39.26a
T. asperelloides T-109
Captan-Carboxin 2 0.71a 30.35b
T. asperelloides T-109
Captan 2 0.63c 26.88b
1Antagonism class determined according to Bell et al., (1982) determined
after 67 hours of culture on PDA; 2Mean values follo wed by the s ame lette r
within each Trichoderma strain and column are not significative different
(LSD,  = 0.05).
Captan and strain T. asperelloides T-19 selected against
Captan-Carboxin. While in the other cases, the IR was
the same or significantly lower than the wild type strain
(Table 3). Taking in account that one of the criteria used
in the selection experiments was the ability of the toler-
ant lines to sporulate, the microscopic study performed
indicates that all the selected Trichoderma lines kept this
characteristic except for T. asperelloides T-19 exposed to
Thiabendazol (data not shown).
3.3. Molecular Analysis
PCR analysis of the Captan-Carboxin lines and the
wild type Trichoderma strains showed different amplifi-
cation patterns such as deletion or addition of DNA
bands. DNA amplified with primer UP-L45 indicated
that the strains T. asperelloides, T-19 and T-84, selected
with the fungicide mixture Captan-Carboxin contain the
same genetic changes compared to the wild type strains,
lost a of 1400 bp DNA band, while the bands of 1150,
500 and 450 bp were new in the fungicide treated lines
(Figure 1).
Although the PCR diagnostic test design ed to identify
Thiabendazole susceptible/resistant genotypes indicated
Figure 1. PCR analysis of Trichoderma strains tolerant to the
fungicide mixture Captan-Carboxin with primer UP-L45. lane
1, molecular weight marker low DNA mass ladder; lane 2,
tolerant T. asperelloides T-19; lane 3, T. asperelloides T-19
wild type; lane 4, tolerant T. asperelloides T-84; lane 5, T. as-
perelloides T-84 wild type; lane 6, negative control.
that there were no changes in the β-tubulin gene (Figure
2(A)), a change at the DNA level was observed when
primer UP-L45 was used. This change is illustrated by
the appearance of a new 400 bp band in both selected
Trichoderma lines (Figure 2(B)). Treatment of the
Trichoderma strains with the chemical fungicide Captan
induced the largest changes at DNA level of the fungi-
cides, primer UP-L45 was used for detection (Figure 3).
4. DISCUSSION
T. asperelloides and T. harzianum contain strains that
could be of importance in biological control of plant
pathogens [20-22]. Trichoderma strains used in this
study were isolated from different geographical areas
and from different sources. All of them were also natu-
rally tolerant to the recommended concentration of the
chemical fungicide Captan, and exposure of Tr icho-
derma strains to increasing concentrations of this fungi-
cide allowed for the selection of tolerant lines. Fun gicide
resistance is a stable, inheritable adjustment by a fungus
to a fungicide, resulting in reduced sensitivity of the
fungus to the fungicide. Resistant isolates are less af-
fected or not inhibited at all by applicatio n of a fungicid e
[23]. The fungicide can in fact still can control sensitive
isolates, causing natural resistant isolates to potentially

A. P. Chaparro et al. / Agricultural Science 2 (2011) 301-307
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
305305
Figure 2. PCR analysis of Trichoderma strains exposed to the
chemical fungicide Thiabendazole compared to the wild type
strains. A. Resistance/susceptibility analysis. Bands in lines 1
to 6 were obtained with the primers designed to detect Thia-
bendazole susceptible genotypes. Bands obtained in lines 8 to
13 were obtained with the primers designed to detect Thiaben-
dazole resistant genotypes. Lane 1, T. asperelloides T-19 wild
type; lane 2, T. asperelloides T-84 wild type; lane 3, T. asper-
elloides T-19 selected with Thiabendazole; lane 4, T. asperel-
loides T-84 selected with Thiabendazole; lane 5, susceptible
Mycosphaerella fijiensis strain (positive control); lane 6, nega-
tive control; lane 7, molecular weight marker low DNA mass
ladder; lane 8, T. asperelloides T-19 wild type; lane 9, T. asper-
elloides T-84 wild type; lane 10, T. asperelloides T-19 selected
with Thiabendazole; lane 11, T. asperelloides T-84 selected
with Thiabendazole; lane 12, Thiabendazole resistant M. fijien-
sis strain (positive control); lane 13, negative control. B. PCR
analysis with primer UP-15 of Trichoderma selected strains
with the fungicide Thiabendazole. Lane 1, T. asperelloides T-
19 wild type; lane 2, T. asperelloides T-84 wild type; lane 3,
molecular weight low DNA mass ladder; lane 4, T. asperel-
loides T-19 selected with Thiabendazole; lane 5, T. asperel-
loides T-84 selected to Thiabendazole; lane 6, negative control.
may become dominant in populations under selection
pressure of fungicide. This phenomenos happens in as-
says, evidencing the fact that Trichoderma has a natural
ability to tolerate fungicides, which is called ‘natural’ or
‘inherent resistance’. Resistance is as a response to re-
peated use of the fungicide, or to the repeated use of
Figure 3. PCR analysis of Trichoderma strains exposed to the
chemical fungicide Captan compared to the wild type strains
with primer UP-L45. Lane 1, T. harzianum T-7 wild type; lane
2, T. asperelloides T-19 wild type; lane 3, T. harzianum T-53
wild type; lane 4, T. asperelloides T-84 wild type; lane 5, T.
asperelloides T-109 wild typo; lane 6, tolerant T. harzianum T-
7; lane 7, tolerant T. asperelloides T-19; l ane 8, tole rant T. har-
zianum T-53; lane 9, tolerant T. asperelloides T-84; lane 10,
tolerant T. asperelloides T-109; lane 11, negative control; lane
12, molecular weight marker low DNA mass ladder.
another chemically related fungicide and/or by a bio-
chemical mechanism of antifungal action [24].
Ruocco et al. [25] explaine d that the ability of Tricho-
derma to withstand relatively high concentrations of a
variety of synthetic and natural toxic compounds, in-
cluding its own antibiotics, depends on efficient cell
detoxification mechanisms supported by a complex sys-
tem of membrane pumps. Now it is well know that the
genome of Trichoderma includes ABC transporters (ATP-
binding cassette (ABC) transporters), which are mem-
bers of a protein superfamily that effluxes drugs from
cells of target organisms. Thus transporters may provide
a mechanism of protection against cytotoxic drugs and
xenobiotic agents. The natural function of ABC trans-
porters in plant pathogenic fungi may relate to transport
of plant-defense compounds or fungal pathogenicity
factors [26]. The ABC transporters may explain the
natural tolerance of fungicides on Trichoderma, and their
ability to successfully to survive in extreme environ-
ments.
Growth of T. asperelloides and T. harzianum strains in
liquid medium with the fungicides Captan and Captan-
Carboxin confirmed that the selected lines have devel-
oped a mechanism to tolerate the exposure to homoge-
nous concentrations of the chemical fungicides. Toler-
ance to the fungicide mixture Captan-Carboxin was ob-
tained in the treated lines of T. asperelloides strains T-19,
T-84 and T-109, while some degree of tolerance to
Thiabendazole was only obtained with the T. asperel-
loides strains T-19 and T-84. These data suggested de-

A. P. Chaparro et al. / Agricultural Science 2 (2011) 301-307
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
306
toxification mechanisms are restricted to particular
strains, and are not present in all the specimens of a taxa.
In some cases, growth rate and IR of the Trichoderma
tolerant lines were affected by the exposure to the
chemical fungicides. The antagonism capacity under in
vitro conditions was only negatively affected in one out
of the 10 tolerant lines obtained. A similar phenomenon
was found in Penicillium on Imazalil resistance and sen-
sible strains on which was no difference in spore pro-
duction and radial growth [27]. In two cases the antago-
nistic capacity was superior in tolerant lines (T. asperel-
loides T109 Captan/Carboxin and T. harzianum T53
Captan). Analogous results were obtained by Mukherjee
et al. [10], with mutants of benomyl-tolerant strains of T.
pseudokoningii, which were superior to the wild type in
biocontrol potential on S. rolfsii. A correlation between
fungicide resistance and antagonistic activity is sug-
gested by Marra et al. [28], affirming that the upregu-
lated expression of ABC transporter genes of T. atro-
viride during the three-way interaction with various
plants and fungal pathogens, possibly supports both an-
tagonistic activity and root colonization.
DNA changes were observed in T. asperelloides lines
T-19 and T-84 treated with Thiabendazole (benzimida-
zole group) (Figure 2(B)). The results of the diagnostic
test designed by Cañas (2004) indicated that there were
not changes in the  -tubulin gene level. Nevertheless
benzimidazole resistance was conferred by point muta-
tions in the β-tubulin gene in most phytopathogenic
fungi. However, exceptions have also been noticed
through via site-directed mutagenesis, a mutation that
confers benomyl tolerance to other fungi does not impart
resistance in T. viride [29]. Kawchuk et al. [30] estab-
lished that the amino acid sequences of the β-tubulin
genes from several thiabendazole-resistant and sensitive
isolates were identical in Gibberella pulicaris. This ana-
lysis confirmed that the β-tubulin gene was not linked to
thiabendazole resistan ce. These results suggest that th ere
must be other genomic regions involved in the resistance
to benzimidazoles, but the exact molecular mechanism
for this resistance still unknown.
Differences in the number of genetic changes ob-
served in the Trichoderma strains treated with chemical
fungicides could be due to their mode of action or to the
approach used for tolerance development. It has been
described that protectant fungicides such as Captan, in-
duce mutations in several genes, contrary to systemic
fungicides in which target a particular gene or gene
product [9,11-13]. This coincides with the results, since
high genetic changes observed in the Captan tolerant
Trichoderma lines as compared to the wild type strains.
The results suggest that it is possible to develop
Trichoderma tolerant lines to some chemical fungicides.
Most importantly, th e changes induced by this tolerance,
in most cases, does not negatively affect the antagonistic
activity of the biological control strains, and in some
other cases, the growth rate and the IR are increased.
The molecular study performed permitted us to recog-
nize changes at the genomic level, which in most cases
are not related to the loss of biological fitness of the
fungal strains.
5. ACKNOWLEDGEMENTS
This research received financial support from COLCIENCIAS
grants No. 2213-12-11593 and 2213-07-12531, and Corporación para
Investigaciones Biologicas (CIB).
REFERENCES
[1] Brunner, K., Zeilinger, S., Ciliento, R., Woo, S.L., Lorito,
M., Kubicek, C.P. and Mach, R. L. (2005) Improvement
of the fungal biocontrol agent Trichoderma atroviride to
enhance both antagonism and induction of plant systemic
disease resistance. Applied Environmental Microbiology,
71, 3959-3965. doi:10.1128/AEM.71.7.3959-3965.2005
[2] Hanson, L.E. and Howell, C.R. (2004) Elicitors of plant
defense responses from biocontrol strains of Tricho-
derma virens. Phytopathology, 94, 171-176.
doi:10.1094/PHYTO.2004.94.2.171
[3] Hoyos, L., Orduz, S. and Bissett, J. (2009b) Growth
stimulation in bean (Phaseolus vulgaris L.) by Tricho-
derma. Biological Control, 51, 409-416.
doi:10.1016/j.biocontrol.2009.07.018
[4] Sahebani, N. and Hadavi, N. (2008) Biological control of
the root knot nematode Meloidogyne javanica by
Trichoderma harzianum. Soil Biology and Biochemistry,
40, 2016-2020. doi:10.1016/j.soilbio.2008.03.011
[5] Lenteren, V. and Woets, J. (1988) Biological and inte-
grated pest control in greenhouses. Annual Review of
Entomology, 33, 239-269.
doi:10.1146/annurev.en.33.010188.001323
[6] Ezzi, I.M. and Lynch, J.M. (2005) Biodegradation of
cyani de by Trichoderma spp. and Fusarium spp. Enzyme
Microbial Technology, 36, 849-954.
doi:10.1016/j.enzmictec.2004.03.030
[7] Tang, J., Liu, L., Hua, S., Chen, Y. and Chen, J. (2009)
Improved degradation of organophosphate dichlorvos by
Trichoderma atroviride transformants generated by re-
striction enzyme-mediated integration (REMI). Biore-
source Technology, 100, 480-483.
doi:10.1016/j.biortech.2008.05.022
[8] Zhou, X., Xu, S., Liu, L. and Chen, J. (2007) Degrada-
tion of cyanide by Trichoderma mutants constructed by
restriction enzyme mediated integration (REMI). Biore-
source Technology, 98, 2958-2962.
doi:10.1016/j.biortech.2006.09.047
[9] Goldman, G., Temmerman, W., Jacobs, D., Contreras, R.,
van Montagu, M. and Herrera-Estrella, A. (1993) A nu-
cleotide substitution in one of the beta-tubulin genes of
Trichoderma viride confers resistance to the antimitotic
drug methyl benzimidazole 2-yl-carbamate. Molecular
and General Genetics, 240, 73-80.

A. P. Chaparro et al. / Agricultural Science 2 (2011) 301-307
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
307307
doi:10.1007/BF00276886
[10] Mukherjee, P.K., Sherkhane, P.D. and Murthy, N.B.
(1999) Induction of stable benomyl-tolerant phenotypic
mutants of Trichoderma pseudokoningii MTCC 3011,
and their evaluation for antagonistic and biocontrol po-
tential. Indian Journal of Experimental Biology, 37, 710-
712.
[11] Yan, K. and Dickman, M. (1996) Isolation of a β-tubulin
gene from Fusarium moniliforme that confers cold-sen-
sitive benomyl resistance. Applied and Environment Mi-
crobiology, 62, 3053-3056.
[12] Deyle, C., Laigret, F. and Corio-Costet, M. (1997) A
mutation in the 14 α-demethylase gene of Uncicula ne-
cator that correlates with resistance to a sterol biosynthe-
sis inhibitor. Applied and Environment Microbiology, 63,
2966-2970.
[13] Yamamoto, E. and Baird, V. (1999) Molecular charac-
terization of four beta-tubulin genes from dinitroaniline
susceptible and resistant biotypes of Eleusine indica.
Plant Molecular Biology, 39, 45-61.
doi:10.1023/A:1006108412801
[14] Hoyos, L., Orduz, S. and Bissett, J. (2009a) Genetic and
metabolic biodiversity of Colombia and adjacent neo-
tropic regions. Fungal Genetics and Biology, 46, 615-
631. doi:10.1016/j.fgb.2009.04.006
[15] Bell, D., Wells, H. and Markham, C. (1982) In vitro an-
tagonism of Trichoderma species against six fungal plant
pathogens. Phytopathology, 72, 379-382.
doi:10.1094/Phyto-72-379
[16] Bulat, S., Lubeck, M., Mironenko, N., Jensen, D. and
Lubeck, P. (1998) UP-PCR analysis and ITS1 ribotyping
of strains of Trichoderma and Gliocladium. Mycological
Research, 102, 933-943.
doi:10.1017/S0953756297005686
[17] Lubeck, M., Alekhina, A., Lubeck, S., Jensen, F. and
Bulat, A. (1999) Delineation of Trichoderma two differ-
ent genotypic groups by a highly robust fingerprinting
method, UP-PCR, and UP-PCR product cross-hybridi-
zation. Mycological Research, 103, 289-298.
doi:10.1017/S0953756298007126
[18] Raeder, U. and Broda, P. (1985) Rapid preparation of
DNA from filamentous fungi. Letters in Applied Micro-
biology, 1, 17-20.
doi:10.1111/j .1472-765X.1985.tb01479.x
[19] Cañas, G. (2004) Identificación de cepas de Mycosphae-
rella fijiensis resistentes al benomyl usando la reacción
en cadena de la polimerasa PCR. Trabajo de grado
Magíster of Science en Biotecnología, Universidad Na-
cional de Colombia, sede Medellín, Colombia.
[20] Harman, G.E. (2006) Overview of mechanisms and uses
of Trichoderma spp. Phytopathology, 96, 190-194.
doi:10.1094/PHYTO-96-0190
[21] Sanz, L., Montero, M., Redondo, J., Llobell, A. and
Monte, E. (2005) Expression of an a-1,3-glucanase dur-
ing mycoparasitic interaction of Trichoderma asperel-
loides. FEBS Journal, 272, 493-499.
doi:10.1111/j .1742-4658.2004.04491.x
[22] Viterbo, A. and Chet, I. (2006) TasHyd1, a new hydro-
phobin gene from the biocontrol agent Trichoderma as-
perelloides, is involved in plant root colonization. Mo-
lecular Plant Pathology, 7, 249-258.
doi/10.1111/j.1364-3703.2006.00335.x
[23] Ma, Z. and Michailides, J. (2005) Advances in under-
standing molecular mechanisms of fungicide resistance
and molecular detection of resistant genotypes in phyto-
pathogenic fungi. Crop Protection, 24, 853-863.
doi:10.1016/j.cropro.2005.01.011
[24] Brent, K.J. and Hollomon, D.W. (1995) Monitoring fun-
gicide resistance in crop pathogens: How can it be man-
aged? FRAC, Brussels.
[25] Ruocco, M., Lanzuise, S., Vinale, F., Marra, R., Turrà, D.,
Woo, S.L. and Lorito, M. (2009) Identification of a new
biocontrol gene in Trichoderma atroviride: The role of an
ABC transporter membrane pump in the interaction with
different plant-pathogenic fungi. Molecular Plant-Mi-
crobe Interactions, 22, 291-301.
doi:10.1094/MPMI-22-3-0291
[26] De Waard, M.A. (1997) Significance of ABC transporters
in fungicide sensitivity and resistance. Pesticide Science,
51, 271-275.
doi:10.1002/(SICI)1096-9063(199711)51:3<271::AID-P
S642>3.0.CO;2-#
[27] Holmes, J.G. and Ecker, J.W. (1995) Relative fitness of
Imazalil-resistant and sensitive biotypes of Penicillium
digitatum. Plant Disease, 79, 1068-1073.
doi:10.1094/PD-79-1068
[28] Marra, R, Ambrosino, P., Carbone, V., Vinale, F., Woo,
S.L., Ruocco, M., Ciliento, R., Lanzuise, S., Ferraioli, S.,
Soriente, I., Gigante, S., Turrà, D., Fogliano, V., Scala, F.
and Lorito, M. (2006) Study of the three-way interaction
between Trichoderma atroviride, plant and fungal patho-
gens by using a proteomic approach. Current Genetics,
50, 307-321. doi:10.1007/s00294-006-0091-0
[29] Mukherjee, M., Hadar, R., Mukherjee, P.K. and Horwitz,
B.A. (2003) Homologous expression of a mutated
beta-tubulin gene does not confer benomyl resistance on
Trichoderma virens. Journal of Applied Microbiology, 95,
861-867. doi:10.1046/j.1365-2672.2003.02061.x
[30] Kawchuk, L.M., Hutchison, L.J., Ve rhaeghe, C.A., Lynch,
D.R., Bains, P.S. and Holley, J.D. (2002) Isolation of the
ß-tubulin gene and characterization of thiabendazole re-
sistance in Gibberella pulicaris. Canadian Journal of
Plant Pathology, 24, 233-238.
doi:10.1080/07060660309507001