Communicative & Integrative Biology

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Evaluation of the sensitivity and synergistic effect

of Trichoderma reesei and mancozeb to inhibit
under in vitro conditions the growth of Fusarium
oxysporum

María Fernanda Gonzalez, Freddy Magdama, Luis Galarza, Daynet Sosa &
Christian Romero

To cite this article: María Fernanda Gonzalez, Freddy Magdama, Luis Galarza, Daynet Sosa
& Christian Romero (2020) Evaluation of the sensitivity and synergistic effect of Trichoderma
reesei and mancozeb to inhibit under in vitro conditions the growth of Fusarium oxysporum ,
Communicative & Integrative Biology, 13:1, 160-169, DOI: 10.1080/19420889.2020.1829267

To link to this article: https://doi.org/10.1080/19420889.2020.1829267

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COMMUNICATIVE & INTEGRATIVE BIOLOGY
2020, VOL. 13, NO. 1, 160–169
https://doi.org/10.1080/19420889.2020.1829267

RESEARCH PAPER

Evaluation of the sensitivity and synergistic effect of Trichoderma reesei and

mancozeb to inhibit under in vitro conditions the growth of Fusarium oxysporum
María Fernanda Gonzaleza,b, Freddy Magdamaa,c, Luis Galarzaa,c, Daynet Sosaa,c, and Christian Romeroa,c
a
Escuela Superior Politécnica del Litoral, ESPOL, Centro de Investigaciones Biotecnológicas del Ecuador (CIBE), Guayaquil, Ecuador; bFacultad
de Ingeniería Química, Universidad de Guayaquil, Guayaquil, Ecuador; cFacultad de Ciencias de la Vida (FCV), Escuela Superior Politécnica del
Litoral, ESPOL, Guayaquil, Ecuador

ABSTRACT ARTICLE HISTORY

Trichoderma: is a saprophytic, soil-borne fungus with a worldwide distribution that has been Received 27 March 2020
extensively studied due to their capacity to synthesize secondary metabolites with antimicrobial Revised 17 September 2020
activity, parasitize other fungi and directly interact with plant roots, inducing resistance to disease Accepted 18 September2020
and tolerance to abiotic stresses. Fusarium wilt caused by the soil-inhabiting fungus Fusarium
KEYWORDS
oxysporum is considered one of the most important diseases that affect banana cultivars. Biological control; fungicide;
Currently, more environmentally friendly alternatives to control this disease are being proposed, Fusarium oxysporum;
these strategies include the application of low doses of synthetic fungicides and the use of Trichoderma reesei;
biocontrol agents such as Trichoderma or Xylaria. Thus, this study aimed to evaluate under minimum inhibitory
in vitro conditions the synergistic effect of the biological control agent T. reesei C2A combined concentration; synergistic
with low doses of mancozeb to inhibit the mycelial growth of F. oxysporum F1. To perform the effect
synergistic essays, 0.1 mg/mL of mancozeb was suspended in PDA plates, then plugs of T. ressei
C2A were placed at the center of the Petri dishes, the plates were incubated for 7 days at 28°C.
Results showed that the mycoparasitic capacity of the biocontrol strain to inhibit the mycelial
growth of F. oxysporum F1 was enhanced approximately 36% compared to the control plates.
Although these results are promising, future studies under greenhouse and field conditions are
necessary to corroborate the effectiveness of this approach.

1. Introduction The main agricultural method to control this patho­

gen is based on the application of synthetic fungicides
Banana is a tropical fruit that grows in more than 130
or sterilants, including mancozeb, methyl bromide and
countries, it ranks amongst the world’s most valuable
quaternary ammonium, which can be applied as pow­
primary agricultural commodity due to its nutritional
dered, emulsions, granulated, or solutions [8].
properties [1,2]. In the sixties, banana industry experi­
Mancozeb is a dithiocarbamate, non-systemic agricul­
enced dramatic losses due to Fusarium wilt caused by
tural fungicide with high spectrum of biological activ­
the soil-borne fungus Fusarium oxysporum [3,4].
ities against a wide range of pathogenic fungi including
Fusarium wilt was detected in Ecuador in 1936 at the
ascomycetes, oomycetes and basidiomycetes [9,10].
United Fruit plantations in Tenguel [5]. F. oxysporum
Although the application of synthetic fungicides to
penetrates the plant through the tertiary roots, then
control the growth of phytopathogens has been satis­
passes into the rhizome vascular system and pseudos­
factory in most cases, evidence suggests that synthetic
tem and invades the xylem vessels. The fungus pro­
compounds including benomyl, chlorothalonil, captan,
duces conidia, which is carried along the vascular
mancozeb, maneb and propiconazole can cause adverse
bundles where they start new areas of infection, causing
health effects on humans and the environment [11,12].
their obstruction preventing water transport and redu­
Thus, more environmentally friendly alternatives to
cing nutrients uptake [6]. It also secretes toxins that
control the growth of phytopathogens are currently
induce wilting by altering cell metabolism [7], with­
being proposed. These strategies include the application
ering by F. oxysporum remains a latent concern as it
of low doses of synthetic fungicides combined with the
represents a threat to the production of banana in
inoculation of biological agents such as nonpathogenic
Ecuador, causing considerable manufacturing and
fungi (Trichoderma, Clonostachys and Xylaria) or bac­
exporting losses.
teria (Bacillus, Lysinibacillus and Solibacillus) [13,14]. It

CONTACT Christian Romero [email protected] Escuela Superior Politécnica del Litoral, ESPOL, Centro de Investigaciones Biotecnológicas del
Ecuador (CIBE), Guayaquil, Ecuador
© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided the original work is properly cited.
 COMMUNICATIVE & INTEGRATIVE BIOLOGY 161

has been documented that these microorganisms exhi­ the presence of conidia, mycelium coloration and pre­
bit strong antagonistic interactions (i.e. antibiosis, para­ sence of hyphae. Three Petri dishes containing PDA
sitism and competition for space and nutrients) with were inoculated with 8 mm plugs of both fungi, the
plant pathogens to maintain they growth at a lower plates were incubated for 7 days at 28°C before per­
density that would occur in the absence of biological forming the macroscopic and microscopic
competitors [14,15]. identification.
Trichoderma is considered as a potential ecological alter­
native to transform agricultural systems highly dependent
on synthetic inputs into sustainable and more productive
2.3. In vitro experimental design
systems [16]. Under in vitro and greenhouse conditions,
several strains of Trichoderma were able to inhibit the 2.3.1. Dual culture plate assay
growth of a wide range of phytopathogens [17–20]. The mycoparasitic capacity of T. reesei C2A to control the
Trichoderma species are also considered important plant growth of F. oxysporum F1 was evaluated by the dual
growth-promoting fungi (PGPF) as they significantly facil­ culture plate essay as described elsewhere [26,27]. The
itate plant growth and development by increasing solubili­ tests consisted of the inoculation of 8 mm plugs containing
zation of organic compounds increasing nutrient efficacy, agar and mycelium of T. reesei C2A on one side of a Petri
releasing plant-growth stimulatory agents and inducing dish containing PDA, on the other side of the plate,
systemic resistance [21–25]. This study aimed to establish a mycelial plug of F. oxysporum F1 was inoculated main­
under in vitro conditions an environmentally friendly alter­ taining a distance of 6 cm between each disc. Plates were
native to control the mycelial growth of the phytopathogen then incubated for 7 days at 28°C. T. reesei C2A myco­
causing banana Fusarium wilt. This strategy consisted of parasitism was measured daily using the antagonistic
evaluating in solid media the synergistic interaction of the capacity index described in Table 1.
biological control agent T. reesei C2A combined with low
doses of mancozeb to inhibit the mycelial growth of
F. oxysporum F1. 2.3.2. Minimum inhibitory concentration assays
The application of different doses of mancozeb against the
beneficial strain T. reesei C2A and the pathogenic strain
2. Materials and methods F. oxysporum F1 was evaluated by establishing the mini­
mum inhibitory concentration. Mancozeb was suspended
2.1. Fungal strains and fungicide used in sterile-distilled water and added to Petri dishes contain­
This study was conducted at the Centro de Investigaciones ing molten PDA to achieve final concentrations of 0.1, 0.01,
Biotecnológicas del Ecuador (CIBE) from the Escuela 0.001 and 0.0001 mg/mL, respectively. Once the agar soli­
Superior Politécnica del Litoral (ESPOL). The fungal strains dified, 8 mm plugs containing agar and mycelium of each
T. reesei C2A (biological control) and F. oxysporum F1, F2 fungus were transferred to the center of the plates. The
and F3 (phytopathogens) were obtained from CIBE’s plates were incubated for 7 days at 28°C, mycelial growth
Microbial Culture Collection. Before carrying out the sen­ of the colonies were measured in two directions (vertically
sitivity and synergistic essays, the strains were reactivated and horizontally) and reported as the average value of the
three times in Potato Dextrose Agar (PDA) and grown for 7 two measurements [19].The experiment was performed in
days at 28°C, or until the mycelium sporulated. The selec­ triplicate and carried out three times to ensure the repro­
tion of the pathogenic strain used in this study was con­ ducibility of the results.
ducted quantitatively using a Kruskal–Wallis test with
multiple comparisons (p < 0.05) finding that F1 diameter
growth was statistically different that F2 and F3 while con­ Table 1. Scale used to measure the antagonistic capacity of T
reesei C2A.
fronted to T. reesei C2A for 2 days (Results not shown).
Antagonistic
Mancozeb was purchased from a certified commercial degreea Antagonistic capacity (Pathogen-Antagonist)
house and was used to perform the sensitivity and syner­ 0 No invasion of the surface of the colony of
gistic assays. F. oxysporum
1 Invasion of 1/4 of the surface of the colony of
F. oxysporum
2 Invasion of 1/2 of the surface of the colony of
2.2. Macroscopic and microscopic characterization F. oxysporum
3 Total invasion of the surface of the colony of
of the pathogenic and beneficial fungi F. oxysporum
4 Total invasion of the surface of the colony of
The identity of the fungal strains was confirmed by F. oxysporum sporulation on it
macroscopic and microscopic characteristics such as a Adapted from [28]
162 M. F. GONZALEZ ET AL.

2.3.3. Determination of the percentage inhibition of culture plate essays was analyzed using the observational
diameter growth (PIDG) ranking reported by [32]. Quantitative measurements from
The percentage inhibition of diameter growth (PIDG) the in vitro essays were recorded in internal records in the
was used to determine the ability of mancozeb to laboratory and then imported to Excel. Measurements of
inhibit the mycelial growth of both the pathogen the growth of the mycelial diameter were reported in cm
F. oxysporum F1 and the beneficial fungus T. reesei and included the 8 mm agar disc. Initial data entry of the
C2A [29]. The PIDG was estimated by measuring the mycelial growth from the macroscopic essays showed that
diameter growth of the fungal strains inoculated in 12 values were missing due to random contamination of the
Petri dishes containing 15 mL of PDA supplemented Petri dishes. The missing values were imputed using the
with 4 different concentrations of mancozeb (0.1, means substitution method [33].
0.01, 0.001 and 0.0001 mg/mL) comparing to the Measurements of the mycelial growth from the dual
growth of the positive controls. Cultures were incu­ culture plate essays were compared to the growth of the
bated for 7 days at 28°C and mycelia measuring positive controls for the first 3 days because as of day 4,
growth was performed every 24 hours. the mycelia of both fungi were overlapped. The myce­
Diameter of the sample diameter of the positive control lial growth reduction was reported as the percentage of
PIDGð%Þ ¼ � 100
diameter of the positive control growth inhibition and compared to the positive control.
The percentage of inhibition of F. oxysporum F1 was
2.3.4. Synergistic assays using mancozeb calculated by comparing the mycelial growth of the
Synergistic inhibition of the mycelial growth of the positive controls to the growth of F. oxysporum F1
pathogen F. oxysporum F1, was assessed based on the when is co-cultured in the same Petri dish with the
protocol described by [30]. In vitro tests were per­ biocontrol strain T. reesei.
formed in PDA Petri dishes supplemented with he minimum inhibitory concentration of mancozeb was
0.1 mg/mL of mancozeb. 15 mL of molten PDA were determined by measuring the mycelial growth of each fungal
transferred to a sterile 15 mL Falcon tube, an aliquot of strain separately for 7 days, comparison of the mycelial
mancozeb from a stock solution (3 mg/mL) was added growth in cm was performed using an ANOVA test in
to the tube to obtain a final concentration of 0.1 mg/mL R Studio [34]. This analysis was conducted independently
(v/v), the agar was then poured into the Petri dish and for the four concentrations of mancozeb (0.1, 0.01, 0.001,
gently shaken to get a homogenous mixture. Once the 0.0001 mg/mL). All mean values were reported with stan­
agar solidified, 8 mm plugs containing agar and mycelia dard deviation in centimeters (cm), and mean plots were
of 5-day fungi cultures of T. reesei C2A and used to illustrate the mycelial growth over time. The variables
F. oxysporum F1 were inoculated at each side of the tested were the mean diameter growth for 7 days using the
Petri dish at 1.5 cm from the edge and maintaining Tukey method with 95% confidence. Different letters were
6 cm of distance between each disc. Plates were then used to show a significant difference over the days, and the
incubated for 7 days at 28°C. The percentage of inhibi­ more distant letters were used to select the concentration
tion of F. oxysporum F1 was calculated using the equa­ that was chosen to carry out the synergy test.
tion described by [30,31]. All experiments were To study the evolution over time of the two co-
performed in triplicate and independently replicated cultured fungal strains in Petri dishes supplemented
three times. with 0.1 mg/mL de mancozeb, the PIDG of T. reesei
against F. oxysporum was calculated by an experimental
A1 A2
%I¼ � 100 factorial design with repetitions using multiple compar­
A1
isons with the Tukey method. All statistical tests were
where %I = inhibition percentage (%), A1 = area of the done with a significance of 5%, using R Studio, P-values
Petri dish in mm2 covered by Fusarium oxysporum F1, lower than 0.05 were considered to show significant
control plate that was not added the commercial fungi­ differences. Finally, growth over time was tested by
cide (mancozeb), A2 = area of the Petri dish in mm2 comparing the diameter growth from day one to day
covered by Fusarium oxysporum F1 co-inoculated with three, and the time in hours from day 1 to day 3, using
T. reesei C2A and 0.1 mg/mL of mancozeb. the equation described by [31]:
C2 C1
TC ¼
2.4. Data analysis T2 T1
Macroscopic and microscopic identification was performed Where: TC = growth rate (cm/h); C1 = initial growth
by examining the morphological characteristics of the (cm); C2 = final growth (cm); T1 = initial time (h);
mycelial growth. Qualitative data obtained from the dual T2 = final time (h)
 COMMUNICATIVE & INTEGRATIVE BIOLOGY 163

3. Results at day 2. In contrast, F. oxysporum F1 showed a steady

mycelial growth (1.50-day 1, 2.92-day 2, 4.24-day 3,
3.1. Macroscopic and microscopic characterization
5.37-day 4, 6.90-day 5, 7.83-day 6 and 8.69-day 7).
of the pathogenic and beneficial fungal strains
Growth inhibition was calculated using radial growth
Results showed that both fungi had well-defined measurements of days 1, 2 and 3, radial growth of days
macroscopic and microscopic characteristics. 4 to 7 could not be measured as Trichoderma mycelia
T. reesei exhibited a rapid growth in PDA and after overgrown in the plates. Inhibition increased over time,
about 60 to 72 hours of being incubated at 28°C, the from 59% on day 1 to 62% on days 2 and 3 (Table 2).
mycelia covered the entire diameter of the 9 cm Petri These results suggest that T. reesei C2A exhibited
dish (Figure 1). Typical growth characteristics of strong antagonistic activity against F. oxysporum F1,
T. reesei C2A included white cottony hyphae without inhibiting mycelial growth and preventing sporulation.
aerial mycelium, yellowish-greenish conidia and Thus, T. reesei C2A could be used as a potential biolo­
change of the medium coloration due to the produc­ gical control to prevent Fusarium wilt in banana
tion of a yellowish pigment. Micromorphology char­ plantations.
acteristics such as hyaline conidiophores, long
primary branch and a short secondary branch were
observed. T. reesei C2A exhibited whitish mycelia
3.3. Minimum inhibitory concentration assays
which grew approximately at a rate of 3.41 cm
a day. After 2 days of incubation, mycelia color Mycelial growth was reported as the average of the
changed from whitish to yellowish and covered the vertical and horizontal measures in centimeters. Using
entire diameter of the Petri dish as of day 3, mycelia the Tukey’s method, we determined that during the
color turned greenish-grayish with a smooth appear­ 7-day assay there were significant differences between
ance exhibiting greenish ellipsoidal conidia. the mycelial growth of the biocontrol agent and the
Macroscopic characteristics of F. oxysporum F1 phytopathogenic strain. The mycelial growth of
started with an initial growth rate of 1.50 cm at day T. reseei C2A was not significantly affected by any of
1, with a steady increase up to 8.69 cm on day 7, it the concentrations used (0.1, 0.01, 0.001 and
did not produce diffusible pigments, its vegetative 0.0001 mg/mL) (P < 0.0001). Although when mancozeb
and aerial mycelia were whitish and pinkish, respec­ concentrations of (0.1, 0.01, 0.001 and 0.0001 mg/mL)
tively, macroconidia and microconidia were analyzed were added to the culture medium, the mycelial growth
to identify the species of the pathogenic strain. of F. oxysporum F1 was reduced by 33.2, 8.3, 6.8 and
7.4%, respectively, compared to the positive control
(Table 3). The growth of the phytopathogen was sig­
3.2. Dual culture plate assay nificantly reduced (33.2%, P < 0.0001) only when
0.1 mg/mL of the fungicide were suspended in the
The antagonistic effect of T. reesei C2A against medium. As the growth of F. oxysporum F1 was sig­
F. oxysporum F1 was observed after 3 of incubation as nificantly affected at 0.1 mg/mL, this concentration was
fungal colonies started competing for space and nutri­ selected to perform the synergistic assays (Figure 3).
ents and the mycelia of the biocontrol strain grew and
sporulated over the pathogen, T. reesei C2A fully cov­
ered the Petri dish including ¼ of F. oxysporum F1
(Figure 2). Trichoderma mycelia changed color from 3.4. Percentage inhibition of diameter growth
greenish to grayish and invaded 50 to 100% of the (PIDG)
Fusarium colony. T. reesei C2A experienced an expo­ The interaction between both fungi was measured by
nential mycelial growth from 3.41 cm at day 1 to 9 cm PIDG in PDA with 4 different concentrations of manco­
zeb. Figure 4, shows that over time the biocontrol agent
grew faster than the pathogen strain in presence of 0.1 mg/
mL of mancozeb (Figure 4). The Figure indicates that only
at 0.1 mg/mL of mancozeb, the percentage of inhibition of
Fusarium had negative values from day 1 till day 7, mean­
ing that the mycelial growth of F. oxysporum F1 is sig­
Figure 1. Macroscopic and microscopic characteristics of T. nificantly inhibited compared to the positive control,
reesei C2A (a and b) and F. oxysporum F1 (c and d) in PDA, whereas since day 4, the growth of T. reesei C2A was not
plates were incubated at 28°C for 7 days. affected by the presence of the fungicide.
164 M. F. GONZALEZ ET AL.

Figure 2. Assessment of antagonistic activity of T. reesei C2A against F. oxysporum F1. a) Dual plate culture essay in PDA exhibiting
the antagonistic capacity of T. reesei C2A to inhibit the growth of F. oxysporum mycelia. b) Growth of positive controls in PDA. C)
Antagonist capability of T. reesei C2A using a 0–4 scale as stated by [28].

Table 2. Diameter growth (30 observations) of T. reesei C2A and F. oxysporum F1 in Petri dishes incubated at 28°C, data were daily
recorded for 3 days.
Mycelial growth (SD) Mycelial growth (SD)
Mean growth (SD) in cm in cm of the positive Mean growth (SD) in cm in cm of the positive Percentage of antagonistic
Day of Trichoderma reesei C2A control of Fusarium oxysporum F1 control effect between C2A and F1 p-valuea
1 3.48 (0.38) 3.41 (0.58) 1.43 (0.1) 1.50 (0.25) 59% 0.08501
2 7.13 (3.31) 9 (0) 2.73 (0.1) 2.92 (0.21) 61% 0.09154
3 9 (0) 9 (0) 3.41 (0.2) 4.24 (0.26) 62% 0.1695
a
difference in mycelial growth of Fusarium oxusporum F1, it was evaluated using a non-parametric hypothesis test

Table 3. Growth diameter in cm (SD) of T. reesei C2A and F. oxysporum F1 in experiments with four different concentra­
tions of mancozeb.
Incubation time Growth in cm
T. reesei C2A Control 0.1 mg/mL 0.01 mg/mL 0.001 mg/mL 0.0001 mg/mL
Day 1 3.41 (0.58) 0.00 (0) a 3.38 (0.14) a 3.98 (0.31) a 4.33 (0.25) a
Day 2 9.00 (0) 0.63 (0.86) a 9.00 (0) b 9.00 (0) b 9.00 (0) b
Day 3 9.00 (0) 4.72 (1.98) b 9.00 (0) b 9.00 (0) b 9.00 (0) b
Day 4 9.00 (0) 9.00 (0) c 9.00 (0) b 9.00 (0) b 9.00 (0) b
Day 5 9.00 (0) 9.00 (0) c 9.00 (0) b 9.00 (0) b 9.00 (0) b
Day 6 9.00 (0) 9.00 (0) c 9.00 (0) b 9.00 (0) b 9.00 (0) b
Day 7 9.00 (0) 9.00 (0) c 9.00 (0) b 9.00 (0) b 9.00 (0) b
F. oxysporum F1 Control 0.1 mg/mL 0.01 mg/mL 0.001 mg/mL 0.0001 mg/mL
Day 1 1.50 (0,25) 0.00 (0) a 1.11 (0.23) a 1.31 (0.05) a 1.36 (0.12) a
Day 2 2.92 (0,21) 1.11 (0.02) b 2.17 (0.23) b 2.68 (0.07) b 2.90 (0.12) b
Day 3 4.24 (0.26) 2.02 (0.1) c 3.48 (0.10) c 3.97 (0.04) c 4.15 (0.38) c
Day 4 5.37 (0.43) 2.02 (0.1) c 4.08 (0.07) d 4.73 (0.20) d 6.46 (0.28) d
Day 5 6.90 (0.78) 4.01 (0.1) d 5.99 (0.30) e 6.56 (0.21) e 7.00 (0.27) e
Day 6 7.83 (0.63) 4.70 (0.1) e 7.21 (0.39) f 7.67 (0.32) f 7.51 (0.05) f
Day 7 8.69 (0.59) 5.80 (0.2) f 7.97 (0.04) g 8.10 (0.23) g 8.05 (0.25) g

3.5. Synergistic tests between T. reesei C2A and

0.1 mg/mL of mancozeb, showed that the first day of
0.1 mg/mL of mancozeb against F. oxysporum F1
the essay, the mycelial growth of the phytopathogen
The synergistic tests of T. reesei C2A against was completely inhibited (100%). As of day 2, growth
F. oxysporum F1 in PDA plates supplemented with inhibition of F. oxysporum F1 decreased to 51% and
 COMMUNICATIVE & INTEGRATIVE BIOLOGY 165

Figure 3. a) Mycelial growth of T. reesei C2A and F. oxysporum F1 in 9 cm Petri dishes supplemented with 4 different concentrations
of mancozeb (0.1, 0.01, 0.001 and 0.0001 mg/mL) after 7 days of incubation at 28°C. b) Plot of means of the mycelial growth of T.
reesei C2A and F. oxysporum F1 after 7 days of incubation at 28°C.

overgrowing toward F. oxysporum F1 till day 7, when

the Petri dish was totally covered by Trichoderma
(Figure 6).
These results suggested that T. reesei C2A could be
used in combination with low doses of the fungicide as
an environmentally friendly alternative to inhibit under
in vitro conditions the growth of banana phytopathogens.

4. Discussion
Trichoderma species are soilborne organisms associated
with the roots of plants that have been widely used in
agriculture for their potential to control plant diseases.
Species of Trichoderma are recognized for their myco­
parasitic and antibiosis capability to inhibit the mycelial
growth of various pathogens including F. oxysporum,
F. solani, Alternaria alternata, Botrytis cinerea and
Figure 4. Graph of percentage inhibition of diameter growth of Rhizoctonia solani [35,36]. In this study, the biocontrol
T. reesei C2A and F. oxysporum F1 in PDA with 4 different strain T. reesei C2A and the pathogenic strain
concentrations of mancozeb. F. oxysporum F1 were characterized based on their
macroscopic and microscopic features as reported else­
where [37–40]. T. reesei C2A showed a typical whitish
T. reesei C2A mycelia started overgrowing toward the cottony mycelium that changed to a photosensitive
pathogenic strain (Figure 5). By day 3, F. oxysporum F1 greenish-grayish color after sporulation (i.e. after 3 or
mycelial growth inhibition was 36% compared to the 4 days of incubation). The production of diffusible
positive control and growth inhibition of T. reesei C2A pigments, which are characteristic of T. reesei isolates,
was reduced only 10% compared to the control. The were detected at the bottom of the plates. Furthermore,
following days, the biocontrol strain continued distinguishing conidiophores, conidia and intercalary
166 M. F. GONZALEZ ET AL.

Figure 5. Synergistic effect of T. reesei C2A against F. oxysporum F1 in PDA plates supplemented with 0.1 mg/mL of mancozeb.

Figure 6. Synergistic effect between the biocontrol strain T. reesei C2A and 0,1 mg/mL of mancozeb to inhibit the growth of F.
oxysporum F1. a) Picture was taken after 3 days of incubation at 28°C. b) Picture was taken after 7 days of incubation at 28°C.

phialides were observed too [41]. F. oxysporum F1 on control, inhibition then increased to 61% and 62% on
the other hand, exhibited concentric rings, hyaline con­ the second and third day, respectively, demonstrating
idiophores with pyramidal ramifications and fixed soli­ the mycoparasitic mechanism of the biocontrol strain
tary accessories as the ones reported by [38,42]. Finally, overgrowing toward the phytopathogen. Similar results
ITS (Internal transcribed spacer) molecular markers were reported by [43–45], who using four different
were used to completed the taxonomic identity of the strains (T29, T1, T2 and T3) of T. reesei demonstrated
fungal strains (data not shown). The dual culture plate that they were able to inhibit in a high percentage the
assays and synergy tests allowed us to monitor for 7 mycelial growth of F. oxysporum f. sp. cubense,
days under in vitro conditions the antagonistic and F. oxysporum f. sp. cicero and F. oxysporum f. sp. mel­
synergistic capacity of T. reesei C2A to inhibit the ongenae. T. reesei isolates are known to activate a large
mycelial growth of F. oxysporum F1. The evolution battery of secondary metabolites and/or enzymes
over time of the percentage of inhibition in the con­ including plant cell wall degrading enzymes (CWDEs)
frontational essays shown during the first 3 days, con­ which degrade the cell wall of soil-plant pathogens
firmed the ability of T. reesei C2A to inhibit the growth [46,47].
of F. oxysporum F1. On the first day, the PIDG of Although the current methods to control Fusarium
F. oxysporum F1 was 59% compared to the positive wilt are based on the application of synthetic fungicides
 COMMUNICATIVE & INTEGRATIVE BIOLOGY 167

such as mancozeb, methyl bromide and quaternary a synergistic effect that inhibits in a higher percen­
ammonium, the pathogen is not always eliminated and tage of the growth of the phytopathogens. Although
might continue sporulating in necrotic tissues due to its these results are promising, future studies under
saprophytic capacity [8,48,49]. The use of these fungicides greenhouse and field conditions are necessary to cor­
is not an economically and environmentally sustainable roborate the effectiveness of this approach.
practice because their overuse have led to serious envir­
onmental pollution problems, generating pathogen resis­
tance and leaving toxic residues on fruits [50]. Thus, more 5. Conclusion
environmentally friendly alternatives to control the devel­
opment of phytopathogenic fungi are currently being The biocontrol strain Trichoderma reesei C2A obtained
proposed to reduce the potentially harmful effects of the from the Microbial Culture Collection held at the Centro
continuous application of synthetic pesticides and fertili­ de Investigaciones Biotecnológicas del Ecuador, exhib­
zers on agricultural lands [51,52]. These strategies involve ited under in vitro conditions mycoparasitic activity,
the synergistic application of low doses of synthetic fun­ reducing 62% the mycelial growth of F. oxysporum
gicides combined with biocontrol agents such as the non­ F1after 3 days of incubation at 28°C. The synergistic
pathogenic fungi Trichoderma, Clonostachys and Xylaria essays demonstrated that when the growth medium is
[13,14]. Recent studies under in vitro conditions have suspended with low concentrations of mancozeb
shown that when mancozeb is added to the culture med­ (0.1 mg/mL) combined with plugs of the biocontrol
ium in concentrations lower than 5 mg/mL, the mycelial strain T. ressei C2A, the mycoparasitic capacity of the
growth of Trichoderma is not significantly inhibited biocontrol strain was enhanced approximately by 36%
[53,54]. Furthermore [55], tested the tolerance of 26 compared to the positive control. These results strongly
Trichoderma isolates against 4 pesticides and evaluated suggest that T. ressei C2A could be used in combination
their antagonistic capacity against the sheath blight patho­ with mancozeb as an environmentally friendly alterna­
gen of rice Rhizoctonia solani. The authors reported that tive to control the growth of F. oxysporum, the causal
the pesticide pyrethroid significantly enhanced the agent of Fusarium wilt.
growth of all Trichoderma isolates and the strains
T. reesei and T. longibrachiatum were the most effective
in inhibiting the growth and the sclerotial formation of Disclosure of potential conflicts of interest
R. solani. Furthermore [30], were able to quantify under The authors of this manuscript declare that the research was
in vitro conditions the synergistic interaction of the syn­ conducted in the absence of any commercial or financial
thetic fungicide Captan 50® and Trichoderma asperelleum relationships that could be construed as a potential conflict
T8a, their results showed that by using this integrated of interest.
alternative, the mycelial growth of Colletotrichum gloeos­
porioides, the causal agent of anthracnose in mango, was Funding
inhibited 99% compared to the positive control. The
authors suggested that this approach could help reduce This work was supported by the Centro de Investigaciones
Biotecnológicas del Ecuador (CIBE) and Escuela Superior
economic and environmental problems in the field as Politécnica del Litoral (ESPOL).
lower amounts of Captan 50® might be used to control
the growth of C. gloeosporioides. The synergistic essays
performed in this study using 0.1 mg/mL of mancozeb
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