Int. J. Biosci.

2014

International Journal of Biosciences | IJB |

ISSN: 2220-6655 (Print) 2222-5234 (Online)
http://www.innspub.net
Vol. 5, No. 9, p. 21-36, 2014

RESEARCH PAPER OPEN ACCESS

Effectiveness of Trichoderma spp. at controlling Fusarium

oxysporum f.sp. phaseoli in bean plants at a greenhouse scale

Dorcas Zúñiga Silgado*, Evelyn Becerra Agudelo

Study Program of Biotechnology, Faculty of Health Science, Mayor College of Antioquia, University
Institution, Career. 78 N ° 65-46 Robledo, Medellín, Colombia

Key words: Trichoderma spp., as a biofungicide in the control of Fusarium oxysporum, Phaseolus vulgaris.

http://dx.doi.org/10.12692/ijb/5.9.21-36 Article published on November 10, 2014

Abstract

Fusarium oxysporum L. is a known etiological agent that causes dieback or root rot in multiple crops. The
symptoms of this fungus are primarily associated with withering and death due to the weakening of the plant.
This research evaluated the effectiveness of Trichoderma spp., at controlling Fusarium, and its ability to improve
the performance of bean plants at a greenhouse scale. The commercial inoculum Fitotripen wp™, which
contained three species of Trichoderma, was evaluated for the assay, and the F. oxysporum f.sp Phaseoli
pathogenic isolate was also evaluated. The greenhouse scale assay had a 6x2 factorial arrangement. The in vivo
experiment was performed by applying the antagonistic fungus and pathogen to bean plants of the Cargamanto
variety (Phaseolus vulgaris). The severity of the disease was assessed using a completely randomized design. The
treatments T6, T9 and T12 were those which generally presented a better control when considering the set of
biometric variables evaluated. The study indicated that the Trichoderma species was able to efficiently control
Fusarium as well as promote bean growth and performance.
* Corresponding Author: Dorcas Zúñiga Silgado  dorcas.zuniga@colmayor.edu.co

21 Silgado and Agudelo

 Int. J. Biosci. 2014

Introduction sp., Rhizoctonia sp., Sclerotium sp., Pythium sp.,

Fusarium oxysporum L is reported as the etiologic Fusarium sp., Verticillium sp., among others
agent that causes root rot or radical putrification in (Gonzalez et al., 2005; Bernal et al., 2000).
multiple crops (Zúniga et al., 2010; Rutkowska -
Krause et al., 2003; Schneider, 1984). The Competitive hyperparasitism is one of the
symptomology of this fungus is primarily associated mechanisms employed by Trichoderma spp., in this
with the wilting and death through the weakening of condition, it produces antifungal metabolites and
the plant (Garcés et al., 2001). Other symptoms hydrolytic enzymes (Martinez et al., 2013; Howell,
include the stunting of growth, yellowing in older 2003 In: Javaid and Ali, 2011). These molecules lead
leaves and adventitious root formation. The vascular to structural changes at the cellular level in the
tissue necrosis also stands as strong evidence of phytopathogens with that the Trichoderma
Fusarium sp., (Zúniga et al., 2010; Appel and antagonize. Among those structural changes are:
Gordon, 1994). It is reported that this pathogen vacuolation, granulation, disintegration of the
attacks more than 100 species of gymnosperms and cytoplasm and cell lysis (Mohamed et al., 2004). In
angiosperms (Hernandez et al., 2011; Garofalo and hyperparasitism, Trichoderma adheres to the hyphae
McMillan, 2003; Bosland., 1988) and can form three of the phytopathogenic through specialized structures
resistant structures: macroconidia (distinctive called appressoria and releases enzymes like
structures of the genere), microconidia and glucanases, chitinases, quitobiosas that degrade or
chlamydospores, the latter are those that allow it to weaken the cell wall and destroy the reproductive
survive as free-living saprophyte in the absence of a structures hyphae such as when hiperparasitar to
host (Hernandez et al., 2011; Nelson, 1981). To Sclerotium cepivorum (Vera et al., 2005). This
control this fungal pathogen the precise use of phenomenon is very important, because many fungi
agrochemicals are required. Often these chemicals are form resistant structures in the ground that allow
used indiscriminately leading to deleterious effects on them to survive under adverse environmental
the environment such as pollution of soil and water conditions for up to 20 years (Higuera et al., 2003).
sources (Capote and Torres, 2004).
Also Trichoderma spp., produces antibiotics such as
The problem about mentioned above requires the viridine, gliotoxin, gliovirina and peptaibols those
search for new biotechnology-based alternatives will who control the proliferation of phytopathogens
lead to suggest the implementation of biological (Howell et al., 1993). The mechanisms described
control methods such as the use of Trichoderma spp., exhibit that Trichoderma spp., as an effective agent
as a biocontrol agent (Hermosa et al., 2001). This is for the control of phytopathogens. Bernal et al.,
fungus that is easy to isolate and grow in natural (2000) reported strains of Trichoderma spp., as an
culture media or semi- natural media (Rey et al., environmentally friendly alternative for the control of
2000). Trichoderma spp., is a free-living fungi found F. oxysporum Schlecht f . sp cubense, with over 70%
in soils and root ecosystems where there are complex of antagonism to the pathogen.
interactions between the host plant, pathogens and
various environmental factors (Harman, 2006; Woo Inhibition of in vitro growth of pathogenic fungi by
et al., 2006). The interest in this fungus is found its antagonistic fungi has been widely described. The
antagonism ofthe soil microorganisms causing most common methodology is to treat both
diseases in plants (Montealegre et al., 2011; Elias et microorganisms equally dual way (Aquino -Martinez,
al., 2009). Different species of Trichoderma spp., are 2007) even though they may or may not be inoculated
used in agriculture for the handling of at the same time, usually as "Plug" or drop. Later its
phytopathogens and for the limiting of the effectiveness is determined by a scale of Degrees of
development of harmful fungi such as Phytophthora Antagonism (Bell et al., 1982) or, using Abbott's

22 Silgado and Agudelo

 Int. J. Biosci.
adapted formula (Aquino - Martinez, 2007).
Fernandez and Suarez (2009) worked at the in vitro
level with native and commercial strains of
Trichoderma harzianum for the control o F.
foxysporum. These researchers suggest that it is likely
to reach an effective control of the pathogen if the
antagonist was inoculated as a preventative measure
right before planting the plant or in the moment of its
planting. This practice could stimulate the
colonization of the rhizosphere by the rapid growth of
the fungus which would prevent the arrival or
development of the pathogen on the plant. It is
favorable that the antagonist is found adapted to the
environmental conditions of the rhizosphere (Bernal
et al., 2000), therefore, more studies are necessary
both in the greenhouse and in the field, not only to
determine their effectiveness in vivo but to find the
spore concentration mL-1 suitable for the application
of Trichoderma spp.
Hernández et al. (2011) in their studies with
Trichoderma in the greenhouse and in the field
report that the fungus favors the ecophysiological
response of plants given to induce plant growth. This
response is because Trichoderma degrades episperm
seed and is involved in respiratory conditions during
germination. This fungus accelerates the development
of primary meristematic tissues, which increases the
volume, height, and dry weight of the plant (Shoresh
and Harman, 2008a; Shoresh and Harman, 2008b;
Gravel et al., 2007). Trichoderma secret Indole Acetic
Acid (IAA) stimulating phytohormone processes as
germination, growth, root development and increases
nutrient absorption, aspects that influence and
enhance vegetative growth of crops such as beans,
potatoes, tomatoes, corn, banana, papaya, passion
fruit and coffee trees (Fernández and Suárez, 2009;
Sánchez- Pérez, 2009; Vinale et al., 2008; Gravel et
al., 2007; Harman, 2006, Harman et al., 2004;
Cupull et al., 2003). This beneficial effect of
Trichoderma promotes the health of crops due to a
well-nourished plant that will exhibit greater
resistance to attack by a pathogen.
In Colombia, several studies reported the antagonistic
23 Silgado and Agudelo
2014
capacity of Trichoderma spp., against fungal
pathogens causal agents of wilt and root rots such as
Rhizoctonia solani, Sarocladium sp., and Sclerotinia
sp., rice, flowers, potatoes, vegetables, fruit and
beans; F. oxysporum in beans and carnations;
Botrytis cinerea in flowers; Ceratocystis fimbriata in
coffee; Rosellinia bunodes in cocoa; Phytophthora
cactorum in apple; Colletotrichum gloeosporioides in
tamarillo (Fernández and Suarez, 2009; Suarez et al.,
2008; Páez and Baquero, 2004; Rico et al., 2001,
Torres et al., 2000). González et al. (2005) also report
the efficiency of Trichoderma spp., as biocontrol
against F. oxysporum in their evaluations with
different inoculum densities, where concentrations of
106 spores mL-1 of Trichoderma were effective at field
level to control Fusarium. Notwithstanding the
details listed, biological control by Trichoderma spp.,
reported contrasting results regarding its
effectiveness and efficiency as antagonistic to
pathogenic microbial populations (Hernandez et al.,
2011; Fernández and Suárez, 2009, Pérez et al., 2009;
Sivan and Chet, 1986).
It can be affirmed that the effectiveness of the
antagonistic capacity of Trichoderma on Fusarium
depends on the stage in which the antagonist was
inoculated into the plant, ie the treatment should be
preventive and not curative. For this reason, it would
be more effective as an antagonist if it is allowed to
grow first in the rhizosphere before the pathogen can
enter. This will improve plant nutrition and stimulate
defense mechanisms of the plant to start early
pathogen inhibition. Based on this premise, the
hypothesis of our work suggests that the biocidal
effect of Trichoderma spp., against Fusarium
oxysporum L, depends on the moment of inoculation
during the actual phenological cycle of the plant and
the dose of inoculum of the mycoparasite that is
employed. The aim of this study was to evaluate the
antagonistic effect of Trichoderma spp., on Fusarium
oxysporum L at different stages of phenological cycle
of Phaseolus vulgaris L.
Materials and Methods
Vegetable material
 Int. J. Biosci.
The plant model used in this study was the Red
Cargamanto variety of bean (Phaseolus vulgaris L),
which has been reported as highly susceptible to
Fusarium oxysporum L fungal attack (Montoya and
Castaño, 2009). Prior to planting, the seeds were
disinfected in a 2% (v/v) sodium hypochlorite
solution for 30 seconds and washed twice with sterile
distilled water. The seeds were planted in 5kg
capacity plastic bags containing a sterile mixture of
peat, sand and vermiculite (3:1:0.5). The bags were
placed in two greenhouse modules at the Institución
Universitaria Colegio Mayor de Antioquia, Colombia.
Fungal material
The UN178 pathogenic strain of F. oxysporum f.sp
Phaseoli was used. This strain was donated by the
Laboratory of Vegetable Health of the University
Nacional of Colombia. Prior to the selection of the
strain, pathogenicity tests were carried out
confirming the high aggressivity of the fungus. The
fungus was cultivated in Potato Dextrose Agar (PDA)
and maintained at 21°C ± 2 until the antagonistic
tests were performed. For the biocontrol assays, the
Fitotripen wp™ bioinput was used. This commercial
inoculum contained three species of Trichoderma (T.
harzianum, T. koningii and T. viridae) at a
concentration of 1x108 SPORES g-1 .
Preparation of the inocula of F. oxysporum and
Fitotripem wp TM
The inoculum of F. oxysporum f.sp Phaseoli was
prepared at a concentration of 1.28 x 109 SPORES mL-
1. In the greenhouse, each experimental unit was
inoculated with 10mL of pathogenic fungus in
accordance with the treatment. From the Fitotripen
wpTM inoculum, 5 doses were prepared plus a control
with no inoculum. In accordance with the treatment,
the doses were 0.25, 0.50, 1.00, 1.25 and 1.50g / 5kg
of soil. For the inoculation of Fitotripen wp™, each
dose was diluted in a litre of sterile water. In each
litre, 250mL were inoculated in each experimental
unit. This procedure was carried out on a weekly basis
for a total of 12 applications.
Greenhouse pathogenicity tests
24 Silgado and Agudelo
2014
The greenhouse experimental design consisted of a
factorial arrangement of 6 x 2. In this case, 6 was the
number of Fitotripen wp™ doses evaluated and 2 the
number of stages in the phenological cycle in which F.
oxysporum f.sp Phaseoli was inoculated. In this way,
12 treatments were obtained, each with 4 replicas, for
a total of 48 experimental units. In the greenhouse
the experimental units were distributed randomly
(Table 1).
On a daily basis, the plants were watered with tap
water. Starting 8 days after being planted, the plants
were fertilized every two weeks with an NPK solution
to avoid symptoms of nutritional deficiency.
Assay of bioinoculants on P. vulgaris plants
From the moment the seedlings were planted,
photograph records were taken on a weekly basis in
each of the experimental units, until day 60 when the
treatment systems were dismantled. In each
experimental unit the following biometric parameters
were analysed: a) the number of leaves (N°): number
of photosynthetically active cotyledon and true leaves
on the different plants; b) the number of fruits (N°):
number of mature pods with seeds in each treatment
replica; c) total height of the plant (cm), measured
from the tip of the root to the most apical leaf of each
of the sample plants; d) length of the stem (cm),
measured from the longest apical leaf to the base of
the stem; e) length of the root (cm), measured from
the tip of the main root to the base of the stem of each
plant sampled; f) dry weight of the plant: drying was
carried out at 60ºC in the stove until a constant dry
weight was achieved (Tables 2 and 3).
Statistical evaluation of treatments
A completely randomized design was employed with a
factorial arrangement of 6 x 2: six levels for the
dosage of Fitotripen wp™, and two levels for the time
of pathogen application. The statistical analysis was
carried out with the SAS program (2003) using the
GLM procedure. Values of P ≤0.05 were considered
statistically significant, and when necessary pairwise
comparisons were performed with the Tukey test.
 Int. J. Biosci. 2014

Results T1 and T7
The results of the biometric analysis can be observed Controls: without Fitotripen wp™ + 1.28x109
in Tables 2 and 3. This data corresponds to average SPORES/mL of F. oxysporum f.sp Phaseoli : T1:
values of the bioassays, each of which have 4 replicas inoculation of the pathogen at the time of planting,
for the different treatments: T7: inoculation of the pathogen after 30 days of
germination.

Table 1. Bean treatments evaluated according to the period of inoculation of Fitotripen wp™ vs F. oxysporum
f.sp Phaseoli.
Treatments Stages in the phenological Dosis of Fitotripen wp™ (g) inoculated Time of inoculation with
cycle of the bean since sowing F. oxysporum
T1 Seeds 0.00
T2 0.25 Time 1:
T3 0.50 While planting
T4 1.00
T5 1.25
T6 1.50
T7 30 days of germination 0.00
T8 0.25
T9 0.50 Time 2:
T10 1.00 30 days after planting
T11 1.25
T12 1.50

T2 and T8 T3 and T9
25% of Fitotripen wp™ (applied at the time of 50% of Fitotripen wp™ (applied at the time of
planting) + 1.28x109 SPORES/mL of F. oxysporum planting) + 1.28x109 SPORES/mL of F. oxysporum
f.sp Phaseoli : T2: inoculation of the pathogen at the f.sp Phaseoli : T3: inoculation of the pathogen at the
time of planting, T8: inoculation of the pathogen after time of planting, T9: inoculation of the pathogen after
30 days of germination. 30 days of germination.

Table 2. Average of the biometric parameters of Phaseolus vulgaris L plants, with different concentrations of
Fitotripen wp™ and inoculation of Fusarium oxysporum f.sp Phaseoli at the time of planting.
Treatments THP TDWP LS DWS LR DWR NL NFr DWFr
(cm) (g) (cm) (g) (cm) (g) HL IL (g)
T1 111.25 8.13 65.00 2.95 46.25 0.40 1.50 17.25 3.75 4.78
T2 107.25 8.93 61.75 4.00 45.50 0.40 1.50 23.75 5.25 4.53
T3 114.50 8.60 62.25 3.73 52.25 0.50 0.50 19.25 4.75 4.38
T4 103.75 7.65 61.75 2.43 42.00 0.30 0.75 16.50 4.75 4.93
T5 99.50 7.75 59.00 4.95 40.50 0.38 1.75 16.25 5.50 2.43
T6 124.25 9.68 79.50 4.20 44.75 0.35 4.75 17.00 4.00 5.13
THP= Total height of the plant; TDWP= Total dry weight of the plant; LS= Length of the stem; DWS= Dry weight
of the stem; LR= Length of the root; DWR= Dry weight of the root; NL= Number of leaves; HL= Healthy leaves;
IL= Infected leaves; NFr= Number of fruits; DWFr= Dry weight of fruits.

T4 and T10 time of planting, T10: inoculation of the pathogen

100% of Fitotripen wp™ (applied at the time of after 30 days of germination.
planting) + 1.28x109 SPORES/mL of F. oxysporum
f.sp Phaseoli : T4: inoculation of the pathogen at the T5 and T11

25 Silgado and Agudelo

 Int. J. Biosci. 2014

125% of Fitotripen wp™ (applied at the time of T6 and T12

planting) + 1.28x109 SPORES/mL of F. oxysporum
f.sp Phaseoli : T5: inoculation of the pathogen at the
time of planting, T11: inoculation of the pathogen
after 30 days of germination.
150% of Fitotripen wp™ (applied at the time of
planting) + 1.28x109 SPORES/mL of F. oxysporum
f.sp Phaseoli : T6: inoculation of the pathogen at the
time of planting, T12: inoculation of the pathogen
after 30 days of germination.

Table 3. Average of the biometric parameters of Phaseolus vulgaris L plants, with different concentrations of
Fitotripen wp™ and inoculation of Fusarium oxysporum f.sp Phaseoli after 30 days of germination.
Treatments THP TDWP LS DWS LR DWR NL NFr DWFr
(cm) (g) (cm) (g) (cm) (g) HL IL (g)
T7 117.00 6.53 69.00 2.73 48.00 0.43 9.00 16.75 2.50 3.38
T8 115.75 6.55 60.75 3.20 55.00 0.48 2.50 27.00 4.00 2.88
T9 122.00 7.08 67.25 2.95 54.75 0.48 4.75 22.75 4.00 3.65
T10 109.25 7.80 60.50 3.00 48.75 0.43 3.75 29.25 4.25 4.38
T11 108.75 8.88 61.75 3.75 47.00 0.48 9.25 20.00 5.50 4.65
T12 116.50 9.88 66.50 4.73 50.00 0.48 22.50 8.75 5.75 4.68
THP= Total height of the plant; TDWP= Total dry weight of the plant; LS= Length of the stem; DWS= Dry weight
of the stem; LR= Length of the root; DWR= Dry weight of the root; NL= Number of leaves; HL= Healthy leaves;
IL= Infected leaves; NFr= Number of fruits; DWFr= Dry weight of fruits.

Number of Leaves (N°) obtained for treatments T5 and T12, in which an

The appearance of cotyledon leaves was seen average of 5 pods per treatment were observed, as
simultaneously in all treatments after 10 days of seen in Figures 1a and 1b.
germination. The greatest growth, development and
production of true leaves by the plants were observed Table 5. Summary of the behaviour of the mean
in treatments T2 and T10. In these treatments, the populations in the Fitotripen wp™ application stage
number of leaves was 25 and 33 respectively, as for the number of healthy leaves (NHL).
shown in Figures 1a and 1b. In the pathogen Dose Mean n
application stage (P<0.01) and the Fitotripen wp™ 1 5.250 8 ab*
application stage (P<0.05), there was a significant 2 2.000 8 b*
effect on the number of healthy leaves (NHL), as seen
3 2.625 8 ab*
in Tables 4 and 5.
4 2.250 8 b*
5 5.500 8 ab*
Table 4. Summary of the behaviour of the mean
6 13.625 8 a*
populations in the pathogen application stage for the
*Different letters indicate significant difference
number of healthy leaves (NHL).
(P<0.05).
Stage Mean n
1 1.792 24 a*
Total length of the plant (cm)
2 8.625 24 b*
The plants evaluated in treatments T6 and T9
*Different letters indicate significant difference presented the greatest average total height. In these
(P<0.01). treatments plants reached values of 124.25 cm and
122 cm respectively (Tables 2 and 3). Nevertheless,
Number of fruits (N°) when measuring the height of the aerial parts of the
The counting of mature pods with seeds was carried plant and the length of roots separately, T7 had a
out when the experiment was dismantled (90 days longer stem than T9. Likewise, in terms of the length
after planting). The greatest number of pods was

26 Silgado and Agudelo

 Int. J. Biosci. 2014

of the roots, T3 and T8 presented the greatest values
(52.25 cm and 55 cm respectively), as seen in Figures
2a and 2b. In the pathogen application stage, a
significant effect was observed for the variables FWS
(fresh weight of the stem) (P<0.0001) and LR (length
of the roots) (P<0.05), as seen in Tables 6 and 7.
Table 6. Summary of the behaviour of the mean
populations in the pathogen application stage for the
variable Fresh Weight of the Stem (FWS).
Stage Mean n It should be noted that for the total dry weight
Total dry weight of the plant (g)
Treatments T6 (9.68 g) and T12 (9.88 g) presented
the greatest average dry weight of the plants (Table 8
and Table 9). However, when measuring the average
dry weight of the stem separately, T5 (4.95 g)
presented greater values than T6 and T12. T3 (0.50 g)
obtained the greatest average dry weight of the roots
compared to the other treatments (Figures 3a and
3b).

1 45.450 24 a* variable (TDW), a 5% significance was not observed

2 23.983 24 b* for the dosage of Fitotripen wp™ or the Fusarium

application stage (P value: 0.0582). However, the P
*Different letters indicate significant difference
value suggests a trend toward significance and
(P<0.0001).
possible differences in terms of population between
the doses and the stage, even though these were not
Table 7. Summary of the behaviour of the mean
found in this assay. Apart from the fact that the P
populations in the pathogen application stage for the
value is closely significant, it is notable that the Tukey
variable Root Length (LR).
test, which is relatively conservative, identified
Etapa Media n
differences between the means of the TDW of the
1 45.208 24 a*
plant for some of the dosage levels. It is expected that
2 50.583 24 b*
an assay with a larger number of repetitions would be
*Different letters indicate significant difference
able to identify these differences.
(P<0.05).

Table 8. Averages of the biometric parameters of the Phaseolus vulgaris L. plants with different concentrations
of Fitotripen wp™ and inoculation of Fusarium oxysporum f.sp Phaseoli at the time of planting.
Treatments TDWP (g) TFWP (g) DWS (g) FWS (g) DWR (g) FWR (g) DWFr (g) FWFr (g)
T1 8.13 36.25 2.95 18.67 0.40 1.15 4.78 16.43
T2 8.93 58.85 4.00 33.88 0.40 1.88 4.53 23.10
T3 8.60 48.13 3.73 28.58 0.50 1.50 4.38 18.05
T4 7.65 37.20 2.43 20.67 0.30 1.10 4.93 15.43
T5 7.75 56.08 4.95 38.80 0.38 1.30 2.43 15.98
T6 9.68 44.50 4.20 28.25 0.35 1.38 5.13 14.88
TDWP= total dry weight of the plant; TFWP= total fresh weight of the plant; DWS= dry weight of the stem;
FWS= fresh weight of the stem; DWR= dry weight of the roots; FWR= fresh weight of the roots; DWFr= dry
weight of the fruits; FWFr= fresh weight of the fruits.

Biocidal effect of Trichoderma spp. on Fusarium
oxysporum f.sp Phaseoli and promotion of bean
growth
The results of this investigation are in agreement with
data reported by Lara et al., 2011 who found that the
most conclusive biometric parameters in their assays
with Rhapanus sativus were length and dry weight.
In our investigation the statistical analysis showed
highly significant differences (P<0.01) between the
control treatments (T1 and T7) with respect to: i) T2,
T3, T5 and T6 simultaneously inoculated with
Fitotripen wp™ and Fusarium oxysporum f.sp
Phaseoli at the time of planting; ii) T9, T10 and T12
also inoculated with Fitotripen wp™ at the time of
planting and with Fusarium oxysporum f.sp Phaseoli
after 30 days of germination. Additionally, it can be
stated that the higher the dosage of inoculation with
Trichoderma spp. (T6 and T12), the better the

27 Silgado and Agudelo

 Int. J. Biosci. 2014

response to the biometric parameters (Figures 4 and
5).
This data suggests that inoculation with Trichoderma
spp., should be considered as a preventative
treatment in the integrated management of bean
production. The application of Trichoderma spp.,
before and while planting stimulates the colonization
of the rhizosphere at a speed at which the fungus
grows (Bernal et al., 2000). This competition of the
microparasite for space and nutrients in the
rhizosphere impedes the arrival and/or colonization
of the pathogen on the plant (Fernandez and Suárez,
2009). It can therefore be said that preventative
inoculation with Trichoderma spp. increases the
health and nutrition of bean crops (Otadoh et al.,
2011). The antagonistic effect of Trichoderma spp.,
against Fusarium oxysporum f.sp Phaseoli for a
better biocontrol of the crop depends on the dosage
with which the antagonistic fungus is applied to the
root of the plant (Filion et al., 2003), and on the stage
at which the soil is inoculated with the fungus.

Table 9. Averages of the biometric parameters of the Phaseolus vulgaris L. plants with different concentrations
of Fitotripen wp™ and inoculation of Fusarium oxysporum f.sp Phaseoli after 30 days of germination.
Treatments TDWP (g) TFWP (g) DWS (g) FWS (g) DWR (g) FWR (g) DWFr (g) FWFr (g)
T7 6.53 30.10 2.73 18.75 0.43 1.08 3.38 10.23
T8 6.55 35.80 3.20 22.43 0.48 1.18 2.88 12.20
T9 7.08 36.48 2.95 20.48 0.48 1.73 3.65 13.98
T10 7.80 45.13 3.00 25.58 0.43 1.80 4.38 17.65
T11 8.88 46.00 3.75 27.20 0.48 1.98 4.65 16.70
T12 9.88 52.95 4.73 29.43 0.48 3.13 4.68 20.00
TDWP= total dry weight of the plant; TFWP= total fresh weight of the plant; DWS= dry weight of the stem;
FWS= fresh weight of the stem; DWR= dry weight of the roots; FWR= fresh weight of the roots; DWFr= dry
weight of the fruits; FWFr= fresh weight of the fruits.

Discussion microscopic analysis showed that microparasites were

Biocidal effect of Trichoderma spp., on Fusarium not present, thereby demonstrating that the
oxysporum f.sp Phaseoli commercial inoculum Trichoderma spp. was not able
The necrosis of vascular bundles of roots is perceived to grow on the surface of the mycelium of F.
as a severe symptom of attack by the fungus (Sharma, Oxysporum. In the microscopic observations, the
2011). In this process, the absorption of nutrients penetration or coiling up of the T. harzianum hyphae
from the roots to the aerial parts of the plant is around the F. Oxysporum hyphae was also not seen.
blocked, and in turn photosynthesis decreases due to This suggests that commercial isolates do not possess
subsequent damage to the biomass of the plant the capacity to parasitize this pathogen.
(Otadoh et al., 2011). In this investigation,

Fig. 1a and 1b. Average numbers of leaves and fruits of P .vulgaris for the different treatments. NF: number of
fruits, TNL: total number of leaves, NHL: number of healthy leaves, NIL: number of infected leaves.

28 Silgado and Agudelo

 Int. J. Biosci. 2014

The above behaviour probably occurs due to the fact chitinases and β-1-3glucaneses. These enzymes allow
that the chitin of the cell wall of F. oxysporum is a site to be established for the penetration of the
covered by a protein layer that prevents its microparasite. After this organism penetrates, it
degradation by the chitinases and β-1-3glucaneses produces antibiotics that permeate the effected hypha
that produce T. harzianum (Inbar and Chet, 1995). and inhibit the re-synthesis of the phytopathogen
This complicates the process of penetration, (Matroudi et al., 2009). Suárez et al., 2008, also
pedration and control. Although in this investigation recorded microparasitism of isolates of Trichoderma
F. oxysporum was not parasitized, there are reports spp. on Fusaium solani. These findings allow it to be
that state that this pathogen can be attacked by assumed that assays with a greater number of replicas
Trichoderma spp., (Salazar et al., 2011). Such an and a longer evaluation time would permit
attack may occur because Trichoderma causes microparasitism processes to be recorded.
degradation of the chitin by the production of

Fig. 2a and 2b. Average lengths of P. Vulgaris in each of the Fitotripen wp™ treatments. THP: total height of
the plant, LS: length of the stem, LR: length of the root.

Biocidal effect of Trichoderma spp., on Fusarium
oxysporum f.sp Phaseoli and promotion of bean
growth
Microparasitism and antibiosis are well known
mechanisms involved in the control of pathogens by
Trichoderma. Competition for nutrients and space is
just as important as the phenomenon of mutualism.
The complete course of interaction between
Trichoderma and Fusarium has been observed in
dual cultures in petri dishes. This interaction can be
divided into three phases: (i) the initial phase, where
interaction is without mycelium contact and instead
only by diffusion of the metabolites of both
microorganisms, this phase is what decides the
interaction; (ii) the intermediate phase, where
Trichoderma may or may not be able to inhibit the
effect of Fusarium, some chemotactic attraction
mechanisms might be active; (iii) the final phase,
where Trichoderma parasitizes Fusarium.
In this research, the short experimentation time may
have been the reason why microparasitism
phenomena could not be seen. However, evidence of
antibiosis was observed, which explains the
emergence of better biometric results for the
treatments where first Trichoderma was inoculated
and then 30 days later Fusarium was inoculated. In
this study, bean plants inoculated with microparasites
and pathogens presented mild symptoms with respect
to withering and foliar infection. In contrast, when
the controls that did not use the biocontrol fungus
were inoculated with the pathogen, they suffered
from more severe symptoms characteristic of the
aforementioned disease. This suggests that although
the species of Trichoderma used as a biocontrol did
not completely protect the plants, there was clear
evidence of the antagonistic effect of Trichoderma
spp.

29 Silgado and Agudelo

 Int. J. Biosci.
Fig. 3a and 3b. Average dry weights of P. vulgaris in the different Fitotripen wp™ treatments. TDWP= total
dry weight of plant; DWS= dry weight of the stem; DWR= dry weight of the root.
Salazar et al., 2011; Acosta and Garcés, 2005, and
Harman, 2000, reported that although not all
Trichoderma isolates can control certain pathogens,
they can improve the performance of plants and
stimulate better root development. This improves the
ability of plants to resist and/or tolerate the
pathogenic effect of the fungus, as seen in the results
obtained in this investigation where treatments
inoculated with Trichoderma spp., showed a greater
root proliferation compared to the control treatments.
Our results agree with those reported by the authors
mentioned above who found that antagonistic isolates
promote greater root growth and allow the damage
caused by pathogenic fungi to be reduced, even
though there is no decrease in the incidence of the
disease. Likewise, Chang et al., 1986, found that T.
harzianum was able to increase root growth in
tomato plants. The results of this investigation also
concur with similar observations described by
Montealegre, 2011; and Salazar et al., 2011, who
specified that applying Trichoderma spp., in a
preventative way is a practice that allows biocontrol
to be exercised on different pathogenic fungi
including F oxysporum, thereby decreasing the
pathogenic effects of such fungi. The ability of
Trichoderma spp., to develop direct exchanges with
pathogens through the application of conidial
suspension severely reduces the diseases caused by F.
oxysporum (Kamal et al., 2009; and Haran et al.,
1996).
The reduction in the incidence of diseases and the
30 Silgado and Agudelo
2014
increase in the protection of bean seedlings against
withering were significant when testing the ability of
the fungus to control F. oxysporum f. sp. Phaseoli.
The antagonistic capacity of Trichoderma spp., is
highly variable and depends on the dosage and time
of application, which is similar to the results
described by Otadoh et al., 2011. These results
suggest that the greatest withering caused by
Fusarium in bean plants may be due to the reduction
in the population density of Tricocherma, which is an
observation supported by Suárez et al., 2008.
The principles of the above effects are related to the
antagonistic properties of Trichoderma. These
properties imply parasitism, lysis of the pathogenic
fungi, and competition for the limiting factors in the
rhizosphere, principally iron and carbon according to
Sivan and Chet, 1985. The capacity of Trichoderma
spp., to control the pathogen that causes withering
helps to induce plant growth and development at a
greenhouse scale (Vinale et al., 2004).
Our results show that bean plants inoculated with
Trichoderma in soils treated at the time of planting,
and inoculated with Fusarium 30 days after
germination, show a significant increase in growth, a
greater number of healthy leaves, and greater dry and
fresh weights. The results of the investigation suggest
that the increase in the growth and development of
the plants due to the effect of the inoculation of
Trichoderma spp., are in response to root
development. This is supported by Yedidia et al.,
 Int. J. Biosci. 2014

1999, who found that plants inoculated with T. in their growth and the when the pathogen was
harzianum absorbed more nutrients when the inoculated at a later stage in their development.
biocontrol fungus was inoculated at a very early stage

Fig. 4. Growth of the Red Cargamanto bean plants (Phaseolus vulgaris L) in the different treatments with
Fitotripen and F. oxysporum applied the time of planting.

Furthermore, Harman, 2000, established that hormones. Otadoh et al., 2011, reported a similar
Trichoderma spp., are colonizers of opportunistic response, saying that due to the capacity of
plants. They affect the growth of plants through the Trichoderma spp., to inhibit less significant
promotion of abundant and healthy leaves and roots, pathogens in the rhizosphere, rotting of the seeds and
possibly as a result of control by the production of premature drowning could be induced.

31 Silgado and Agudelo

 Int. J. Biosci. 2014

In conclusion, this study demonstrated that the
commercial inoculum based on Trichoderma spp. has
the potential to be used as a biocontrol agent to
protect bean plants from F. oxysporum f. sp.
Phaseoli. However, further studies are recommended
regarding the use of native isolates as biocontrol
agents and the evaluation of these strains of
Trichoderma at a greater concentration and longer
experimentation time, both at a greenhouse and field
scale.

Fig. 5. Growth of the Red Cargamanto bean plants (Phaseolus vulgaris L) in the different treatments with
Fitotripen and F. oxysporum applied 30 days after planting.

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