Effectiveness of Trichoderma spp. at controlling Fusarium ... · Effectiveness of Trichoderma spp....

21 Silgado and Agudelo

Int. J. Biosci. 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 [email protected]

International Journal of Biosciences | IJB |

ISSN: 2220-6655 (Print) 2222-5234 (Online)

http://www.innspub.net

Vol. 5, No. 9, p. 21-36, 2014

http://dx.doi.org/10.12692/ijb/5.9.21-36

mailto:[email protected]

http://www/



Introduction

Fusarium oxysporum L is reported as the etiologic

agent that causes root rot or radical putrification in

multiple crops (Zúniga et al., 2010; Rutkowska -

Krause et al., 2003; Schneider, 1984). The

symptomology of this fungus is primarily associated

with the wilting and death through the weakening of

the plant (Garcés et al., 2001). Other symptoms

include the stunting of growth, yellowing in older

leaves and adventitious root formation. The vascular

tissue necrosis also stands as strong evidence of

Fusarium sp., (Zúniga et al., 2010; Appel and

Gordon, 1994). It is reported that this pathogen

attacks more than 100 species of gymnosperms and

angiosperms (Hernandez et al., 2011; Garofalo and

McMillan, 2003; Bosland., 1988) and can form three

resistant structures: macroconidia (distinctive

structures of the genere), microconidia and

chlamydospores, the latter are those that allow it to

survive as free-living saprophyte in the absence of a

host (Hernandez et al., 2011; Nelson, 1981). To

control this fungal pathogen the precise use of

agrochemicals are required. Often these chemicals are

used indiscriminately leading to deleterious effects on

the environment such as pollution of soil and water

sources (Capote and Torres, 2004).

The problem about mentioned above requires the

search for new biotechnology-based alternatives will

lead to suggest the implementation of biological

control methods such as the use of Trichoderma spp.,

as a biocontrol agent (Hermosa et al., 2001). This is

fungus that is easy to isolate and grow in natural

culture media or semi- natural media (Rey et al.,

2000). Trichoderma spp., is a free-living fungi found

in soils and root ecosystems where there are complex

interactions between the host plant, pathogens and

various environmental factors (Harman, 2006; Woo

et al., 2006). The interest in this fungus is found its

antagonism ofthe soil microorganisms causing

diseases in plants (Montealegre et al., 2011; Elias et

al., 2009). Different species of Trichoderma spp., are

used in agriculture for the handling of

phytopathogens and for the limiting of the

development of harmful fungi such as Phytophthora

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

Fusarium sp., Verticillium sp., among others

(Gonzalez et al., 2005; Bernal et al., 2000).

Competitive hyperparasitism is one of the

mechanisms employed by Trichoderma spp., in this

condition, it produces antifungal metabolites and

hydrolytic enzymes (Martinez et al., 2013; Howell,

2003 In: Javaid and Ali, 2011). These molecules lead

to structural changes at the cellular level in the

phytopathogens with that the Trichoderma

antagonize. Among those structural changes are:

vacuolation, granulation, disintegration of the

cytoplasm and cell lysis (Mohamed et al., 2004). In

hyperparasitism, Trichoderma adheres to the hyphae

of the phytopathogenic through specialized structures

called appressoria and releases enzymes like

glucanases, chitinases, quitobiosas that degrade or

weaken the cell wall and destroy the reproductive

structures hyphae such as when hiperparasitar to

Sclerotium cepivorum (Vera et al., 2005). This

phenomenon is very important, because many fungi

form resistant structures in the ground that allow

them to survive under adverse environmental

conditions for up to 20 years (Higuera et al., 2003).

Also Trichoderma spp., produces antibiotics such as

viridine, gliotoxin, gliovirina and peptaibols those

who control the proliferation of phytopathogens

(Howell et al., 1993). The mechanisms described

exhibit that Trichoderma spp., as an effective agent

for the control of phytopathogens. Bernal et al.,

(2000) reported strains of Trichoderma spp., as an

environmentally friendly alternative for the control of

F. oxysporum Schlecht f . sp cubense, with over 70%

of antagonism to the pathogen.

Inhibition of in vitro growth of pathogenic fungi by

antagonistic fungi has been widely described. The

most common methodology is to treat both

microorganisms equally dual way (Aquino -Martinez,

2007) even though they may or may not be inoculated

at the same time, usually as "Plug" or drop. Later its

effectiveness is determined by a scale of Degrees of

Antagonism (Bell et al., 1982) or, using Abbott's



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

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



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

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.



Results

The results of the biometric analysis can be observed

in Tables 2 and 3. This data corresponds to average

values of the bioassays, each of which have 4 replicas

for the different treatments:

T1 and T7

Controls: without Fitotripen wp™ + 1.28x109

SPORES/mL of F. oxysporum f.sp Phaseoli : T1:

inoculation of the pathogen at the time of planting,

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

cycle of the bean

Dosis of Fitotripen wp™ (g) inoculated

since sowing

Time of inoculation with

F. oxysporum

T1 Seeds 0.00

Time 1:

While planting T2 0.25

T3 0.50

T4 1.00

T5 1.25

T6 1.50

T7 30 days of germination 0.00

Time 2:

30 days after planting

T8 0.25

T9 0.50

T10 1.00

T11 1.25

T12 1.50

T2 and T8

25% of Fitotripen wp™ (applied at the time of

planting) + 1.28x109 SPORES/mL of F. oxysporum

f.sp Phaseoli : T2: inoculation of the pathogen at the

time of planting, T8: inoculation of the pathogen after

30 days of germination.

T3 and T9




time of planting, T9: inoculation of the pathogen after

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

(cm)

TDWP

(g)

LS

(cm)

DWS

(g)

LR

(cm)

DWR

(g)

NL NFr DWFr

(g) HL IL

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

after 30 days of germination.

T5 and T11








T6 and T12






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

(cm)

TDWP

(g)

LS

(cm)

DWS

(g)

LR

(cm)

DWR

(g)

NL NFr DWFr

(g) HL IL

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°)

The appearance of cotyledon leaves was seen

simultaneously in all treatments after 10 days of

germination. The greatest growth, development and

production of true leaves by the plants were observed

in treatments T2 and T10. In these treatments, the

number of leaves was 25 and 33 respectively, as

shown in Figures 1a and 1b. In the pathogen

application stage (P<0.01) and the Fitotripen wp™

application stage (P<0.05), there was a significant

effect on the number of healthy leaves (NHL), as seen

in Tables 4 and 5.

Table 4. Summary of the behaviour of the mean

populations in the pathogen application stage for the

number of healthy leaves (NHL).

Stage Mean n

1 1.792 24 a*

2 8.625 24 b*

*Different letters indicate significant difference

(P<0.01).

Number of fruits (N°)

The counting of mature pods with seeds was carried

out when the experiment was dismantled (90 days

after planting). The greatest number of pods was

obtained for treatments T5 and T12, in which an

average of 5 pods per treatment were observed, as

seen in Figures 1a and 1b.


populations in the Fitotripen wp™ application stage

for the number of healthy leaves (NHL).

Dose Mean n

1 5.250 8 ab*

2 2.000 8 b*

3 2.625 8 ab*

4 2.250 8 b*

5 5.500 8 ab*

6 13.625 8 a*


(P<0.05).

Total length of the plant (cm)

The plants evaluated in treatments T6 and T9

presented the greatest average total height. In these

treatments plants reached values of 124.25 cm and

122 cm respectively (Tables 2 and 3). Nevertheless,

when measuring the height of the aerial parts of the

plant and the length of roots separately, T7 had a

longer stem than T9. Likewise, in terms of the length



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.



variable Fresh Weight of the Stem (FWS).

Stage Mean n

1 45.450 24 a*

2 23.983 24 b*


(P<0.0001).



variable Root Length (LR).

Etapa Media n

1 45.208 24 a*

2 50.583 24 b*


(P<0.05).

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).

It should be noted that for the total dry weight

variable (TDW), a 5% significance was not observed

for the dosage of Fitotripen wp™ or the Fusarium

application stage (P value: 0.0582). However, the P

value suggests a trend toward significance and

possible differences in terms of population between

the doses and the stage, even though these were not

found in this assay. Apart from the fact that the P

value is closely significant, it is notable that the Tukey

test, which is relatively conservative, identified

differences between the means of the TDW of the

plant for some of the dosage levels. It is expected that

an assay with a larger number of repetitions would be

able to identify these differences.

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



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

Biocidal effect of Trichoderma spp., on Fusarium

oxysporum f.sp Phaseoli

The necrosis of vascular bundles of roots is perceived

as a severe symptom of attack by the fungus (Sharma,

2011). In this process, the absorption of nutrients

from the roots to the aerial parts of the plant is

blocked, and in turn photosynthesis decreases due to

subsequent damage to the biomass of the plant

(Otadoh et al., 2011). In this investigation,

microscopic analysis showed that microparasites were

not present, thereby demonstrating that the

commercial inoculum Trichoderma spp. was not able

to grow on the surface of the mycelium of F.

Oxysporum. In the microscopic observations, the

penetration or coiling up of the T. harzianum hyphae

around the F. Oxysporum hyphae was also not seen.

This suggests that commercial isolates do not possess

the capacity to parasitize this pathogen.

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.



The above behaviour probably occurs due to the fact

that the chitin of the cell wall of F. oxysporum is

covered by a protein layer that prevents its

degradation by the chitinases and β-1-3glucaneses

that produce T. harzianum (Inbar and Chet, 1995).

This complicates the process of penetration,

pedration and control. Although in this investigation

F. oxysporum was not parasitized, there are reports

that state that this pathogen can be attacked by

Trichoderma spp., (Salazar et al., 2011). Such an

attack may occur because Trichoderma causes

degradation of the chitin by the production of

chitinases and β-1-3glucaneses. These enzymes allow

a site to be established for the penetration of the

microparasite. After this organism penetrates, it

produces antibiotics that permeate the effected hypha

and inhibit the re-synthesis of the phytopathogen

(Matroudi et al., 2009). Suárez et al., 2008, also

recorded microparasitism of isolates of Trichoderma

spp. on Fusaium solani. These findings allow it to be

assumed that assays with a greater number of replicas

and a longer evaluation time would permit

microparasitism processes to be recorded.

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.



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

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.,



1999, who found that plants inoculated with T.

harzianum absorbed more nutrients when the

biocontrol fungus was inoculated at a very early stage

in their growth and the when the pathogen was

inoculated at a later stage in their development.

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

Trichoderma spp., are colonizers of opportunistic

plants. They affect the growth of plants through the

promotion of abundant and healthy leaves and roots,

possibly as a result of control by the production of

hormones. Otadoh et al., 2011, reported a similar

response, saying that due to the capacity of

Trichoderma spp., to inhibit less significant

pathogens in the rhizosphere, rotting of the seeds and

premature drowning could be induced.



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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