**2.3. Antagonistic activity of the strains of** *Trichoderma spp.* **on** *F. oxysporum in vitro*

To evaluate the antagonistic activity of *Trichoderma* spp., Cherif and Benhamou technique was used [29]. For each of the treatments that were performed in Petri dishes with PDA (Agar, Dextrose and Potato) culture medium, was place at one end of the Petri dish a 5 mm diameter agar disc with mycelium of the pathogenic fungus, in this case *F. oxysporum,* due to its slow growth was allowed to develop for 2 days and then another 5 mm disc with mycelium of *Trichoderma* spp. was inoculated at the opposite end, (natives) at a distance of approximately 5 cm between them [30, 31]. The controls consisted of mycelium of the pathogens and the antagonist, separately cultured in Petri dishes with PDA medium described above. Petri dishes were incubated at a temperature of 26°C. To measure the inhibitory effect of antagonistic fungus to the pathogen, measures colony diameter was recorded every 24 h; until standoff and formation of an inhibition zone between the colonies was formed. Ten repetitions were considered for each comparison, in this case will be three strains of *Trichoderma* spp., a single strain of *F. oxysporum*, evaluating a total of 30 experimental units. The percentage growth inhibition are calculated using the formula given by [32]:

$$\text{PICR} = \left[ (\mathbf{R1} - \mathbf{R2}) / \mathbf{R1} \right] \times \mathbf{100} \tag{2}$$

The variables evaluated were incidence and severity of disease at 30 days after sowing, both for the radical part and for the aerial part (**Table 2**) using the scale proposed by Amaro-Leal [34]. The percentage of mortality and survival of tomato seedlings at 45 days, as well as height,

c Dry necrosis approx 1.5 cm, suberized, adventitious roots. C Plants with all leaves with loss of

Biological Control of *Fusarium oxysporum* in Tomato Seedling Production with Mexican Strains…

d Root presenting lesions with necrosis of 2 cm. D Plants with withered leaves. Planta

B Plant with some leaves with loss of

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159

turgor.

turgor.

sana.

**Code Description Code Description** a Circular dry injury. A Healthy plant.

b Light necrosis at the base of the root, lesion greater than

e Lesions with necrosis of 2 cm, roots begin to detach. f Lesions with necrosis of 2 cm, large amount of roots are

g It gives off epidermis leaving vascular tissue, loss of 50%

h Detachment 80% roots expose tissue loss of epidermis,

**Table 2.** Severity scale of *F. oxysporum* attack on tomato culture.

The results obtained were subjected to an analysis of variance (ANOVA) and test of separation of means by Tukey (p < 0.05), sing the SPSS version 17 (Statistical Package for Social Sciences)

The Tv-T3 (1) and Th-T4 (3) strains of *Trichoderma* spp. presented a cottony texture with abundant density and abundant mycelium, a dark green and white coloration, whereas the Tav-T7 (2) strain presented a regular density, regular mycelium and a green/yellow coloration. For the *F. oxysporum* strain in this case (Fo-A) presented a velvety texture with a regular density and mycelium of pink white color, as described by Guzman [35], in addition its mycelium is formed by septate hyphae and the conidiophores present clusters of macroconidia where chlamydospores are observed.

*T. harzianum* presented the highest growth rate with a mean of 1.25 cm/day, followed by *T. viridae* with 0.75 cm/day, with the *T. atroviridae* strain being the lowest growth rate with 0.64 cm/day. For *F. oxysporum* the growth was 0.83 mm/day, results similar to those found by Amaro-Leal [36], with a speed between 70 and 73 mm/day in PDA, results similar to those of the present investigation.

stem thickness and total dry biomass were evaluated.

to determine differences between treatments.

**3.1. Growth rate and rate of development**

**3. Results and discussion**

**Scale of severity in root of plants**

0.5 cm.

released.

of roots.

necrotic roots.

*where:*

PICR: Percent inhibition of radial growth.

R1: Diameter of token.

R2: Diameter of organism tested.

Additionally, the strain was compared respect to the antagonistic capacity according to the scale proposed by Bell [33] in 1982 (**Table 1**).

#### **2.4. Greenhouse antagonism tests**

Seeds of tomato (*L. esculentum* Mill) var. Ramses were used, these were sterilized in 20% (v/v) sodium hypochlorite solution for 20 min, followed by three 5-minute washes in sterile distilled water. Subsequently the sowing was carried out in disinfected trays of 27 × 17 × 4 cm, containing 60 g of sterilized vermiculite in an autoclave at 121°C for 1 h. In each tray 100 tomato seeds were sown. Three strains of *Trichoderma* spp. were selected in the laboratory for their antagonistic response [Th-T4 (3), Tav-T7 (2), Tv-T3 (1)] plus two commercial strains (Perkins-C21 and Tricovel-25) and a control inoculated with *F. oxysporum* without *Trichoderma* spp. plus an absolute control; for a total of seven treatments, where each tray with 100 tomato seeds represented an experimental unit. The inoculations were carried out at the time of planting the seeds in the trays with 1 mL of a suspension of *F. oxysporum* at a concentration of 5 × 106 conidia mL−1 per well. The trays were installed in a culture chamber at 25 ± 2°C, were irrigated with 80 mL of distilled water per tray every 2 days. After 15 days, 1 mL of the five strains of *Trichoderma* spp. was applied at a total concentration of 83 × 104 conidia with a viability percentage of 96%.


**Table 1.** Class and characteristics of *Trichoderma* spp. antagonistic capacity.


the antagonist, separately cultured in Petri dishes with PDA medium described above. Petri dishes were incubated at a temperature of 26°C. To measure the inhibitory effect of antagonistic fungus to the pathogen, measures colony diameter was recorded every 24 h; until standoff and formation of an inhibition zone between the colonies was formed. Ten repetitions were considered for each comparison, in this case will be three strains of *Trichoderma* spp., a single strain of *F. oxysporum*, evaluating a total of 30 experimental units. The percentage growth

158 Fusarium - Plant Diseases, Pathogen Diversity, Genetic Diversity, Resistance and Molecular Markers

PICR = [(**R1** − **R2**)/**R1**] × **100** (2)

Additionally, the strain was compared respect to the antagonistic capacity according to the

Seeds of tomato (*L. esculentum* Mill) var. Ramses were used, these were sterilized in 20% (v/v) sodium hypochlorite solution for 20 min, followed by three 5-minute washes in sterile distilled water. Subsequently the sowing was carried out in disinfected trays of 27 × 17 × 4 cm, containing 60 g of sterilized vermiculite in an autoclave at 121°C for 1 h. In each tray 100 tomato seeds were sown. Three strains of *Trichoderma* spp. were selected in the laboratory for their antagonistic response [Th-T4 (3), Tav-T7 (2), Tv-T3 (1)] plus two commercial strains (Perkins-C21 and Tricovel-25) and a control inoculated with *F. oxysporum* without *Trichoderma* spp. plus an absolute control; for a total of seven treatments, where each tray with 100 tomato seeds represented an experimental unit. The inoculations were carried out at the time of planting the seeds in

per well. The trays were installed in a culture chamber at 25 ± 2°C, were irrigated with 80 mL of distilled water per tray every 2 days. After 15 days, 1 mL of the five strains of *Trichoderma*

1 Overgrowth of *Trichoderma* that colonized the entire medium surface and reduced colony pathogen.

4 Pathogenic fungus colonized at least 2/3 of the medium surface and resist invasion by *Trichoderma*.

3 *Trichoderma* and pathogen colonized medium to medium (more than 1/3 and less than 2/3).

5 Overgrowth of the pathogenic fungus that colonized the entire surface of the medium.

conidia mL−1

conidia with a viability percentage of 96%.

the trays with 1 mL of a suspension of *F. oxysporum* at a concentration of 5 × 106

inhibition are calculated using the formula given by [32]:

PICR: Percent inhibition of radial growth.

scale proposed by Bell [33] in 1982 (**Table 1**).

spp. was applied at a total concentration of 83 × 104

2 Overgrowth *Trichoderma* colonized at least 2/3 of the medium surface.

**Table 1.** Class and characteristics of *Trichoderma* spp. antagonistic capacity.

*where:*

R1: Diameter of token.

**Class Characteristics**

R2: Diameter of organism tested.

**2.4. Greenhouse antagonism tests**

**Table 2.** Severity scale of *F. oxysporum* attack on tomato culture.

The variables evaluated were incidence and severity of disease at 30 days after sowing, both for the radical part and for the aerial part (**Table 2**) using the scale proposed by Amaro-Leal [34]. The percentage of mortality and survival of tomato seedlings at 45 days, as well as height, stem thickness and total dry biomass were evaluated.

The results obtained were subjected to an analysis of variance (ANOVA) and test of separation of means by Tukey (p < 0.05), sing the SPSS version 17 (Statistical Package for Social Sciences) to determine differences between treatments.
