*2.3. Pathogenicity Tests*

Fifty peanut plants of the "Virginia Champs" variety provided by local farmers were used. Each two-month-old plant was individually planted in a 1 L plastic pot, containing a sterilized mixture of Peatmoss and Agrellite (1:1 *v*/*v*). The inoculation of "PUE 4.0" strain (*M. phaseolina*) was by the toothpick method three days after sowing [31]. Development of the plants was done under greenhouse conditions (70% relative humidity and 28 ◦C) until the appearance of symptoms of disease.

Toothpicks were previously sterilized and then placed in Petri dishes with the *M. phaseolina* colony until 100% colonization was reached. Using toothpicks with mycelium, small 2 mm wounds were made on the roots. Sterile toothpicks were used for control group plants, tests were carried out in duplicate. After three weeks, inoculated plants showed symptoms of wilting chlorosis on leaves and discoloration of the vascular ring, from brown to dark brown: characteristic symptoms of charcoal rot, while control plants remained healthy, fulfilling Koch's postulates.

#### *2.4. In Vitro Assessment of Antagonistic Capacity of Trichoderma spp.*

For the evaluation of the antagonism test in vitro, the group of strains of *T. harzianum* (T-H3), *T. asperellum* (T-AS1), *T. hamatum* (T-A12), *T. koningiopsis* (T-K11), and an endemic strain of the *T. harzianum* (T-Ah) study region were used, whose sequences are found in the database of the National Center for Biological Information (NCBI) with access numbers MK780094, MK778890, MK791650, and MK791648, respectively. The dual confrontations were carried out with the strain "PUE 4.0" of *M. phaseolina* (MW585378) in a completely randomized experimental design with five treatments and three repetitions in duplicate.

For the evaluation of mycelial development, 5 mm diameter fragments of the *Trichoderma* strains as well as *M. phaseolina* were inoculated in Petri dishes with PDA (Potato and Dextrose Agar) and incubated in dark conditions at 28 ◦C for 10 days. The diameter of the mycelium was measured every 12 h with a digital vernier (CD-6 Mitutoyo) to estimate the growth speed (cm/d−1), which was calculated with the linear growth function [32] Equation (1).

$$\mathbf{y} = \mathbf{m}\mathbf{x} + \mathbf{b} \tag{1}$$

where:

y = is the distance

m = slope x = is time

b = the constant factor.

Antagonism and percentage of inhibition were evaluated considering the mycelial growth radius of *Trichoderma* spp. and *M. phaseolina* (with their respective controls). PDA discs (5 mm in diameter) with mycelia of *Trichoderma* spp. and *M. phaseolina* were placed at the extremes of Petri plates containing PDA and incubated at 28 ◦C for 240 h. Then, mycelial growth was scored every 12 h until the first contact between the mycelia of each antagonist with *M. phaseolina* occurred [33].

The percentage of radial growth inhibition (PIRG) was calculated based on the formula of Equation (2).

$$\text{PIRG}\% = (\text{R1} - \text{R2}) / \text{R1} \times 100 \tag{2}$$

where:

PIRG = Percent inhibition of radial growth.

R1 = Radial growth (mm) of *M. phaseolina* without *Trichoderma* spp.

R2 = Radial growth (mm) of *M. phaseolina* with *Trichoderma* spp.

Invasion of the antagonist or colonization on the surface of the *M. phaseolina* mycelium was taken as the index of antagonism with the scale proposed by Bell [34] (Table 1).

**Table 1.** *Trichoderma* strain antagonism evaluated in vitro using Bell's scale [34], considering the invasion of the surface.

