**4. Discussion**

*M. phaseolina* is considered an important necrotrophic phytopathogen for various crops of agricultural interest, affecting at least 500 plant species in more than 100 families [12,38], where it has been reported to be a causal agent of charcoal rot, affecting the root and stem of peanut crops [9].

According to the morphological characteristics and the amplification of the 5.8S rDNA gene region [39], the identity of "PUE 4.0" strain was confirmed, corresponding to *M. phaseolina*, which coincides with the descriptions reported by Pandey [40] and Márquez [12].

The presence of microsclerotia in the Xylem caused the development of necrotic roots, chlorotic leaves, and premature death of the plants, symptoms that coincide with those reported in strawberry and sunflower crops [41]. This may be due to the presence of phytotoxic metabolites of *M. phaseolina* such as patulin, phaseolinon, and botryodiplodin, which play an important role in the early stages of charcoal rot [42,43]. As far as we know, this is the first report of this pathogen that causes charcoal rot in peanut (*Arachis hypogea* L.) crop in the variety "Virginia Champs" in Puebla, Mexico.

Species of the genus *Trichoderma*, known as green spore fungi, have been widely reported as biological control agents against diseases caused mainly by soil-borne pathogens [33].

In the present study, a parasitism greater than 50% was observed for all *Trichoderma* strains at 240 h against *M. phaseolina*, where there were areas of interaction in the dual culture assays. This may be due to the action capacity of the different *Trichoderma* species, as competition for space and nutrients [18] decreases the growth and development of the strain "PUE 4.0" in vitro.

Cubilla-Ríos [44] evaluated the antagonistic capacity of three *Trichoderma* species: *T*. *arundinaceum*, *T. brevicompactum*, and *T. harzianum* T34 on two isolates of *M. phaseolina* present in sesame and soybean, and found a greater antagonist activity exerted by *T. harzianum* T34. Likewise, Sreedevi [45] reported that *T. harzianum* reduced growth by 64.4% in *M. phaseolina*, isolated from the root rot of peanuts, results like those obtained in the present investigation with "T-H3" strain from *T. harzianum* (63.55%). According to the literature [46], the properties of *T. harzianum* can be attributed to the production of various volatile substances such as acetaldehyde; isocyanide derivatives, terpenes; derivatives of alpha-pyrone; piperazine, hydrazone derivatives as well as polyketides and alcohols [47]; and responsible for the degradation of the cell wall of fungi. This may be present in the inhibition process against *M. phaseolina* [48].

In Figure 2b,c, an interaction zone surrounding the mycelium of *M. phaseolina* could be observed. It could be rational to infer those volatile substances, enzymes that degrade the cell wall (Chitinase and Glucanase) and a large amount of antibiotics [49,50], exert a greater inhibition on the growth and development of "PUE 4.0" strain. In this sense, the strain of *T. koningiopsis* (T-K11) exerted a greater inhibition of radial growth (PICR) on *M. phaseolina* from the tenth day and a class I classification, according to the scale established by Bell [34]. Ruangwong [51] mentions that *T. koningiopsis* (PSU3-2) contains azetidine, 2-phenylethanol, and ethyl hexadecanoate; compounds that may be associated with antibiosis and suppression of growth on the mycelial of *M. phaseolina* in the present investigation.

The biological control of *M. phaseolina* under field conditions through different isolates of *Trichoderma* reduced the charcoal rot for the peanut crop. In addition, it was possible to observe reddish brown lesions in the total surface of the root in the group of plants inoculated only with "PUE 4.0" strain, characteristic symptoms of *M. phaseolina* mentioned by Ghosh [9] for the peanut crop, and Etebarian [52] for the melon crop.

Of the different microbial antagonists used in the present investigation, *T. koningiopsis* (T-K11) and *T. harzianum* (native) showed more efficient results in reducing charcoal rot and the incidence of the disease. The same finding was found by Hussain [22] and Khan [53], who observed that *T. harzianum* is effective in controlling *M. phaseolina* for mung beans. Likewise, Dubey [54] observed that *T. harzianum* increased the frequency of healthy plants, results similar to those obtained in the present investigation by *T. koningiopsis* (T-K11), which managed to reduce the incidence of charcoal rot by 76% in the field.

If we consider the average of China as the largest peanut producer in the world (1.45 t/ha−1), the average yield obtained in the present investigation with *T. koningiopsis* (T-K11) showed higher production (1.60 ± 0.01 t/ha−1) [3]. This can be explained by the fact that the genus *Trichoderma* can inhibit the growth of pathogenic fungi and exert positive effects on the absorption of nutrients, plant growth, and yield, in addition to protecting them against biotic and abiotic stress. [55–57]. In a study presented by Osman [58], it was observed that *T. harzianum* alone and combined with yeast improved the yield and quality of peanuts under field conditions. On the other hand, Dania [59], found the highest number of peanut pods per plant (15.67) in a combination of *T. hamatum* and cattle manure, results similar to those obtained in the present investigation by *T. koningiopsis* (16.01) and *T. harzianum* (15.35).
