*2.1. Area of Isolation*

Vegetable tissue samples with rot at the stem and root level were collected in a plot of 3144.3 m<sup>2</sup> of peanut crop with a history of high charcoal rot incidence [7] during the summer–fall 2020 production. Agricultural plot corresponds to the Buenavista de Benito Juárez community, belonging to the municipality of Chietla in the state of Puebla-Mexico, with a warm desert climate (Bwh) and average rainfall of 700 mm [23]. Sampling was directed towards individuals with symptoms associated with genus *Macrophomina*; all samples were kept in plastic bags in a cooler until they were transferred to laboratory, to be processed.

The samples were cut into small 5 mm pieces, disinfected with 1% sodium hypochlorite for 3 min, and washed with sterile water. Finally, they were wrapped with sterile paper towels and placed in a laminar flow chamber at 20 ◦C for 15 min [24]. Subsequently, the samples were placed upright in Petri dishes with potato dextrose agar medium (PDA, Dioxon) modified with chloramphenicol (20 mg/mL<sup>−</sup>1) and incubated at 28 ◦C for 5 days. The identification of fungal colonies associated with the genus *Macrophomina* was carried out by the observation of reproductive structures under a microscope and employing taxonomic keys of Barnett and Hunter [25]. For the microscopic observation, thin layer PDA cultures (microculture technique) were used. The mycelial cultures were observed

after eight days of incubation at 28 ◦C employing lactophenol. The microscopic morphology of the fungi was examined under an optic microscope (Carl Zeiss, Jena, Germany) [26].
