Botrytis cinerea is the causal agent of gray mold, a disease that attacks many plants worldwide. This phytosanitary problem was reported in orchards in the State of Mexico, Mexico, specifically in pomegranate crops. This study isolated this phytopathogen from fruits collected in the municipality of Chilcuautla, state of Hidalgo, Mexico, indicating that the disease is spreading across the country. Two strains of phytopathogens, MIC and MID, were obtained after conducting Koch’s postulates. These strains were identified molecularly by amplifying and analyzing the concatenated sequence of the regions that correspond to the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (G3PDH), and DNA-dependent RNA polymerase subunit II (RPB2). In vitro confrontation tests demonstrated that Bacillus velezensis 160 inhibited the development of both strains of B. cinerea under study through the action of nonvolatile, thermostable, and volatile metabolites. Applying this bacterium to pomegranate orchards for two consecutive years (2022 and 2023) decreased the incidence, severity, and progression of the disease. We conclude that this bacterium can be used as a fungicide to control gray mold in pomegranate crops. [ ] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Gray mold disease, caused by the fungus Botrytis cinerea, affects a wide variety of plants. In this study, we conducted several in vitro tests and genomic analyses on three strains of this fungus (BcPgIs-1, BcPgIs-3, MIC) previously isolated from diseased pomegranate fruits, collected at two geographic locations in Mexico. Our goal was to identify possible differences among these strains. The development of the three strains in distinct culture media, the production of extracellular enzymes, and their effect on the progression of infection in pomegranate fruits were evaluated. The genomes were sequenced using the Illumina platform and analyzed with various bioinformatics tools. All strains possess genetic determinants for virulence and cell wall polymer degradation, but MIC exhibited the highest pectinolytic activity in vitro. This strain also produced sclerotia in a shorter time (7 days) in PDA medium. BcPgls-3 demonstrated the highest conidia production across all the culture media used. Both BcPgls-3 and MIC damaged all the pomegranate fruits 8 days after inoculation, while the BcPgls-1 required up to 9 days. Sequencing of the three strains yielded high-quality sequences, resulting in a total of 17 scaffolds and genomes that exceed 41 million bp, with a GC content of approximately 42%. Phylogenomic analysis indicated that the MIC strain is situated in a group separate from BcPgIs-1 and BcPgIs-3. BcPgIs-3 possesses more coding sequences, but MIC has more genes for CAZymes and peptidases. The three strains share 10,174 genes, while BcPgIs-3 and MIC share 851. These findings highlight the differences among the strains studied, which may reflect their adaptive capacities to their environment. Results contribute to our understanding of the biology of gray mold in pomegranates and could assist in developing more effective control strategies.