Antagonistic microorganisms produce antimicrobials to inhibit the growth of competitors. Although water-soluble antimicrobials are limited to proximal interactions via aqueous diffusion, volatile antimicrobials are able to act at a distance and diffuse through heterogeneous environments. Here, we identify the mechanism of action of Muscodor albus, an endophytic fungus known for its volatile antimicrobial activity toward a wide range of human and plant pathogens and its potential use in mycofumigation. Proposed uses of the Muscodor species include protecting crops, produce, and building materials from undesired fungal or bacterial growth. By analyzing a collection of Muscodor isolates with varying toxicity, we demonstrate that the volatile mycotoxin, N-methyl-N-nitrosoisobutyramide, is the dominant factor in Muscodor toxicity and acts primarily through DNA methylation. Additionally, Muscodor isolates exhibit higher resistance to DNA methylation compared with other fungi. This work contributes to the evaluation of Muscodor isolates as potential mycofumigants, provides insight into chemical strategies that organisms use to manipulate their environment, and provokes questions regarding the mechanisms of resistance used to tolerate constitutive, long-term exposure to DNA methylation.
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