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Brown spot disease, caused by C ochliobolus miyabeanus, is currently considered to be one of the most important yield reducers of rice ( O ryza sativa L.). Despite its agricultural importance, little is known about the virulence mechanisms deployed by the fungus. Therefore, we set out to identify novel virulence factors with a role in disease development. This article reports, for the first time, the production of tentoxin by C . miyabeanus as a virulence factor during brown spot disease and the identification of the non-ribosomal protein synthetase ( NRPS) CmNps3, responsible for tentoxin biosynthesis. We compared the chemical compounds produced by C . miyabeanus strains differing in virulence ability using ultra-high-performance liquid chromatography ( UHPLC) coupled to high-resolution Orbitrap mass spectrometry ( HRMS). The production of tentoxin by a highly virulent strain was revealed by principal component analysis of the detected ions and confirmed by UHPLC coupled to tandem-quadrupole mass spectrometry ( MS/ MS). The corresponding NRPS was identified by in silico genome analysis and confirmed by gene deletion. Infection tests with wild-type and C mnps3 mutants showed that tentoxin acts as a virulence factor and is correlated with chlorosis development during the second phase of infection. Although rice has previously been classified as a tentoxin-insensitive plant species, our data demonstrate that tentoxin production by C . miyabeanus affects symptom development. [ABSTRACT FROM AUTHOR]