Comparative chemical screening and genetic analysis reveal tentoxin as a new virulence factor in C ochliobolus miyabeanus , the causal agent of brown spot disease on rice

Summary 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 ) CmNps 3, 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 O rbitrap 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.