Botrydial (BOT) is a non-host specific phytotoxin produced by the polyphagous phytopathogenic fungus Botrytis cinerea. The genomic region of the BOT biosynthetic gene cluster was investigated and revealed two additional genes named Bcbot6 and Bcbot7. Analysis revealed that the G + C/A + T-equilibrated regions that contain the Bcbot genes alternate with A + T-rich regions made of relics of transposable elements that have undergone repeat-induced point mutations (RIP). Furthermore, BcBot6, a Zn(II)(2)Cys(6) putative transcription factor was identified as a nuclear protein and the major positive regulator of BOT biosynthesis. In addition, the phenotype of the Delta Bcbot6 mutant indicated that BcBot6 and therefore BOT are dispensable for the development, pathogenicity and response to abiotic stresses in the B. cinerea strain B05.10. Finally, our data revealed that B. pseudocinerea, that is also polyphagous and lives in sympatry with B. cinerea, lacks the ability to produce BOT. Identification of BcBot6 as the major regulator of BOT synthesis is the first step towards a comprehensive understanding of the complete regulation network of BUT synthesis and of its ecological role in the B. cinerea life cycle.