Dothistromin is a broad-spectrum mycotoxin produced by the Dothideomycete pine needle pathogen Dothistroma septosporum. It accumulates in lesions, causing characteristic red bands on needles infected with this fungus. Dothistromin is similar in structure to the aflatoxin precursor versicolorin B and the biosynthetic pathways of these toxins share many common gene products. Although dothistromin is not essential for pathogenicity in dothistroma needle blight, its presence in infected needles suggests it might have a role in the disease process. We tested the hypothesis that dothistromin is a virulence factor by studying Pinus radiata infected with dothistromin-deficient mutants of D. septosporum. The mutants were able to infect pine needles and complete their life cycle as previously shown, and were unaffected in spore germination, epiphytic growth or needle penetration. However, colonization of the mesophyll by the mutants was restricted compared to the wild type. Correspondingly, lesions produced by the mutants were smaller and produced significantly fewer spores than lesions produced by wild-type strains. Interestingly, ‘green islands’ in which chlorophyll was maintained at a higher level than in adjacent chlorotic and necrotic regions, surrounded early-appearing lesions caused by both wild-type and mutant strains. At a later stage of disease development green islands were still present in the mutant but appeared to be masked by the extended dothistromin-containing lesions in the wild type. Overall the results support a role for dothistromin in virulence in dothistroma needle blight.This article is protected by copyright. All rights reserved.