SUMMARY Sirodesmin PL is a non-host-selective phytotoxin produced by Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus). Previous studies have shown that sirodesmin PL biosynthesis involves a cluster of 18 co-regulated genes and that disruption of the two-module non-ribosomal peptide synthetase gene (sirP) in this cluster prevents the production of sirodesmin PL. Loss of sirodesmin PL did not affect the growth or fertility of the sirP mutant in vitro, but this mutant had less antibacterial and antifungal activity than the wild-type. When the sirP mutant was inoculated on to cotyledons of B. napus, it caused similar-sized lesions on cotyledons as the wild-type isolate, but subsequently caused fewer lesions and was half as effective as the wild-type in colonizing stems, as shown by quantitative PCR analyses. However, no significant difference was observed in size of lesions when either wild-type or mutant isolates were injected directly into the stem. The expression of two cluster genes, sirP and an ABC transporter, sirA, was studied in planta. Fungal isolates containing fusions of the green fluorescent protein gene with the promoters of these genes fluoresced after 10 days post-inoculation (dpi). Transcripts of sirP and sirA were detected after 11 dpi in cotyledons by reverse transcriptase PCR, and expression of both genes increased dramatically in stem tissue. This expression pattern was consistent with the distribution of sirodesmin PL in planta as revealed by mass spectrometry experiments.