Following Agrobacterium tumefaciens-mediated mutagenesis in Leptosphaeria maculans, we identified the mutant 210, displaying total loss of pathogenicity towards its host plant (Brassica napus). Microscopic observations showed that m210 is unable to germinate on the host leaf surface and is thus blocked at the pre-penetration stage. The pathogenicity phenotype is linked with a single T-DNA insertion into the promoter region of a typical plasma membrane H(+)-ATPase-encoding gene, termed Lmpma1, thus leading to a twofold reduction in Lmpma1 expression. Since LmPMA1 is involved in intracellular pH homeostasis, we postulate that reduction in LmPMA1 activity disturbs the electrochemical transmembrane gradient in m210, thus leading to conidia defective in turgor pressure generation on leaf surface. Whole genome survey showed that L. maculans possesses a second plasma membrane H(+)-ATPase-encoding gene, termed Lmpma2. Silencing experiments, expression analyses and phylogenetic studies allowed us to highlight the essential role assumed by the Lmpma1 isoform in L.maculans pathogenicity.