The research aimed to determine the phenotypic characteristic, pathogenicity and molecular characterization of Hypomyces perniciosus isolates responsible for wet bubble disease of the white button mushroom Agaricus bisporus. Phenotypic characteristics such as colony appearance, mycelium texture, and pigmentation were studied on four different agar media, i.e., potato dextrose agar (PDA), malt extract agar (MEA), Czapek-Dox yeast agar (CYA), and Sabouraud dextrose agar (SDA), after eight days of incubation. Additionally, the growth rate of the tested isolates was studied depending on the pH of the medium. Fungal isolates showed the highest mycelial growth on MEA and SDA at pH 6.0. However, on CYA at pH 7.0, the mycelium exhibited the worst growth. Isolate identification and genetic relationship analysis were carried out using internal transcribed spacer region sequencing and the random amplified polymorphic DNA method. The research confirmed that all Polish isolates belong to the species H. perniciosus, and the genetic diversity is relatively low. Phylogenetic analyses revealed three subgroups of H. perniciosus isolates. The first group included three genetically distinct isolates with a similarity coefficient in the range of 0.76–0.85 to isolates CBS 815.73 and CBS 322.52. The second group was divided into two subgroups and included 16 isolates with a genetic similarity range of 0.91 to 1.0 to CBS 815.73 and CBS 322.52 isolates. Furthermore, the eight genetically similar isolates exhibited the greatest pathogenicity towards A. bisporus.
