A reliable in vitro fruiting system for Armillaria mellea for evaluation of Agrobacterium tumefaciens transformation vectors

• We established a reliable in vitro fruiting method for heterothallic Armillaria mellea.
• Temperature decrease is crucial for primordia induction.
• We constructed a versatile Agrobacterium tumefaciens transformation vector.
• Truncated and full length hph genes conferred hygromycin resistance in A. mellea.
• The Phanerochaete chrysosporium gpd promoter was able to drive hph in A. mellea.

Armillaria mellea is a serious pathogen of horticultural and agricultural systems in Europe and North America. The lack of a reliable in vitro fruiting system for heterothallic A. mellea has hindered research and required dependence on intermittently available wild-collected basidiospores of endemic genotypes, necessitating the use of variable genetic material in transformation studies. Here we describe a reliable, reproducible in vitro fruiting method for heterothallic A. mellea from the western US. Isolates and growth conditions were evaluated to determine effective fruiting conditions. Following medium colonisation for 4 weeks, cultures were incubated under warm/bright conditions for 4–6 weeks before incubation in dim/cool conditions. Primordia emerged within 3–4 weeks following a temperature decrease and this was most efficient when coupled with a light reduction. Basidiocarps matured within 3–4 weeks and produced viable basidiospores. Agrobacterium tumefaciens and vectors were evaluated by transformation of in vitro-produced basidiospores and a versatile transformation vector was constructed to simplify promoter and marker gene exchange using homologous recombination in yeast. Fruiting bodies and viable basidiospores of A. mellea have been reliably produced in vitro which, coupled with the enhanced knowledge of suitable A. tumefaciens strains and vectors for transformation, will assist future genetic research into this important pathogen.