title: A reliable in vitro fruiting system for Armillaria mellea for evaluation of Agrobacterium tumefaciens transformation vectors.
abstract: 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.
authors.affiliation: School of Biological Sciences, Life Sciences Building, University of Bristol, 24 Tyndall Avenue, Bristol BS8 1TQ, United Kingdom. Electronic address: Kathryn.Ford@bristol.ac.uk.
authors.forename: Kathryn L
authors.initials: KL
authors.lastname: Ford
authors.affiliation: United States Department of Agriculture-Agricultural Research Service, 363 Hutchison Hall, University of California, One Shields Avenue, Davis, CA 95616, United States. Electronic address: K.Baumgartner@ucdavis.edu.
authors.forename: Kendra
authors.initials: K
authors.lastname: Baumgartner
authors.affiliation: The Royal Horticultural Society, Wisley, Woking, Surrey GU23 6QB, United Kingdom. Electronic address: Beatrice.henricot@forestry.gsi.gov.uk.
authors.forename: Béatrice
authors.initials: B
authors.lastname: Henricot
authors.affiliation: School of Biological Sciences, Life Sciences Building, University of Bristol, 24 Tyndall Avenue, Bristol BS8 1TQ, United Kingdom. Electronic address: Andy.Bailey@bristol.ac.uk.
authors.forename: Andy M
authors.initials: AM
authors.lastname: Bailey
authors.affiliation: School of Biological Sciences, Life Sciences Building, University of Bristol, 24 Tyndall Avenue, Bristol BS8 1TQ, United Kingdom. Electronic address: Gary.Foster@bristol.ac.uk.
authors.forename: Gary D
authors.initials: GD
authors.lastname: Foster
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D055392:Armillaria / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification; D048690:Fruiting Bodies, Fungal / Q000254:growth & development*; D018014:Gene Transfer Techniques; D022761:Gene Transfer, Horizontal; D005827:Genetics, Microbial / Q000379:methods*; D066298:In Vitro Techniques / Q000379:methods; D008027:Light; D009172:Mycology / Q000379:methods*; D013696:Temperature; D014170:Transformation, Genetic; D014481:United States
keywords: Basidiocarp; Basidiomycete; Fungal transformation; Honey fungus; Mushroom