A reliable in vitro fruiting system for armillaria mellea for evaluation of agrobacterium tumefaciens transformation vectors

Authors: FORD, KATHRYN - University Of Bristol; Baumgartner, Kendra; HENRICOT, BEATRICE - Royal Horticultural Society; BAILEY, ANDY - University Of Bristol; FOSTER, GARY - University Of Bristol

Submitted to: Fungal Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/22/2015
Publication Date: 6/30/2015
Publication URL: http://dx.doi.org/10.1016/j.funbio.2015.06.007
Citation: Ford, K.L., Baumgartner, K., Henricot, B., Bailey, A.M., Foster, G.D. 2015. A reliable in vitro fruiting system for armillaria mellea for evaluation of agrobacterium tumefaciens transformation vectors. Fungal Biology. 119:859-869. doi: 10.1016/j.funbio.2015.06.007.

Interpretive Summary: Armillaria mellea is a serious fungal pathogen of horticultural and agricultural plants in Europe and North America. We are unable to cultivate the mushrooms in the laboratory, and this limits the types of studies we can do on the spread of the fungus. Here we describe a reliable, reproducible fruiting method to produce mature mushrooms in the laboratory. We evaluated different isolates of the fungus from around the world, which were grown on different mixtures of grains, sawdust, and fruit. Then they were grown under different light and temperature conditions to determine which combinations of growth medium and conditions produced mushrooms. Following colonisation of rice, sawdust and tomato medium at room temperature for four weeks, cultures were incubated in growth rooms under warm/bright conditions (four to six weeks) before being placed in dim/cool conditions. Mushrooms began to emerge within three to four weeks following a temperature decrease of at least 8°C, and this was most efficient when coupled with a light reduction. Mushrooms matured within three to four weeks, and produced viable spores. Strains of the bacterium Agrobacterium tumefaciens and transformation vectors were constructed and evaluated by transformation of spores form the lab-produced mushrooms. A versatile transformation vector, producing transformation efficiencies similar to the previous vector pBGgHg, was constructed to simplify promoter and marker gene exchange using the yeast homologous recombination system. In addition, mushrooms were induced in cultures established from spores of these transgenic isolates. This reliable procedure for mushroom cultivation fills an important gap in the suite of study tools available for Armillaria research.

Technical 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 has hindered research, and necessitated dependence on intermittently available wild-collected basidiospores. Here we describe a reliable, reproducible fruiting method to produce mature basidiocarps with viable basidiospores. We evaluated isolates (heterothallic vs. homothallic, wild-type vs. transgenic), media, and light and temperature conditions to determine the most effective conditions. Following colonisation of rice, sawdust and tomato medium at room temperature for four weeks, cultures were incubated in growth rooms under warm/bright conditions (four to six weeks) before being placed in dim/cool conditions. Primordia emerged within three to four weeks following a temperature decrease of at least 8°C and this was most efficient when coupled with a light reduction. Basidiocarps of both heterothallic and homothallic isolates matured within three to four weeks, and produced viable basidiospores. Strains of Agrobacterium tumefaciens and transformation vectors containing the hygromycin resistance gene (hph) were constructed and evaluated by transformation of in vitro-produced basidiospores from a heterothallic isolate. A versatile transformation vector, producing transformation efficiencies similar to the archetypal basidiomycete vector pBGgHg, was constructed to simplify promoter and marker gene exchange using the yeast homologous recombination system. In addition, fruiting was induced in mycelial cultures established from transgenic fruiting bodies. This reliable in vitro fruiting system fills an important gap in the suite of study tools available for Armillaria genetics and pathogenesis.