Volume 17 Part 1 Article 42: A Nested PCR for the Detection of Mycogone Perniciosa Causing Wet bubble Disease of White Button Mushrooms

Volume 17 Part 1 Article 42
Year 2008
Title: A Nested PCR for the Detection of Mycogone Perniciosa Causing Wet bubble Disease of White Button Mushrooms
Authors: L. Meyer and L. Korsten


Wet bubble disease of white button mushrooms (Agaricus bisporus (Lange) Imbach) is caused by the fungus Mycogone perniciosa (Magnus) Delacroix and has been recorded worldwide in button mushroom production. This contagious disease is difficult to eradicate, since chlamydospores can survive for more than three years in various substrates. A comprehensive knowledge of the dispersal, survival and primary inoculum sources of the pathogen will add to effective control and eradication of the pathogen. A nested polymerase chain reaction (PCR) test on different mushroom material was developed to unequivocally identify the wet bubble disease pathogen. This includes the isolation of DNA from various mushroom samples and the design of a primer set based on internal transcribed spacer (ITS) sequences. A portion of the ITS region of the ribosomal DNA operon was amplified using universal primers. These fragments were sequenced and the data used to design a primer set MYC5-MYC2R. In order to confirm the specificity of the primers, they were analysed using the BLAST program, and sequenced isolates were used to evaluate the efficacy of the primer set. Other isolates, including known fungal contaminants and direct DNA isolations from mushroom tissue, were included in the evaluation process. The consistent positive identification found in this study proves the sensitivity of these primers in detecting the presence of Mycogone in mushroom tissue. It is now possible to extract DNA directly from casing material and to perform the PCR successfully with very specific and robust M. perniciosa primers all within a day. The primer set and test method described in this paper provides an effective, rapid, accurate protocol for one-day analysis of mushroom material suspected of wet bubble disease infection.

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