Molecular and Microbiological Methods for the Detection and Measurement of Dry Bubble Disease caused by Lecanicillium (Verticillium) fungicola on Mushroom Farms

Agaricus bisporus (Lange) Imbach – button mushrooms is a commonly cultivated mushroom throughout Europe which has very significant agricultural production. Mushroom cultivation is a monoculture which is exposed to different pathogens and pests. The most economically significant mushroom pathogens is Lecanicillium fungicola, the causative agent of Dry Bubble disease. This mycoparasite is responsible for severe losses of cultivated mushrooms and can terminate all mushroom production. The purpose of this study was to investigate sources of dry bubble disease on mushroom farms using microbiological and molecular approaches. The main focus of the research was to develop a selective medium, to modify the existing selective method and molecular method – Real Time PCR for detection from samples originating from mushroom farms. The first task of this research was evaluate DNA extractions methods from pure cultures of L. fungicola and optimise PCR conditions using a know sets of primers. All tested DNA extraction method gave good genomic DNA useful for PCR. The next part of this research was identify and detect of L. fungicola in samples originating from mushroom farms. A PCR assay was developed and optimised for the detection of L. fungicola in casing soil and other mushroom farm debris. Four different methods were evaluated for the isolation of DNA from soil containing different concentrations of conidia of L. fungicola including manual extraction and commercially available kits. Only two methods succeeded extracted L. fungicola DNA from samples containing soil and casing. The primers for detection of L. fungicola designed by Zijlstra et al. (2007, 2008 and 2009) gave a 102 bp amplification product and this primer set was tested in PCR reactions for A. bisporus and other mushroom pathogens such as Cladobotryum mycophilum, Mycogone perniciosa and Trichoderma sp. and also Aspergillus fumigatus. On this research also was designed a selective primers for Lecanicillium fungicola detection from mushroom farms using ITS and MAT1-2-1 region. It was not possible to find truly specific primers for this purpose but some of the sets of primers generated can be used for in-vitro test for detection and identification L. fungicola from A. bisporus tissues. This study also succeeded in designing selective media for detection of L. fungicola from mushroom farm samples. Lecanicilium fungicola selective medium already exists (Rinker et al., 1993), but the growth of L. fungicola is very slow due to the inhibitive nature of the ingredients on fungal growth. A modified selective medium and novel selective medium were developed to enable rapid and consistent detection of L. fungicola from contaminated soil and casing samples after 6 days of incubation. Mushroom farms visits were performed for detection of L. fungicola from different locations and stages of the crop cycle from spawn running to 3rd flush. Lecanicillium fungicola was detected by microbiological tests using novel and modified selective media and molecular method Real Time PCR – TaqMan test using the above mentioned primers