Development of species-specific PCR detection for three Mycogone species causing wet bubble disease in white button mushroom

Abstract

Agaricus bisporus, commonly known as white button mushroom, is a major agricultural crop worldwide. However, wet bubble disease in Agaricus bisporus, caused by three Mycogone species, namely Mycogone perniciosa, M. rosea and M. xinjiangensis, leads to tremendous economic losses in China. The three species are difficult to distinguish based on morphology and pathogenicity. An early, fast and accurate method to detect and differentiate among these species is essential for controlling the disease. Based on differences in Tef-1α gene sequences, therefore, three sets of species-specific primers, T-6-3/AT-4, T-2/AT-2 and T-7-1/AT-3, were successfully designed for the detection and identification of M. perniciosa, M. rosea and M. xinjiangensis, respectively, using an endpoint PCR assay. Subsequently, the specificities of the three primers were demonstrated using 43 isolates, including Mycogone spp., A. bisporus, Trichoderma harzianum, Trichothecium roseum, Cladobotryum dendroides, Lecanicillium fungicola, Fusarium solani and other soil-borne pathogens. The detection limits for M. perniciosa, M. rosea and M. xinjiangensis were 0.5 pg/μL, 0.2 ng/μL and 20 pg/μL of DNA template, respectively, suggesting strong amplification with the three primer pairs. Moreover, the species-specific primers efficiently detecteerd the target species in artificially inoculated mushrooms and soils. This is the first report to identify and distinguish each of the three Mycogone species. Besides, this PCR-based molecular method could provide a sensitive, quick and highly specific tool to discriminate among the three Mycogone species. Collectively, the assay developed in this study could have great application value for early detection of Mycogone species in soil and effective control of wet bubble disease.