Comparing Approaches for Capturing Bacterial Assemblages Associated with Symptomatic (Bacterial Blotch) and Asymptomatic Mushroom (Agaricus bisporus) Caps

Bacterial blotch is a complex of diseases, caused by Pseudomonas tolaasii and other Pseudomonas spp., whose growth may be influenced by other microorganisms associated with mushroom caps. The objectives of this work were to (i) compare methods for extracting DNA from mushroom caps to assess bacterial composition, and (ii) describe and quantify the composition of bacteria in and on symptomatic and asymptomatic mushroom caps. Symptomatic and asymptomatic mushrooms were collected at an organic farm in Pennsylvania and processed. Bacteria were removed from mushrooms by sonication to target surface-associated bacteria (washate), from whole mushrooms postsonication to target bacteria within or tightly adhering to the mushrooms (post-washate), or from whole macerated mushrooms to target all mushroom-associated bacteria in one sample (whole). Bacterial composition was determined through sequencing the V4 hypervariable region of the 16S rRNA gene on an Illumina MiSeq. Bacterial composition differed significantly based on mushroom health status and community removal method. Regardless of sampling approach, Proteobacteria and Bacteroidetes appeared as the most dominant phyla. In comparing the three extraction methods, higher Shannon diversity and Pielou’s evenness were observed for the washate and post-washate methods compared with whole mushroom sampling. Additionally, higher Shannon diversity, operational taxonomic unit richness, and Pielou’s evenness were found for the asymptomatic mushrooms when compared with symptomatic mushrooms. Pseudomonas spp. were overrepresented on mushroom surfaces (washate method), regardless of health status. This research made strides in describing the mushroom cap microbiome and established sampling methods that will allow researchers to target studies and isolation of specific bacterial taxa.