An improved sequence-aided T-RFLP analysis of bacterial succession during oyster mushroom substrate preparation

While oyster mushroom (Pleurotus spp.) is one of the most popular cultivated edible mushrooms, there is scanty information about the microbial community taking part in mushroom substrate production. In this study, an improved sequence-aided terminal restriction fragment length polymorphism (T-RFLP) was used to identify and (semi-)quantify the dominant bacteria of oyster mushroom substrate preparation. The main features of the improved T-RFLP data analysis were the alignment of chromatograms with variable clustering thresholds, the visualization of data matrix with principal component analysis ordination superimposed with cluster analysis, and the search for stage-specific peaks (bacterial taxa) with similarity percentage (analysis of similarity) analysis, followed by identification with clone libraries. By applying this method, the dominance of the following bacterial genera was revealed during oyster mushroom substrate preparation: Pseudomonas and Sphingomonas at startup, Bacillus, Geobacillus, Ureibacillus, Pseudoxanthomonas, and Thermobispora at the end of partial composting, and finally several genera of Actinobacteria, Thermus, Bacillus, Geobacillus, Thermobacillus, and Ureibacillus in the mature substrate. As the proportion of uncultured bacteria increased during the process, it is worth establishing strain collections from partial composting and from mature substrate for searching new species.