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An Improved Sequence-aided T-RFLP Analysis of Bacterial Succession During Oyster Mushroom Substrate Preparation

  • Environmental Microbiology
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Abstract

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.

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Acknowledgments

This study was supported by the grant OMFB-00977/2005 from the Ministry of Agriculture and Rural Development (Hungary) and by the Hungarian Scientific Research Fund (OTKA K 83764). Balázs Vajna was supported by a scholarship from the Ministry of Education and Culture, Hungary (DFÖ 0054/2009).

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Correspondence to Balázs Vajna.

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Supplementary Figure 1aSupplementary Figure 1bSupplementary Figure 1c

Different ordination of the T-RFLP data sets. White, gray and black points mark stage 1, 3 and 7 samples, respectively. The superimposed groups derive from cluster analysis. Samples surrounded with dotted, dashed and continuous lines refer to groups derived from cluster analysis (UPGMA method, Bray–Curtis similarity) at 20 % similarity. (A) PCA, (B) CA, (C) NMDS based on Bray–Curtis similarities (GIF 34 kb)

(GIF 32 kb)

(GIF 39 kb)

High-resolution image (EPS 5,593 kb)

High-resolution image (EPS 4,657 kb)

High-resolution image (EPS 4,882 kb)

Supplementary Figure 2

Five parallel T-RFLP chromatograms of sample 6-061-7 with Hin6I digestion. At short fragments (on the left) the width of a peak corresponding to equivalent T-RFs (black-coloured) is much smaller than at longer fragments (on the right) (GIF 26 kb)

High-resolution image (EPS 12,128 kb)

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Vajna, B., Szili, D., Nagy, A. et al. An Improved Sequence-aided T-RFLP Analysis of Bacterial Succession During Oyster Mushroom Substrate Preparation. Microb Ecol 64, 702–713 (2012). https://doi.org/10.1007/s00248-012-0063-5

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  • DOI: https://doi.org/10.1007/s00248-012-0063-5

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