Succession and potential role of bacterial communities during Pleurotus ostreatus production

Abstract

There is an increasing interest in studying bacterial-fungal interactions (BFIs), also the interactions of Pleurotus ostreatus, a model white-rot fungus and important cultivated mushroom. In Europe, P. ostreatus is produced on a wheat straw-based substrate with a characteristic bacterial community, where P. ostreatus is exposed to the microbiome during substrate colonisation. This study investigated how the bacterial community structure was affected by the introduction of P. ostreatus into the mature substrate. Based on the results obtained, the effect of the presence and absence of this microbiome on P. ostreatus production in an experimental cultivation setup was determined. 16S rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) and amplicon sequencing revealed a definite succession of the microbiome during substrate colonisation and fruiting body production: a sharp decrease in relative abundance of Thermus spp. and Actinobacteria, and the increasing dominance of Bacillales and Halomonas spp. The introduced experimental cultivation setup proved the protective role of the microbial community against competing fungi without affecting P. ostreatus growth. We could also demonstrate that this effect could be attributed to both living microbes and their secreted metabolites. These findings highlight the importance of bacterial-fungal interactions during mushroom production.

Keywords: Pleurotus ostreatus; T-RFLP; amplicon sequencing; bacterial community structure; bacterial-fungal interactions; succession and spatial heterogeneity.

Figure 1.

Stages of the P. ostreatus cultivation process and the 11 sampling days. Sampling days are denoted with numbers and arrows on the horizontal axis. ‘NGS’ shows samples processed for amplicon sequencing. Stage 1: 1/1. vegetative growth; 1/2. fruiting body induction; 1/3. first flush and harvest; Stage 2: 2/1. second vegetative growth; 2/2. second fruiting body induction; 2/3. second flush and harvest; columns: biological efficiency/day in the production house; black line: temperature during the investigation period. Biological efficiency was calculated as ratio of weight of fresh fruiting bodies per weight of dried substrate on day 1. Figure modified from Bánfi et al. (2015) with the permission of Fungal Biology.

Figure 2.

Scheme of applied treatments and sampling regions in the experimental cultivation setup. (A), Scheme of applied treatments (see details in text and Supplementary Fig. 1) A: autoclaved; AW: autoclaved and sterile water injected; AA: autoclaved and inoculated with suspension of an antagonistic bacterium; AF: autoclaved and inoculated with cell-free filtrate of microbial suspension; AM: autoclaved and inoculated with microbial suspension; N: normal, untreated, not autoclaved. (B), Sampling regions at the end of the 1^st and 2^nd periods of experiment. ‘nc’: noncolonised region, ‘fh’: region of front hyphae, ‘col’: three-week colonised region sampled before induction of the fruiting bodies, ‘colio’: older colonised region sampled after induction of fruiting bodies, ‘colir’: recently colonised region sampled after induction of fruiting bodies.

Figure 3.

Bacterial community composition changes according to 16S rRNA gene based T-RFLP fingerprints during substrate colonisation and fruiting body production of P. ostreatus. (A) NMDS ordination using Bray-Curtis distances for samples. Different small symbols denote samples from different mushroom blocks (A–E). Filling of symbols is lightened from black to white in parallel with sampling date (day 1 to day 71). Blue filling shows samples subjected to pyrosequencing. Ellipses mark weekly mean ± SD area based on samples’ NMDS coordinates; whereas, arrows point from weekn mean to weekn+1 mean. Filling of ellipses and their respective sampling date label denote significantly different groups of samples according to CCDA method (Supplementary Fig. 3). (B) The same NMDS ordination as in Fig. 3A though only ellipses for each week are plotted. Red arrows show significantly fitted (p < 0.05) P. ostreatus lignocellulose-degrading enzyme activities of the same samples. Enzyme activity data are derived from Bánfi et al. (2015). Enzyme names are abbreviated: β-gluc- β-glucosidase, Cbh- cellobiohydrolase, Chit- N-acetyl-β-glucosaminidase, Lcc- laccase, and MnP- manganese peroxidase. (C) NMDS ordination comparing bacterial community changes in P. ostreatus substrate preparation production series (data derived from Vajna et al. 2012) and samples from the present study. Substrate preparation is the first part of the P. ostreatus mushroom production, where the mature substrate also used in this study was produced. Ellipses correspond to mean ± SD area as in the previous parts. Stages 1, 3 and 7 in darkening green colours correspond to (S1) chopped, wetted wheat straw, (S3) end of partial composting, and (S7) mature substrate (Fig. S2, Supporting Information). For samples from the present study, only weekly ellipses of Fig. 3A are plotted.

Figure 4.

Bacterial diversity based on 16S rRNA gene amplicon sequencing during substrate colonisation and fruiting body production of P. ostreatus(A) Observed number of OTUs and (B) Shannon diversity calculated from the OTU (defined at 97% similarity level) numbers.

Figure 5.

Phylogenetic composition of bacterial communities based on 16S rRNA gene amplicon sequencing during substrate colonisation and fruiting body production of P. ostreatus. Each node represents a taxon from kingdom to family. Tree topology is the same for every sample, whereas colour of the nodes goes from blue (100% relative abundance) through red to yellow (0% relative abundance) according to taxa abundance in each sample. Small numbers on the edges give relative abundance of the taxa, which follow the given edge. Only OTUs having >1% relative abundance at least in one sample were used for calculations. ‘unc’ stands for unclassified bacteria. The original size figure also complemented with genus level is available as Fig. S6 (Supporting Information).

Figure 6.

Effects of different treatments (for codes see Fig. 2A) on the (A) speed of P. ostreatus hyphal colonisation in the experimental cultivation setup and (B) contamination of the tubes after the end of the 1^st period. Values show estimated marginal means ± standard error calculated from the linear model. Upper letters show significantly different groups based on Tukey's post-hoc test.

Figure 7.

Enzymatic activities in the experimental cultivation setup: Effects of regions and different treatment types on the new PCA-derived summary variables in (A-C) 1^st and (D) 2^nd period. To understand which enzymes have a large impact on the respective principal components, see loadings in Table 3. ‘Treatment type’ was introduced based on LM for contamination and has two categories: treatments having higher (A, AA, and AW) and lower (AM, AF, and N) contamination ratios (For codes of the original treatments see Fig. 2A). Values show estimated marginal means ± standard error calculated from linear mixed-effects model. Upper letters show significantly different groups based on Tukey's post-hoc test. Lcc stands for laccase and MnP for manganese peroxidase.