Bacterial Profiling and Dynamic Succession Analysis of Phlebopus portentosus Casing Soil Using MiSeq Sequencing

Abstract

Phlebopus portentosus (Berk. and Broome) Boedijin is a popular edible mushroom found in China and Thailand. To date, P. portentosus is the only species in the order Boletales that can be successfully cultivated worldwide. The use of a casing layer or casing soil overlaying the substrate is a crucial step in the production of this mushroom. In this study, bacterial profiling and dynamic succession analyses of casing soil during the cultivation of P. portentosus were performed. One hundred and fifty samples were collected, and MiSeq sequencing of the V3-V4 region of the 16S rRNA gene was conducted. After performing a decontamination procedure, only 38 samples were retained, including 6 casing soil-originated samples (OS), 6 casing soil samples (FHCS) and 5 upper substrate samples (FHCU) from the period of complete colonization by mycelia; 6 casing soil samples (PCS) and 5 upper substrate samples (PCU) from the primordium period; and 6 casing soil samples (FCS) and 4 upper substrate samples (FCU) from fruit body period. The results revealed that bacterial diversity increased sharply from the hyphal to the primordium stage and then decreased during harvesting. The non-metric multidimensional scaling (NMDS) ordination and analysis of similarities (ANOSIM) analysis suggested that the community composition during different stages was significantly different in casing soil. The most abundant phyla in all of the samples were Proteobacteria, Chloroflexi, Acidobacteria, Actinobacteria, Saccharibacteria, and Bacteroidetes. Burkholderia was the most abundant genus in all the samples except the OS samples. The relative abundance of Burkholderia in the FHCS samples (55.79%) decreased to 35.14% in the PCS samples and then increased to 45.60% in the FCS samples. The abundances of Acidobacterium, Rhizobium, Acidisphaera, Bradyrhizobium, and Bacillus increased from the FHCS to PCS samples. The linear discriminant analysis (LDA) effect size (LEfSe) suggested that Acidobacterium and Acidisphaera are micromarkers for PCS, whereas Bradyrhizobium, Roseiarcus, and Pseudolabrys were associated with fruit body stages. The network analyses resulted in 23 edges, including 4 negative and 19 positive edges. Extensive mutualistic interactions may occur among casing soil bacteria. Furthermore, these bacteria play important roles in mycelial elongation, primordium formations, and the production of increased yields.

Keywords: MiSeq; bacterial diversity; casing soil; decontamination; dynamics.

Figure 1

The P. portentosus cultivation process.

Figure 2

The cultivation of P. portentosus in the three treatments. (A) No covering casing soil; (B) sterilized casing soil; (C) normal covering casing soil.

Figure 3

Microbial diversity and richness. The Shannon and ACE indices were calculated at a depth of 7,096 reads in each sample. The bars indicate the mean; the error bars denote standard deviation; and the lowercase letters above the bars indicate significant differences. FHCS, casing soil from stage V (casing soil completely colonized by hyphae); PCS, casing soil from stage VI (primordial stage); FCS, casing soil from stage VII (fruiting body stage); FHU, upper portion of the substrates from stage V; PU, upper portion of the substrates from stage VI; FU, upper portion of the substrates from stage VII. (A) Shannon, (B) ACE.

Figure 4

Non-metric multidimensional scaling (NMDS) ordination of the bacterial community composition at different stages of P. portentosus cultivation. Ellipses in the plots denote 95% confidence intervals for the centroids of the CS, CU, and OS samples. FHCS: casing soil from stage V (casing soil completely colonized by hyphae); PCS, casing soil from stage VI (primordial stage); FCS, casing soil from stage VII (fruiting body stage); FHU, upper portion of the substrates from stage V; PU, upper portion of the substrates from stage VI; FU, upper portion of the substrates from stage VII. (A) NMDS for CS, CU and OS, (B) NMDS for FCS, FHCS, OS and PCS, (C) NMDS for FCU, FHCU and PCU.

Figure 5

The dynamics of some phyla in the casing soil and upper substrate samples during the development of P. portentosus. (A) Proteobacteria; (B) Chloroflexi; (C) Acidobacteria; (D) Actinobacteria; (E) Saccharibacteria; (F) Bacteroidetes. CS, samples from casing soil; CU, samples from the upper substrate. FHCS, casing soil from stage V (casing soil completely colonized by hyphae); PCS, casing soil from stage VI (primordial stage); FCS, casing soil from stage VII (fruiting body stage); FHU, upper portion of the substrates from stage V; PU, upper portion of the substrates stage VI; FU, upper part of the substrates stage VII.

Figure 6

The dynamics of some genera in the casing soil and upper substrate samples during the development of P. portentosus. (A) Burkholderia; (B) Acidobacterium; (C) Acidobacteria; (D) Acidisphaera; (E) Thermogemmatispora; (F) Actinoallomurus; (G) Bradyrhizobium; (H) Bacillus; (I) Sphingomonas. CS, samples from the casing soil; CU, samples from the upper substrate.

Figure 7

Identification of crucial bacteria associated with different stages. LEfSe reports the taxonomic representation of statistically and biologically consistent differences between the OS, FHCS, PCS, and FCS bacterial communities. OS, original casing soil; FHCS, casing soil completed with colonized hyphae; PCS, casing soil during the primordial period; FCS, casing soil during the fruiting body period.

Figure 8

Microbial networks in the casing soil and upper substrate samples based on the top 20 OTUs. The red color edges were positive associations, and the blue edges were negative associations (p < 0.05). A connection indicates a strong positive Spearman’s correlation coefficient if p > 0.75 and a negative Spearman’s correlation coefficient occurred if p < −0.75.