Metaprofiling of the Bacterial Community in Colonized Compost Extracts by Agaricus subrufescens

Abstract

It is well-known that bacteria and fungi play important roles in the relationships between mycelium growth and the formation of fruiting bodies. The sun mushroom, Agaricus subrufescens, was discovered in Brazil ca. 1960 and it has become known worldwide due to its medicinal and nutritional properties. This work evaluated the bacterial community present in mushroom-colonized compost extract (MCCE) prepared from cultivation of A. subrufescens, its dynamics with two different soaking times and the influence of the application of those extracts on the casing layer of a new compost block for A. subrufescens cultivation. MCCEs were prepared through initial submersion of the colonized compost for 1 h or 24 h in water followed by application on casing under semi-controlled conditions. Full-length 16S rRNA genes of 1 h and 24 h soaked MCCE were amplified and sequenced using nanopore technology. Proteobacteria, followed by Firmicutes and Planctomycetes, were found to be the most abundant phyla in both the 1 h and 24 h soaked MCCE. A total of 275 different bacterial species were classified from 1 h soaked MCCE samples and 166 species from 24 h soaked MCCE, indicating a decrease in the bacterial diversity with longer soaking time during the preparation of MCCE. The application of 24 h soaked MCCE provided increases of 25% in biological efficiency, 16% in precociousness, 53% in the number of mushrooms and 40% in mushroom weight compared to control. Further investigation is required to determine strategies to enhance the yield and quality of the agronomic traits in commercial mushroom cultivation.

Keywords: 16S rDNA; Agaricus blazei; metagenomics; microbiomics; mushroom production; nanopore sequencing.

Figure 1

Steps involved in the preparation of mushroom-colonized compost extract (MCCE) and its addition over the casing of compost blocks for the production of Agaricus subrufescens. The fully colonized compost block with the mycelium was ground by hand and soaked in water for 1 h or 24 h (step 1), followed by filtration (step 2), dilution and storage of the MCCE in the refrigerator (steps 3 to 5). Each bottle obtained with 1 h or 24 h soaking time was then added to another colonized compost block in the casing on the 3rd, 6th, 9th and 15th days after casing (step 6). Full-length 16S rRNA genes of the 1 h and 24 h soaked MCCEs were amplified and sequenced using nanopore technology (step 7). A. subrufescens mushrooms were grown in a chamber under semi-controlled conditions (steps 8 and 9).

Figure 2

Sankey visualization of taxonomic classification at domain (D), phylum (P), class (C), order (O), family (F), genus (G) and species (S) levels based on the 16S rRNA gene amplicons from samples of mushroom-colonized compost extracts (MCCEs) soaked in water for either 1 h (A) or 24 h (B).

Figure 3

Most abundant phyla (A) and genera (B) of bacteria present in the Agaricus subrufescens mushroom-colonized compost extracts (MCCE) soaked in water for either 1 h or 24 h. Others figures refer to Bacteroidetes, BRC 1, Chloroflexi, Cyanocateria, OD 1 (genera) and Verrucomicrobia (phyla).

Figure 4

Agronomic parameters of mushrooms cultivated with the control and application of 1 h- and 24 h soaked MCCE over the casing, which (A) indicate biological efficiency, (B) precociousness, (C) number of mushroom, and (D) weight of mushroom.

Figure 5

Biological efficiency of the Agaricus subrufescens mushroom cultivation after the application of 1 h or 24 h soaked MCCE on the 3rd, 6th, 9th, 12th and 15th days after casing. The red line corresponds to the control treatment. The highlighted red area includes the control mean value and standard deviation (20 ± 5%, n = 4).