Identification of contaminants as members of the Bacillus pumilus group and analysis of contamination sources in the cultivation of Pleurotus geesteranus mushrooms

Oyster mushroom (Pleurotus geesteranus Singer) is grown widely in China withut problems, but occasionally severe bacterial contamination occurred in cultivation bags of Pleurotus geesteranus during high summer temperatures in some companies in the Chunan county of Zhejiang province in 2018, belonging to the so-called Bacillus pumilus group by morphological characteristics, 16S rRNA, GyrB and aroE genes sequencing, fatty acid analysis and MALDI analysis. The inoculation tests confirmed that Bacillus pumilus strains were contaminants causing mycelial disappearance of P. geesteranus. The dual culture tests demonstrated that several strains of B. pumilus were able to inhibit mycelial growth of P. geesteranus. In order to find the sources of contamination the, process of mushroom cultivation was investigated. A 41.0% contamination rate occurred in cultivation bags with sponge plugs, as opposed to a very low contamination rate (<4%) in cultivation bags with cotton plugs. It was also shown that a 13.0% bacterial isolation rate was obtained from autoclaved sponge pieces, as opposed to 2.0% or 0.0% from cotton pieces, respectively. Fifteen strains isolated all were identified by MALDI-Biotyper as Bacillus spp., belonging to four species (Bacillus circulans, B. pumilus, B.cereus and B. halosaccharovorans). Dual culture tests showed that B. circulans, B. cereus and B. halosaccharovorans strains had not inhibitory effect on mycelial growth of P. geesteranus. This study furthermore shows that sponge plugs containing B. pumilus contaminants were the contamination source in cultivation bags of P. geesteranus.