Biological Control Of Rhizopus Stolonifer In The Cultivation Of Flammuilna Velutipes

During cultivation of Flammulina velulipes in the factory, sometimes there is a kind of fungus growing in the medium, which can contaminate F. velutipes and reduce the production of F. Velulipes. Through purification, we got a mold and named it R1. When growing on PDA medium, the characteistics of fungus R1 was observed. Its loose and transparent mycelium radiate to the surrounding in beginning.In interim the white immauature conidia sac is gradually formed in the top of the mycelium. Later, the color of conidia sac turns black which means that the spores become mature. Through morphology and ITS sequence analysis, this fungi is identified as Rhizopus stolonifer.Now there is little research about the prevention and treatment of R. stolonifer using the biological method. We screened a bacteria from the air of develop room by the means of plate sedimentation method. This bacteria can inhibit the growth of R. stolonifer when cultured together. And its metabolites exhibit a strong inhibitory effect on R. stolonifer, Neurospora crassa, Trichoderma viride and Mucor. It indicates that the metabolites has a broad-spectrum antimicrobial activity. We named the bacteria B26. Through observe the strain’s morphology characteistics, physiological and biochemistry experiment and identify the strain’s gyrA and 16SrDNA sequence, the results indicate that strain B26 is Bacillus amyloliquefaciens.We get the metabolites produced by B. amyloliquefaciens B26 by the means of acid precipitation and methanol extraction. We found the metabolites can withstand higher temperature, indicates that it has a excellent temperature stability. Through silica gel column chromatography, HPLC purification and HPLC-MS identification, we obtained two active ingredients from the metabolites produced by B. amyloliquefaciens B26, and the molecular weights were 1044.547 and 1058.567. This two components were preliminary identified as Bacillomycin D with C15 or C16, which belongs to iturin.In 300 μg/mL, the metabolites produced by B. amyloliquefaciens B26 can respectively inhibit the growth of R. stolonifer, T. viride, N. crassa and F. velutipes at the rate of 70%, 61%,81% and 31%. In the fruiting period, it can inhibit the growth of R. stolonifer while ensuring the normal fruiting of F. velutipes by spraying 300 μg/mL metabolites crude extract three times. When thiophanate methyl is in the concentration of 2.4 mg/mL, it can inhibit the growth of R. stolonifer at the rate of 69.17%. At the concentration of 10.842 mg/mL, Methylpartricin Sodium Lauryl sulfate damage the growth of R. stolonifer at the rate of 69.83%. At the concentration of 333.6 μg/mL, chlorothalonil suppress the growth of R. stolonifer at the rate of 71.24%. While the three pesticide thiophanate methyl, Methylpartricin Sodium Lauryl sulfate and chlorothalonil inhibit R. stolonifer also affect the growth the Flammulina velutipes strongly. Therefore the metabolites produced by Bacillus amyloliquefaciens B26 has the better efficacy than 3 kinds of chemical fungicide.