Inhibition And Destruction Of The Antifungal Protein Produced By Bacillus Licheniformis W10 To Sclerotinia Sclerotiorum And Control Of Rape Stem Rot By The Protein

Isolate W10 of Bacillus licheniformis and its culture filtrate were strongly antagonistic to Sclerotinia sclerotiorum causing rape stem rot. After the culture filtrate was precipitated with 30% amonium sulfate and dialysed, the crude antifungal protein was obtained. The hyphae of the pathogen were deformed and cracked after being treated by the antifungal protein, leading to cytoplasm leakage and electrical conductivity increase. The antifungal protein could significantly inhibit the mycelial growth of the pathogen. The mycelial growth was decreased by over 90% when the protein concentration was 100μg/mL. The sclerotial formation and germination could be inhibited by the antifungal protein. No sclerotia would be produced on the media supplemented with the protein concentration of 100μg/mL. The antifungal protein with the concentration of 200μg/mL could obviously hinder the ascospore germination and the tube stretch, with the inhibition rate of 46.0% and 65.6%, respectively.The toxicity tests of carbendazim and procymidone to S. sclerotiorum were taken in vitro. EC50 values of procymidone ranged from 0.0126μg/ml to 0.5662μg/mL. According to the frequency distribution of EC50 values of 32 wild isolates, the sensitivity baseline of the pathogen to procymidone was 0.1981±0.022μg/mL (EC50) and 1.1±0.1595μg/mL (MIC). According to the discriminatory concentration in PSA amended with the fungicide of 5μg/mL (MIC), there were 15 isolates resistant to carbendazim among 168 wild isolates, with the mean resistance ratio of 8.93%, in which 3 isolates were highly resistant (EC50>100μg/mL). But no procymidone-resistant wild isolate was found. The mutants YD38-6, YD38-8 resistant moderately to procymidone, it's EC50 is 41.33μg/mL and 39.08μg/mL, respectively. The mutant YD21a resistant highly to procymidone (EC50>100μg/mL) and carbendazim (EC50>100μg/mL) were obtained from sensitive isolates by the treatment of ultraviolet and procymidone, respectively.EC50 values of the antifungal protein ranged from 20.94μg/ml to 33.83μg/mL, which showed that there were no significant differences in the toxicity to carbendazim or procymidone resisitant and sensitive isolates. Most mixtures of the antifungal protein with the fungicides was found to be addition effects in the toxicity, but the mixture with the ratio 3:1 of the antifungal protein and procymidone could obviously increase the toxicity to procymidone-resistant isolate YD38-6, with the synergistic response (SR) of 1.63.The test indicated that the antifungal protein could well control rape stem rot, with the control effect of 71.8% at the concentration of 3000μg/mL, as carbendazim and procymidone did at the concentration of 1000μg/mL. The mixure (3:1) of the antifungal protein with carbendazim or procymidone at the concentration of 1000μg/mL had the control effect of 67.9%~70.1% on the disease. Therefore, the antifungal protein produced by B. licheniformis W10,as a new bio-fungicide,could be used to control rape stem rot.