Isolation And Partial Characterization Of An Antifungal Protein Produced By Bacillus Licheniformis HS10

Cucumber downy mildew is a serious fungal diseases of cucumber caused by Pseudoperonospora cubensis (Berk.et Curt.) Rostov, which is occurring in 70 countries in the world. At present, the control of cucumber downy mildew mainly depends on chemical pesticides, such as aliette, chlorothalonil, Jia Shuangling, propamocarb or metalaxyl, mancozeb, but its control effect is poor that brings much serious pollutions in environment. Biological control is a green and potential prevention that has a promising future.Bacillus is a dominant microbial population around plant, soil and their microecology.It has good resistance and antibacterial effects against plant disease control. Bacillus licheniformis is one of the potential applications of strain, which has complex metabolic system and rich extracellular antibacterial substances.The strain HS10 isolated from the rhizosphere of healthy cucumber in Huaian, Jiangsu province. HS10 was identified as Bacillus licheniformis (NCBI GENBANK accession number is Bacillus licheniformis HM006898.1) based on its 16S rRNA gene. Bacillus licheniformis HS10 was a potential biocontrol bacterium against cucumber downy with high control efficacy of 61.96%,62.31% in 2010 and 2011. HS10 had good protease, chitinase, cellulase activity.Its secreted protein has significantly inhibition on eight kinds of plant pathogenic fungi:Fusarium graminearum, Sclerotinia sclerotiorum, Botrytis cinerea, Bipolaris sorokinianum, Phytophthora capsici, Rhizoctonia cerealis, Bipolaris maydis, Gaeumannomyces graminis, Pseudoperonospora cubensis. To research the characteristics of the antifungal proteins isolated from HS10, it is purified by using 30%-60% ammonium sulfate precipitation, column chromatography on DEAE Sepharose Fast Flow, RESOURCE Q and Sephadex G-75. SDS-PAGE analysis demonstrate that the protein was a monomer with molecular weight of about 55 kDa. The inhibitory activity was still stable at 100℃ for 30 min and in pH value ranged from 6 to 10. It also negatively affected by Protease K and lOMm metal cations excepted Ca2+. The protein could restrain Phytophthora capsici that had the obvious malformation of hyphal tip and the lysis. This protein was most likely identified as putative carboxypeptidase (Protein ID:gi| 510141549),which is dissociated 39 peptides by MALDI-TOF/TOF-MS.