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journal1 ›› 2016, Vol. 32 ›› Issue (3): 406-413.DOI: 10.16409/j.cnki.2095-039x.2016.03.017

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Culture Condition and Characterization of Factors Affecting Activity of the Extracellular Proteases Produced by Mycoparasite Coniothyrium minitans

XIE Xiaoli1,2, YANG Long1, WU Mingde1, ZHANG Jing1, LI Guoqing1   

  1. 1. Hubei Key Laboratory of Plant Pathology/College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China;
    2. Xi'an Yanta District Food and Drug Administration, Xi'an 710061, China
  • Received:2015-10-26 Online:2016-06-08 Published:2016-06-07

Abstract: The mycoparasite Coniothyrium minitans is an important biocontrol agent of Sclerotinia sclerotiorum. In order to elucidate the role of the extracellular proteases (EPs) produced by C. minitans in mycoparasitism on S. sclerotiorum, a study was carried out to detect the activity of the EPs produced by C. minitans using the methods of gelatin-displying and the Folin phenol reagent quantification, and to clarify the role of the EPs in mycoparasitism of C. minitans. Results showed that the protease activity was detected in C. minitans-infected sclerotia of S. sclerotiorum, implying that the EPs may get involved in mycoparasitism of C. minitans. Results also showed that S. sclerotiorum sclerotia extracts significantly enhanced production of the EPs by C. minitans, compared to the media without S. sclerotiorum sclerotia extracts. In the time-course experiment, production of the EPs by C. minitans in the shake cultures (20 ℃, 200 r/min) was peaked at 5 days post incubation and the protease activity reached up to 0.22 U/mL. The optimum enzymatic reaction conditions were: temperature at 60 ℃, pH at 7.0. The activity of the EPs was stable bellow 40 ℃. Presence of Mg2+, Zn2+, Ca2+, Cu2+, Mn2, Li+, K+ (5 mmol/L each) had no significant (P>0.05) effect on the activity of the EPs. However, the presence of Fe2+ at 5 mmol/L significantly (P<0.05) enhanced the EP activity. The EPs were highly sensitive to phenylmethyl sulfony fluride (PMSF), implying that C. minitans EPs might belong to the serine protease family. These results will be useful for the future study to purify the C. minitans EPs and to clarify the role of the EPs in biocontrol of C. minitans against S. sclerotiorum.

Key words: Coniothyrium minitans, Sclerotinia sclerotiorum, mycoparasite, protease

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