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萎缩芽孢杆菌XW2胞外蛋白的抑菌活性及其特性
黄华毅1,2, 于地美1, 梁英梅,等3
1.北京林业大学 省部共建森林培育与保护教育部重点实验室;2.广东省林业科学研究院;3.北京林业大学 博物馆
摘要:
【目的】研究萎缩芽孢杆菌(Bacillus atrophaeus)XW2胞外蛋白的抑菌活性及其特性,探索植物病害生物防治的新途径。【方法】采用滤纸片法和叶盘法,检测XW2胞外蛋白对杨树炭疽病菌的最小抑菌浓度(MIC)和叶盘抑菌活性,以及对该病菌菌丝和孢子萌发的影响;研究其对另外5种植物病原真菌(尖孢镰刀菌、金黄壳囊孢、犁头霉、细极链格孢和黑腐皮壳菌)的抑菌活性,并分析其抑菌活性在不同的pH(2,3,4,5,6,7,8,9,10和11)、温度(30,40,50,60,70,80,90,100,110 和121 ℃)、蛋白酶(木瓜蛋白酶、胃蛋白酶、胰蛋白酶和蛋白酶K)、紫外线作用时间(1,2,3和4 h)、金属离子(Li、Na、K、Ca2+、Mg2+、Fe3+、Mn2+、Zn2+、Cu2+和Ag)、表面活性剂(Tween 20和SDS)、抑制剂(EDTA)、氧化剂(H2O2)和常温保存时间(0.5,1,2,3,4,5和6个月)下的稳定性,最后分析XW2产细胞壁裂解酶的特性。【结果】萎缩芽孢杆菌XW2胞外蛋白对杨树炭疽病菌的最小抑菌浓度为0.01 mg/disc(5号滤纸片),能明显抑制杨树炭疽病菌在平板和杨树叶片上的生长,有效抑制孢子的萌发和附着孢的形成,并导致菌丝和萌发芽管产生膨大畸形;对尖孢镰刀菌、金黄壳囊孢、犁头霉、细极链格孢和黑腐皮壳菌均有明显的拮抗活性。XW2胞外蛋白在110 ℃下处理30 min及pH 6.0~9.0下均能保持较高的抑菌活性;对蛋白酶、紫外光照、金属离子(Li、Na、K、Ag、Ca2+、Mg2+、Zn2+和Cu2+)、SDS和EDTA均不敏感,但Mn2+和Fe3+会抑制蛋白的抑菌活性,而Tween 20和H2O2则能提高其抑菌活性;在常温条件下保存6个月后,其抑菌活性并未发生明显丧失。细胞壁裂解酶检测发现,XW2能产生蛋白酶和β-1,3-葡聚糖酶。【结论】萎缩芽孢杆菌XW2胞外蛋白的抑菌活性强、稳定性好且抑菌谱广,具有潜在的工业开发和田间应用前景。
关键词:  萎缩芽孢杆菌  胞外蛋白  抑菌活性
DOI:
分类号:
基金项目:国家自然科学基金项目(31470647);中国博士后科学基金面上项目(215M581004);北京市支持中央高校共建项目(2050205);林业公益性行业科研专项经费资助项目(201204501)
Characteristics and antifungal activity of extracellular proteins of Bacillus atrophaeus XW2
HUANG Huayi,YU Dimei,LIANG Yingmei,et al
Abstract:
【Objective】The characteristics and antifungal activity of extracellular proteins secreted by Bacillus atrophaeus XW2 were investigated to explore new pathways for biocontrol of plant diseases.【Method】Filtering paper method and leaf disc method were used to determine the minimum inhibitory concentrations (MIC) of the proteins against Colletotrichum gloeosporioides,the antifungal activity of the proteins against C.gloeosporioides on poplar leaves,and the effects of the proteins on hyphae and spore germination of C.gloeosporioides.The antifungal activities of proteins against other 5 fungal phytopathogen (Fusarium oxysporum,Cytospora chrysosperma,Absidia sp.,Alternaria tenuissima,and Cytospora mali)were also studied,and the effects of pH (2,3,4,5,6,7,8,9,10 and 11),temperature (30,40,50,60,70,80,90,100,110 and 121 ℃),proteases (Papain,Pepsin,Trypsin and Protease K),UV radiation (1,2,3 and 4 h),metal ions (Li,Na,K,Ca2+,Mg2+,Fe3+,Mn2+,Zn2+,Cu2+and Ag),detergents (Tween 20 and SDS),inhibitor (EDTA),oxidants (H2O2),and storage under room temperatures (0.5,1,2,3,4,5 and 6 month) on antifungal activity were analyzed.At last,the properties of XW2 producing cell wall lysis enzymes were analyzed.【Result】The proteins had good antifungal activity against C.gloeosporioides,and the MIC was 0.01 mg/disc.Proteins could also significantly suppress C.gloeosporioides on poplar leaves,inhibit spore germination and aspersorium formation,and lead to hyphae and germ tube of germinated spore developed vacuoles and swelling.Moreover,proteins could significantly inhibit F.oxysporum,C.chrysosperma,Absidia sp.,A.tenuissima,and C.mali.Characterization of proteins indicted that the proteins were stable with good antifungal activity at 110 ℃ for 30 min and with pH of 6.0-9.0,and they were not sensitive to proteases,UV radiation,metal ions (Li,Na,K,Ag,Ca2+,Mg2+,Zn2+,Cu2+),SDS,and EDTA.Mn2+and Fe3+ inhibited the antifungal activity of proteins,while Tween 20 and H2O2 activated the antifungal activity of proteins.Antifungal proteins kept high active after stored 6 months under room temperature.XW2 could also produce protease and β-1,3-glucanase.【Conclusion】Extracellular proteins secreted by XW2 had strong antifungal activity,good stability,and wide antifungal spectrum.Thus,they have potential prospects for industrial development and field application.
Key words:  Bacillus atrophaeus  extracellular proteins  antifungal activity