【作者】 于婷；

【导师】 刘峰；

【作者基本信息】 山东农业大学 ， 农药学， 2009， 硕士

【摘要】 为进一步了解短小芽孢杆菌BSH-4菌株的拮抗作用机制,以黄瓜菌核病菌Sclerotinia sclerotiorum为指示菌测定了该菌株抗菌蛋白的抗菌活性,优化了发酵条件,对抗菌蛋白进行了抗菌谱测定、理化性质分析,初步研究了抗菌蛋白的分离条件,从而为进一步生防应用打下基础。1.为明确BSH-4菌株及其抗菌蛋白的抑菌谱,采用平板对峙法和平板打孔法分别测得此菌株及其抗菌蛋白的抗菌活性。结果表明,此菌株及其抗菌蛋白对黄瓜菌核病菌、黄瓜猝倒病菌、黄瓜蔓枯病菌、黄瓜枯萎病菌、黄瓜立枯病菌、番茄早疫病菌、辣椒疫病病菌、茄子绵疫病菌等8种常见蔬菜病原真菌均具有抑菌活性。且经多次转接后,此菌株的抑菌活性基本不变。2.为提高短小芽孢杆菌BSH-4产生抗菌蛋白的能力,采用单因素和正交试验设计法对BSH-4发酵培养基的组成及其发酵条件进行了优化。结果表明,此菌株的最佳发酵培养基组成为:玉米淀粉2 %,酵母浸膏2 %,CaCl2 0.4 %;最佳发酵条件为:发酵时间48 h,摇瓶转速180 r/min,培养温度37℃,初始pH值6.0,接种量6 %,装液量25 mL(50 mL三角瓶)。在此优化条件下,BSH-4经发酵产生的抗菌蛋白的浓度为16.8μg/mL。3.为明确短小芽孢杆菌BSH-4抗菌蛋白的理化性质,研究了温度、pH值、紫外照射、有机溶剂、金属离子及蛋白酶对其稳定性的影响,并以不同培养基为底物对该蛋白的功能进行初步判断。结果表明,该抗菌蛋白粗提液具有良好的热稳定性,100℃处理1 h后仍具有对照活性的90%左右;对酸碱较敏感,在pH值6-9时活性较高;对紫外线稳定,在紫外光下照射10 h以上时,其活性基本不受影响;对乙醚、丙酮、乙酸乙酯和氯仿不敏感,其活性基本不变,而对甲醇较敏感,其活性下降20%以上;对重金属离子敏感,浓度为10mmol/L的Ag+、Cu2+、Zn2+能够降低活性的40%左右;对蛋白酶较稳定。底物特异性试验表明该蛋白不是几丁质酶、羧甲基纤维素酶和β-1,3-葡聚糖酶。4.初步研究了短小芽孢杆菌BSH-4抗菌蛋白对核盘菌的抑菌机制,采用菌丝生长速率法测定了其对黄瓜菌核病菌Sclerotinia sclerotiorum菌丝生长的毒力,及其对菌丝形态、菌核形成、菌核萌发和菌体细胞膜透性的影响。结果表明,此抗菌蛋白粗提物对核盘菌菌丝生长的EC50值为10.13μg/mL,明显低于对照药剂乙霉威及腐霉利;经此抗菌蛋白处理后,菌丝形态出现黄化、畸形等现象,但尚未发现细胞质外渗;菌核形成的时间有所延长,数量有所减少,且重量略有降低;在此抗菌蛋白粗提物浓度为250μg/mL时不能完全抑制菌核萌发;对菌体细胞膜渗透势的影响不大。5.BSH-4菌株抗菌粗蛋白经Sephadex G-100分子筛柱层析后得两个活性洗脱峰,取活性高的组分进行浓缩,SDS-PAGE电泳检测蛋白纯度得两条电泳图带,分别将条带切下,缓冲液提取后平板打孔法检测,其中一条小分子带有抑菌活性。更多还原

【Abstract】 The antifungal activity, fermentation medium,fermentation conditions and inhibitory mechanisms against cucumber sclerotium of antifungal protein of Bacillus pumilus strain BSH-4 was primarily studied.And for further application of this strain,the isolation and characteristic of its antifungal crude protein were also studied.The method of in dual culture was used to ascertain inhibiting spectrum of BSH-4 and disk diffusion method was applied to test the antifungal activity of antifungal protein of BSH-4. The result showed that BSH-4 strain and its antifungal protein had a broad inhibition spectrum. Both bacteria strain and antifungal protein had antifungal activity against Sclerotinia sclerotiorum,Pythium spp.,Mycosphaerella melonis,Fusarium oxysporum,Rhizoctonia solani,Alternaria solani,Phytophthora capsici and Phytophthora parasitica et al.After several transferred,its antifungal activity was not changed.In order to improve the activity of BSH-4 to producing antifungal protein, the fermentation medium and conditions of the strain were studied via single factor test and orthogonal design. The results showed that the optimal fermentation medium was composed of corn starch 2%, yeast extract 2%, CaCl2 0.4%.And the optimal fermentation condition was fermentation period of 48 hours,rotation speed of 180r/min,cultural temperature of 37℃, initial pH value of 6. 0, inoculation quantity of 6 %,and medium volume of 25mL in 50mL flask. Under such conditions, the concentration of the antifungal protein which were produced from BSH-4 was about 16.8μg/mL.To gain physical and chemical properties of the antifungal protein, the effects of heat, pH, UV irradiation, organic solvents, metal ions prolease were studied,and also used different medium to ascertain its species.The result showed that this crude antifungal proteins were thermal stable, about 90% of the initial activity remained after heating at 100°C for 1 h; they were stable in the pH range of 6–9; the antifungal activity was almost not be destroyed when they were under UV irradiation for 10h; they were stable to ether,acetone, ethyl acetate and chloroform,but methanol could reduce above 20% of the initial activity; Ag+、Cu2+ and Zn2+ ions at the concentration of 10mmol/L could reduced their 40% initial activity and they were not sensitive to proteinase. The result also showed this protein was not chitinase, carboxymethyl cellulase andβ-1,3– glucanase.To ascertain the antagonistic mechanisms of this crude antifungal protein to S. sclerotiorum,the inhibition activities on mycelium growth against S. sclerotiorum were determined by mycelium growth rate method in lab. And its effect on this pathogen’s biology characteristics, such as mycelium shape, sclerotium formation, sclerotium germination and membrane osmotic potential were also studied. The results showed that its EC50 value was 10.13μg/mL, which was significantly lower than Diethofencarb and Procymidone;After treated with this crude antifungal protein, the mycelia became etiolation and malformation, but had not yet found in the cytoplasm extravasation;This crude antifungal protein could delay the formation of sclerotium, the sclerotium numbers decreased contrasted that of the water control, and the weight reduced slightly;At the concentration of 250μg/mL of this crude antifungal protein,it could not restrain the sclerotium germination completely;It had no significant effect on the membrane osmotic potential.Two antagonistic peaks were obtained from the crude antifungal protein after Sephadex G-100 column chromatography. Condece the higher inhibition activity component and showed two band on SDS-PAGE. Antifungal activity was detected after distilling the divided band with Tris-HCl buffer. Clear inhibition zone was appeared on colonies of S. sclerotiorum of Low Molecular Weight protein.更多还原