地衣芽孢杆菌（BL）抗菌蛋白对油菜菌核病菌的抑制与破坏作用及防病效果
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摘要
地衣芽孢杆菌(Bacillus licheniformis)W10培养菌液和滤液对油菜菌核病菌(Sclerotinia sclerotiorum)具有较强的拮抗能力。其培养滤液经硫酸铵沉淀和透析提取的抗菌蛋白处理病菌菌丝后,菌丝出现形态异常甚至断裂;细胞膜透性改变,电解质渗漏而电导率增加。该抗菌蛋白能强烈抑制病菌菌丝生长,当蛋白浓度为100μg/mL时,抑制率达90%以上;能显著抑制菌核形成,当蛋白浓度为100μg/mL时,菌核不能形成,且萌发推迟;能明显阻止子囊孢子萌发和芽管伸长,当蛋白浓度为200μg/mL时,孢子萌发和芽管伸长抑制率分别达46.0%和65.6%。
对油菜菌核病菌进行室内药剂毒力测定。结果表明,腐霉利对病菌的EC50值为0.0126~0.5662μg/mL。根据32个野生菌株敏感性(EC50)频率分布,病菌对腐霉利的敏感基线为0.1981±0.022μg/mL(EC50)和1.1±0.1595μg/mL(MIC)。以5μg/mL为检测标准(MIC),油菜菌核病菌168个野生菌株中有15个抗多菌灵菌株,其中高抗(EC50>100μg/mL)菌株3个,其抗性频率为8.93%,但未检测到抗腐霉利菌株。经紫外线和药剂诱导获得抗药突变株YD38-6、YD38-8和YD21a,其中YD38-6、YD38-8菌株中抗腐霉利,EC50分别为41.33μg/mL和39.08μg/mL;YD21a菌株高抗腐霉利(EC50>100μg/mL)和多菌灵(EC50>100μg/mL)。
试验指出,地衣芽孢杆菌抗菌蛋白对油菜菌核病菌的EC50值为20.94~33.83μg/mL,其毒力对抗、感多菌灵菌株和抗、感腐霉利菌株均无明显差异。抗菌蛋白与两种药剂复配后,大多数配比表现相加作用,但抗菌蛋白与腐霉利3 : 1配比对抗腐霉利菌株YD38-6则有明显的增效作用(SR=1.63)。
盆栽试验显示,抗菌蛋白对油菜菌核病有较好的防治效果,当蛋白浓度为3000μg/mL时,防效达71.8%,与多菌灵和腐霉利1000μg/mL相当;抗菌蛋白与多菌灵或腐霉利3 : 1复配剂在1000μg/mL时,防效达67.9%~70.1%。因此,地衣芽孢杆菌W10抗菌蛋白作为一种新型生物农药可用于油菜菌核病的防治。
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.
对油菜菌核病菌进行室内药剂毒力测定。结果表明,腐霉利对病菌的EC50值为0.0126~0.5662μg/mL。根据32个野生菌株敏感性(EC50)频率分布,病菌对腐霉利的敏感基线为0.1981±0.022μg/mL(EC50)和1.1±0.1595μg/mL(MIC)。以5μg/mL为检测标准(MIC),油菜菌核病菌168个野生菌株中有15个抗多菌灵菌株,其中高抗(EC50>100μg/mL)菌株3个,其抗性频率为8.93%,但未检测到抗腐霉利菌株。经紫外线和药剂诱导获得抗药突变株YD38-6、YD38-8和YD21a,其中YD38-6、YD38-8菌株中抗腐霉利,EC50分别为41.33μg/mL和39.08μg/mL;YD21a菌株高抗腐霉利(EC50>100μg/mL)和多菌灵(EC50>100μg/mL)。
试验指出,地衣芽孢杆菌抗菌蛋白对油菜菌核病菌的EC50值为20.94~33.83μg/mL,其毒力对抗、感多菌灵菌株和抗、感腐霉利菌株均无明显差异。抗菌蛋白与两种药剂复配后,大多数配比表现相加作用,但抗菌蛋白与腐霉利3 : 1配比对抗腐霉利菌株YD38-6则有明显的增效作用(SR=1.63)。
盆栽试验显示,抗菌蛋白对油菜菌核病有较好的防治效果,当蛋白浓度为3000μg/mL时,防效达71.8%,与多菌灵和腐霉利1000μg/mL相当;抗菌蛋白与多菌灵或腐霉利3 : 1复配剂在1000μg/mL时,防效达67.9%~70.1%。因此,地衣芽孢杆菌W10抗菌蛋白作为一种新型生物农药可用于油菜菌核病的防治。
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.
引文
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