白藜芦醇对核桃细菌性黑斑病菌的抑制作用

doi:10.13560/j.cnki.biotech.bull.1985.2020-1267

摘要/Abstract

摘要：

旨为核桃细菌性黑斑病天然杀菌剂的开发提供理论依据,同时为白藜芦醇在农林作物病害防治上的应用奠定基础。测定了白藜芦醇对核桃细菌性黑斑病菌(Xaj)的最低抑菌浓度(MIC)和最低杀菌浓度(MBC)。在白藜芦醇的亚抑菌浓度下,开展了以下实验:(1)通过结晶紫染色法检测了Xaj生物膜的形成;(2)测定了胞外多糖的产生量;(3)用生物化学方法在相应的固体培养基上分析了Xaj胞外酶的分泌情况;(4)在swarming培养基上检测了Xaj的运动能力。结果显示,白藜芦醇对Xaj的MIC为20 μg/mL,MBC为45 μg/mL。在亚抑菌浓度为15 μg/mL的条件下,白藜芦醇能够显著抑制Xaj生物被膜的形成;对该菌EPS的产生也有明显抑制作用;能显著抑制Xaj蛋白酶的分泌,对淀粉酶的分泌有促进作用,对纤维素酶的分泌没有影响;对Xaj运动性没有影响。白藜芦醇对核桃黑斑病菌有抑制作用,影响该菌的毒力因子,有望为核桃黑斑病的防治开发出合适的植物源杀菌剂。

关键词: 白藜芦醇, 核桃细菌性黑斑病菌, 抑菌效果, 植物源杀菌剂

Abstract:

It aims to provide theoretical basis for the development of natural bactericide for walnut bacterial black spot,and to lay a foundation for the application of resveratrol in the prevention and control of agricultural and forestry crop diseases. The minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of resveratrol against Xanthomonas arboricola pv. juglandis(Xaj)were determined. The following experiments were carried out under the sub-inhibitory concentration of resveratrol:1)The formation of Xaj biofilm was detected by crystal violet staining. 2)The production of exopolysaccharide(EPS)was determined. 3)The secretion of extracellular enzyme was analyzed on the corresponding solid medium by biochemical method. 4)The motility of Xaj was detected on swarming medium. The results revealed that the MIC of resveratrol against Xaj was 20 μg/mL,and MBC was 45 μg/mL. The formation of Xaj biofilm was effectively inhibited by resveratrol at a sub-inhibitory concentration of 15 μg/mL. Under this concentration,the production of EPS was also significantly inhibited. Resveratrol effectively inhibited the secretion of Xaj protease,promoted the secretion of amylase,but had no effect on the secretion of cellulase. There was no effect on motility(swarming). Resveratrol has an inhibitory effect on walnut bacterial black spot pathogen,affecting the virulence factors of this bacterium,and is expected to develop a suitable plant-derived fungicide for the prevention and treatment of walnut black spot disease.

Key words: resveratrol, walnut bacterial black spot pathogen, antibacterial effect, botanical fungicides

李峰, 陈雯雯, 邓江丽, 毛清黎, 权文利, 毛雅慧. 白藜芦醇对核桃细菌性黑斑病菌的抑制作用[J]. 生物技术通报, 2021, 37(6): 58-65.

LI Feng, CHEN Wen-wen, DENG Jiang-li, MAO Qing-li, QUAN Wen-li, MAO Ya-hui. Inhibitory Effect of Resveratrol on Walnut Bacterial Black Spot Pathogen[J]. Biotechnology Bulletin, 2021, 37(6): 58-65.

图/表 7

图1 白藜芦醇对Xaj的抑菌活性检测 A:滤纸片法定性检测平板。1为滴加DMSO的滤纸片,2、3、4为滴加有白藜芦醇的滤纸片;B:浓度梯度稀释法测定MIC及MBC值。采用t检验进行显著性分析。P < 0.01,差异显著(**);P < 0.001,差异极显著(***);P > 0.05,差异不显著(NS),下同

Fig. 1 Detection of the antibacterial activity of resveratrol against Xaj A: The plate of filter paper for qualitative analysis, No.1 was the filter paper with DMSO, No.2, 3 and 4 were the filter papers with resveratrol. B: MIC and MBC values were determined by concentration gradient dilution method. The t test was used for significance analysis, NS indicates no significant difference (P>0.05), **P<0.01, ***P<0.001, The same below

图2 白藜芦醇对Xaj生物被膜形成的影响 A:指形管表面生物膜形成情况;B:生物膜产量柱形图

Fig. 2 Effect of resveratrol on Xaj biofilm formation A: Biofilm formation on the surface of finger tube. B: Histogram of biofilm yield

图3 白藜芦醇对Xaj产胞外多糖的影响

Fig. 3 Effect of resveratrol on extracellular polysaccharide production by Xaj

图4 白藜芦醇对Xaj分泌胞外蛋白酶的影响 A:含有1%脱脂牛奶的检测平板;B:胞外蛋白酶产量柱形图

Fig. 4 Effect of resveratrol on extracellular protease secreted by Xaj A: Test plate containing 1% skim milk. B: Histogram of extracellular protease yield

图5 白藜芦醇对Xaj分泌胞外淀粉酶的影响 A:含有0.1%可溶性淀粉的检测平板;B:胞外淀粉酶产量柱形图

Fig. 5 Effect of resveratrol on extracellular amylase secreted by Xaj A: Test plate containing 0.1% soluble starch. B: Histogram of extracellular amylase yield

图6 白藜芦醇对Xaj分泌胞外纤维素酶的影响 A:含有0.5%羧甲基纤维素的检测平板;B:胞外纤维素酶产量柱形图

Fig. 6 Effect of resveratrol on extracellular cellulase secreted by Xaj A: Test plate containing 0.5% carboxymethyl cellulose. B: Histogram of extracellular cellulase yield

图7 白藜芦醇对Xaj运动性的影响 A:运动性检测平板;B:菌落直径统计柱形图

Fig.7 Effect of resveratrol on Xaj motility A: Test plate of motility. B: Histogram of colony diameter statistics

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