中华散尾鬼笔提取物对灰葡萄孢霉菌的作用机制
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摘要
为探究中华散尾鬼笔提取物对灰葡萄孢霉菌的抑制作用及抑菌机理,通过抑菌试验、酶活试验、电导率试验及菌丝微观结构观测,确定中华散尾鬼笔提取物对灰葡萄孢霉菌的抑制作用,分析提取物对相关酶活性、培养液电导率和菌丝结构变化的影响。结果表明:中华散尾鬼笔提取物对灰葡萄孢霉菌菌丝生长的EC50为4. 428 mg/m L;提取物对灰葡萄孢霉菌的过氧化物酶(POD)、超氧化物歧化酶(SOD)、多酚氧化酶(PPO)、果胶酶(Pectinase)、纤维素酶(CL)活性均表现出抑制效果,其中第2天的SOD和PPO活性抑制率分别可达到65. 48%和53. 86%,POD的抑制效果在第4天达到峰值,抑制率为68. 65%;提取物对灰葡萄孢霉菌菌丝形态产生显著影响,导致菌丝生长异常、形态干瘪、菌丝分隔处出现内凹,并伴有菌丝破裂的现象产生。提取物处理的菌体细胞膜透性增强,致各时间点培养液电导率显著高于对照,其中提取物处理的第2天电导率为1 230. 579μS/cm,为对照电导率的1. 3倍。这表明中华散尾鬼笔提取物对灰葡萄孢霉菌有抑制作用,且提取物能抑制与菌丝生长及致病相关的几种酶的活性,影响细胞膜渗透性及菌丝体形态。
The purpose of this study was to investigate the inhibitory effect and antifungal mechanism of extracts from Lysurus mokusin against Botrytis cinerea,through antifungal test,enzyme activity test,electrical conductivity test,and mycelial microstructure observation. The inhibitory effect of the material on Botrytis cinerea was analyzed and the effects of the extracts on activity of the related enzymes,conductivity of the culture solution and changes of the mycelial structure were analyzed. The results showed that EC50 of the extracts from Lysurus mokusin was 4. 428 mg/m L for the mycelial growth of Botrytis cinerea; The extracts showed inhibitory effects on POD,SOD,PPO,Pectinase and CL activities of Botrytis cinerea,and the inhibition rates of SOD and PPO activities on the 2 nd day were 65. 48% and 53. 86%,respectively. The inhibitory effect of POD reached the peak on the4 th day,and the inhibitory rate was 68. 65%. The extracts had a significant effect on mycelial morphology of Botrytis cinerea,resulting in abnormal mycelial growth,dry form,and indentation at the hyphae partition,accompanied by the occurrence of mycelial rupture. Cell membrane permeability of the extract-treated cells was enhanced,and conductivity of the culture fluid was significantly higher than that of the control at various time points. Conductivity of the 2 d treatment was 1 230. 579 μS/cm,which was 1. 3 times that of the control. The above results indicate that the extracts of Lysurus mokusin have inhibitory effects on Botrytis cinerea,and the extracts inhibit the activities of several enzymes associated with growth and pathogenicity,affecting cell membrane permeability and mycelial morphology.
The purpose of this study was to investigate the inhibitory effect and antifungal mechanism of extracts from Lysurus mokusin against Botrytis cinerea,through antifungal test,enzyme activity test,electrical conductivity test,and mycelial microstructure observation. The inhibitory effect of the material on Botrytis cinerea was analyzed and the effects of the extracts on activity of the related enzymes,conductivity of the culture solution and changes of the mycelial structure were analyzed. The results showed that EC50 of the extracts from Lysurus mokusin was 4. 428 mg/m L for the mycelial growth of Botrytis cinerea; The extracts showed inhibitory effects on POD,SOD,PPO,Pectinase and CL activities of Botrytis cinerea,and the inhibition rates of SOD and PPO activities on the 2 nd day were 65. 48% and 53. 86%,respectively. The inhibitory effect of POD reached the peak on the4 th day,and the inhibitory rate was 68. 65%. The extracts had a significant effect on mycelial morphology of Botrytis cinerea,resulting in abnormal mycelial growth,dry form,and indentation at the hyphae partition,accompanied by the occurrence of mycelial rupture. Cell membrane permeability of the extract-treated cells was enhanced,and conductivity of the culture fluid was significantly higher than that of the control at various time points. Conductivity of the 2 d treatment was 1 230. 579 μS/cm,which was 1. 3 times that of the control. The above results indicate that the extracts of Lysurus mokusin have inhibitory effects on Botrytis cinerea,and the extracts inhibit the activities of several enzymes associated with growth and pathogenicity,affecting cell membrane permeability and mycelial morphology.
引文
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[2] Sasanuma I,Suzuki T. Effect of calcium on cell-wall degrading enzymes of Botrytis cinerea[J]. Bioscience Biotechnotechnology and Biochemistry,2016,80(9SI):1730-1736.
[3]李培芬,赵福鑫,董丽萍,等.灰葡萄孢BcKMO在病菌生长、发育和致病过程中的功能[J].中国农业科学,2014(15):2971-2979.
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[5]刘正慧,李丹,SOSSAH Frederick Leo,等.食用菌主要病原真菌和细菌[J].菌物研究,2018,16(3):158-163.
[6]图力古尔,包海鹰,李玉.中国毒蘑菇名录[J].菌物学报,2014(3):517-548.
[7]林连男,王瑞琦,张国珍,等.中华散尾鬼笔提取工艺响应面优化及其粗提物毒力测定[J].吉林农业大学学报,2018,40(1):80-84.
[8]巩婷,温艳华,陈尔东,等.担子菌类真菌新产物研究进展[J].菌物研究,2017,15(2):89-111.
[9]高琴琴,包海鹰.药用真菌抗肿瘤活性成分研究进展[J].菌物研究,2018,16(2):115-121.
[10]贺新生,黄倩倩,潘家莲,等.从鬼笔菌丝体和子实体中提取天然防腐物质研究[J].四川食品与发酵,2004(4):40-42.
[11]王瑞琦,张国财,林连男,等.基于ITS中华散尾鬼笔的分类及抑菌活性研究[J].黑龙江畜牧兽医,2017(23):178-181.
[12]邹莉,谭昀,邹晓俏,等.鲍氏层孔菌发酵产物抑菌活性的研究[J].吉林农业大学学报,2013,35(1):19-23.
[13]杨璟,林连男,王瑞琦,等.中华散尾鬼笔培养基筛选及培养条件优化[J].西北林学院学报,2017(6):145-149.
[14]朴春红,霍越,李想,等.荞麦壳水提取物的抑菌活性[J].吉林农业大学学报,2015,37(4):482-487.
[15]韩璐,赵成爱,马炳阳,等.地肤子水提液及大孔树脂分离物的抑菌作用[J].吉林农业大学学报,2014(4):442-446.
[16]方哲,傅俊范,周如军,等.丁香与苦参复配剂对人参锈腐病菌的抑菌活性[J].吉林农业大学学报,2009(1):13-15.
[17] Marcus L,Barash I,Sneh B,et al. Purification and characterization of pectolytic enzymes produced by virulent and hypovirulent isolates of Rhizoctonia solani kuhn[J]. Physiological and Molecular Plant Pathology,1986,29:325-326.
[18] Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.[J]. Analytical biochemistry,1976,72:248-254.
[19]刘芳芳,张爱华,雷峰杰,等.人参茎叶总皂苷对腐皮镰刀菌的抑制作用及抑菌机理[J].吉林农业大学学报,2018,36(1):85-91.
[20]秦玉坤.新型含氮、硫、磷壳聚糖衍生物的制备、抑菌活性与机理初探[D].青岛:中国科学院研究生院(海洋研究所),2012.
[21]金勤,周国英,刘君昂,等.细胞壁降解酶在油茶炭疽病菌致病过程中的作用研究[J].植物保护,2017(3):97-102.
[22] Yang Y,Yang M,Li M,et al. Cloning and functional analysis of an endo-PG-encoding gene Rrspg1 of Rhizoctonia solani,the causal agent of rice sheath blight[J]. Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie,2012,34(3):436-447.
[23] Paul S,Dubey R C,Maheswari D K,et al. Trachyspermum ammi(L.)fruit essential oil influencing on membrane permeability and surface characteristics in inhibiting food-borne pathogens[J]. Food Control,2011,22(5):725-731.
[24]刘梦茵,刘芳,周涛,等.乌梅提取物对蜡状芽孢杆菌的抑菌机理研究[J].食品科学,2012(1):103-105.
[25] Nazer A I,Kobilinsky A,Tholozan J L,et al. Combinations of food antimicrobials at low levels to inhibit the growth of Salmonella sv. Typhimurium:a synergistic effect?[J]. Food Micrbiology,2005,22(5):391-398.
[26]张赟彬,刘笑宇,姜萍萍,等.肉桂醛对大肠杆菌和金黄色葡萄球菌的抑菌作用及抑菌机理研究[J].现代食品科技,2015(5):31-35.
[27] Lu M,Han Z,Xu Y,et al. Effects of essential oils from Chinese indigenous aromatic plants on mycelial growth and morphogenesis of three phytopathogens[J]. Flavour and Fragrance Journal,2013,28(2):84-92.
[28]崔凯娣,黄学屏,何磊鸣,等.微生物源挥发性化合物苯并噻唑对灰霉病菌的抑制效应[J].中国农业科学,2017(19):3714-3722.
[29]吴洁云,纪兆林,徐敬友,等.灰葡萄孢胞壁降解酶对番茄植株致病作用的分析[J].扬州大学学报(农业与生命科学版),2010,31(3):70-74.
[30]毛胜凤,林海萍,陈安良,等.喜树碱对番茄灰霉病菌的病理反应[J].中国生物防治学报,2014(1):143-148.
[2] Sasanuma I,Suzuki T. Effect of calcium on cell-wall degrading enzymes of Botrytis cinerea[J]. Bioscience Biotechnotechnology and Biochemistry,2016,80(9SI):1730-1736.
[3]李培芬,赵福鑫,董丽萍,等.灰葡萄孢BcKMO在病菌生长、发育和致病过程中的功能[J].中国农业科学,2014(15):2971-2979.
[4] Calvo J,Calvente V,de Orellano M E,et al. Biological control of postharvest spoilage caused by Penicillium expansum and Botrytis cinerea in apple by using the bacterium Rahnella aquatilis[J]. International Joural of Food Microbiology,2007,113(3):251-257.
[5]刘正慧,李丹,SOSSAH Frederick Leo,等.食用菌主要病原真菌和细菌[J].菌物研究,2018,16(3):158-163.
[6]图力古尔,包海鹰,李玉.中国毒蘑菇名录[J].菌物学报,2014(3):517-548.
[7]林连男,王瑞琦,张国珍,等.中华散尾鬼笔提取工艺响应面优化及其粗提物毒力测定[J].吉林农业大学学报,2018,40(1):80-84.
[8]巩婷,温艳华,陈尔东,等.担子菌类真菌新产物研究进展[J].菌物研究,2017,15(2):89-111.
[9]高琴琴,包海鹰.药用真菌抗肿瘤活性成分研究进展[J].菌物研究,2018,16(2):115-121.
[10]贺新生,黄倩倩,潘家莲,等.从鬼笔菌丝体和子实体中提取天然防腐物质研究[J].四川食品与发酵,2004(4):40-42.
[11]王瑞琦,张国财,林连男,等.基于ITS中华散尾鬼笔的分类及抑菌活性研究[J].黑龙江畜牧兽医,2017(23):178-181.
[12]邹莉,谭昀,邹晓俏,等.鲍氏层孔菌发酵产物抑菌活性的研究[J].吉林农业大学学报,2013,35(1):19-23.
[13]杨璟,林连男,王瑞琦,等.中华散尾鬼笔培养基筛选及培养条件优化[J].西北林学院学报,2017(6):145-149.
[14]朴春红,霍越,李想,等.荞麦壳水提取物的抑菌活性[J].吉林农业大学学报,2015,37(4):482-487.
[15]韩璐,赵成爱,马炳阳,等.地肤子水提液及大孔树脂分离物的抑菌作用[J].吉林农业大学学报,2014(4):442-446.
[16]方哲,傅俊范,周如军,等.丁香与苦参复配剂对人参锈腐病菌的抑菌活性[J].吉林农业大学学报,2009(1):13-15.
[17] Marcus L,Barash I,Sneh B,et al. Purification and characterization of pectolytic enzymes produced by virulent and hypovirulent isolates of Rhizoctonia solani kuhn[J]. Physiological and Molecular Plant Pathology,1986,29:325-326.
[18] Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.[J]. Analytical biochemistry,1976,72:248-254.
[19]刘芳芳,张爱华,雷峰杰,等.人参茎叶总皂苷对腐皮镰刀菌的抑制作用及抑菌机理[J].吉林农业大学学报,2018,36(1):85-91.
[20]秦玉坤.新型含氮、硫、磷壳聚糖衍生物的制备、抑菌活性与机理初探[D].青岛:中国科学院研究生院(海洋研究所),2012.
[21]金勤,周国英,刘君昂,等.细胞壁降解酶在油茶炭疽病菌致病过程中的作用研究[J].植物保护,2017(3):97-102.
[22] Yang Y,Yang M,Li M,et al. Cloning and functional analysis of an endo-PG-encoding gene Rrspg1 of Rhizoctonia solani,the causal agent of rice sheath blight[J]. Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie,2012,34(3):436-447.
[23] Paul S,Dubey R C,Maheswari D K,et al. Trachyspermum ammi(L.)fruit essential oil influencing on membrane permeability and surface characteristics in inhibiting food-borne pathogens[J]. Food Control,2011,22(5):725-731.
[24]刘梦茵,刘芳,周涛,等.乌梅提取物对蜡状芽孢杆菌的抑菌机理研究[J].食品科学,2012(1):103-105.
[25] Nazer A I,Kobilinsky A,Tholozan J L,et al. Combinations of food antimicrobials at low levels to inhibit the growth of Salmonella sv. Typhimurium:a synergistic effect?[J]. Food Micrbiology,2005,22(5):391-398.
[26]张赟彬,刘笑宇,姜萍萍,等.肉桂醛对大肠杆菌和金黄色葡萄球菌的抑菌作用及抑菌机理研究[J].现代食品科技,2015(5):31-35.
[27] Lu M,Han Z,Xu Y,et al. Effects of essential oils from Chinese indigenous aromatic plants on mycelial growth and morphogenesis of three phytopathogens[J]. Flavour and Fragrance Journal,2013,28(2):84-92.
[28]崔凯娣,黄学屏,何磊鸣,等.微生物源挥发性化合物苯并噻唑对灰霉病菌的抑制效应[J].中国农业科学,2017(19):3714-3722.
[29]吴洁云,纪兆林,徐敬友,等.灰葡萄孢胞壁降解酶对番茄植株致病作用的分析[J].扬州大学学报(农业与生命科学版),2010,31(3):70-74.
[30]毛胜凤,林海萍,陈安良,等.喜树碱对番茄灰霉病菌的病理反应[J].中国生物防治学报,2014(1):143-148.