地肤子提取物对苹果树腐烂病菌的抑制作用及对其菌体结构和功能的影响
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摘要
为开发新型安全高效的植物源苹果树腐烂病防治剂,采用菌丝生长速率法测定中草药地肤子乙醇提取物及各层析流分L1～L9对苹果树腐烂病菌Valsa mali的抑制作用,同时通过室内常规分析方法观测流分L7对该菌菌丝形态结构、细胞膜通透性及物质吸收和代谢的影响。结果表明,地肤子乙醇粗提物浓度为2 mg/mL时对苹果树腐烂病菌具有较强的离体抑制活性,处理后96 h的抑菌率达到92.16%。分别用石油醚、氯仿、正丁醇萃取醇提物,石油醚萃取物对苹果树腐烂病菌的抑制效果最为明显,其EC_(50)为0.07 mg/mL;在石油醚萃取物通过硅胶柱层析分离得到的9种流分中,流分L7对苹果树腐烂病菌的抑制作用显著,离体抑菌率高达96.73%;且在流分L7处理下,病菌菌丝体出现肿胀、膨大或畸形等现象,细胞膜通透性增大,可溶性蛋白、还原糖以及丙酮酸的含量随流分L7浓度的升高而持续降低,当浓度为100μg/mL时,菌丝体可溶性蛋白、还原糖及丙酮酸的含量分别降低70.78%、71.74%和78.68%。表明在离体培养条件下地肤子乙醇提取物能明显抑制苹果树腐烂病菌的生长,并对菌丝体细胞膜结构和功能稳定性产生显著影响,具有进一步开发为新型植物源苹果树腐烂病防治剂的潜力。
To develop novel botanical fungicides against apple canker pathogen, Valsa mali, the inhibitory activity of both the crude ethanol extract and sub-fractions(L1-L9) of fructus kochiae was tested against V. mali with measuring mycelium growth rate. The mechanism of L7, the most effective subfraction, was preliminarily studied by using conventional analysis methods, and the effect of L7 on mycelial morphology, cell membrane permeability, and rate of essential substance absorption and metabolism was determined. The results showed that the ethanol extracts of fructus kochiae strongly inhibited the growth of V. mali at the concentration of 2 mg/mL, with an in vitro inhibitory rate of 92.16% after 96 h. Crude extraction was performed with petroleum ether, chloroform and n-butanol, followed by inhibition assay with each extract fraction. Petroleum ether extract displayed significantly higher inhibition activity against V. mali with the EC50 at 0.07 mg/mL. Silica gel column chromatography was used to further purify petroleum ether extract and a total of nine sub-fractions(L1-L9) were obtained. The most pronounced antifungal activity was detected in L7 fraction with an inhibitory rate of 96.73%. Morphological alterations of V. mali such as swelling, deformity induced by L7 were observed. Cell membrane permeability was positively reacted to the increase of L7 fraction concentration, while the content of soluble protein, reducing sugar, and pyruvate in mycelia were decreased with the increase of the concentration of L7, which were reduced by 70.78%, 71.74%, and 78.68%, respectively, at the concentration of 100 mg/mL. The study indicated that the ethanol extracts of fructus kochiae significantly inhibited the growth of V. mali in vitro, and negatively impacted cell membrane structure and functional stability of the mycelium. These extracts from plants could be developed as potential fungicides against apple canker.
To develop novel botanical fungicides against apple canker pathogen, Valsa mali, the inhibitory activity of both the crude ethanol extract and sub-fractions(L1-L9) of fructus kochiae was tested against V. mali with measuring mycelium growth rate. The mechanism of L7, the most effective subfraction, was preliminarily studied by using conventional analysis methods, and the effect of L7 on mycelial morphology, cell membrane permeability, and rate of essential substance absorption and metabolism was determined. The results showed that the ethanol extracts of fructus kochiae strongly inhibited the growth of V. mali at the concentration of 2 mg/mL, with an in vitro inhibitory rate of 92.16% after 96 h. Crude extraction was performed with petroleum ether, chloroform and n-butanol, followed by inhibition assay with each extract fraction. Petroleum ether extract displayed significantly higher inhibition activity against V. mali with the EC50 at 0.07 mg/mL. Silica gel column chromatography was used to further purify petroleum ether extract and a total of nine sub-fractions(L1-L9) were obtained. The most pronounced antifungal activity was detected in L7 fraction with an inhibitory rate of 96.73%. Morphological alterations of V. mali such as swelling, deformity induced by L7 were observed. Cell membrane permeability was positively reacted to the increase of L7 fraction concentration, while the content of soluble protein, reducing sugar, and pyruvate in mycelia were decreased with the increase of the concentration of L7, which were reduced by 70.78%, 71.74%, and 78.68%, respectively, at the concentration of 100 mg/mL. The study indicated that the ethanol extracts of fructus kochiae significantly inhibited the growth of V. mali in vitro, and negatively impacted cell membrane structure and functional stability of the mycelium. These extracts from plants could be developed as potential fungicides against apple canker.
引文
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Cantrell CL,Dayan FE,Duke SO.2012.Natural products as sources for new pesticides.Journal of Natural Products,75(6):1231-1242
Cao H,Wang YN,Zhang TQ,Li YS,Liu SQ,Li XH,Shi GL.2007.Acarocidal activity of different parts of Kochia scoparia against Tetranychus viennensis.Scientia Silvae Sinicae,43(9):27-30(in Chinese)[曹挥,王有年,张铁强,李奕松,刘素琪,李向花,师光禄.2007.地肤不同部位提取物对山楂叶螨活性的研究.林业科学,43(9):27-30]
Cao KQ,Guo LY,Li BH,Sun GY,Chen HJ.2009.Investigations on the occurrence and control of apple canker in China.Plant Protection,35(2):114-116(in Chinese)[曹克强,国立耘,李保华,孙广宇,陈汉杰.2009.中国苹果树腐烂病发生和防治情况调查.植物保护,35(2):114-116]
Gao KX,Liu XG.1995.Review of researches on the Valsa canker of apple trees.Journal of Hebei Forestry College,10(1):87-91(in Chinese)[高克祥,刘晓光.1995.苹果树腐烂病研究概况.河北林学院学报,10(1):87-91]
Gao ZP,Ke XW,Wei JL,Chen YC,Kang ZS,Huang LL.2009.Biocontrol efficacy of apple tree Valsa canker by endophytic actinomycetes.Journal of Plant Protection,36(5):410-416(in Chinese)[郜佐鹏,柯希望,韦洁玲,陈银潮,康振生,黄丽丽.2009.七株植物内生放线菌对苹果树腐烂病菌的防治作用.植物保护学报,36(5):410-416]
Lee HJ,Choi GJ,Cho KY.1998.Correlation of lipid peroxidation in Botrytis cinerea caused by dicarboximide fungicides with their fungicidal activity.Journal of Agricultural and Food Chemistry,46(2):737-741
Li SH,Cao H,Liu SQ.2009.Study on antifungal activities of the petroleum ether extract from Euphorbia fischeriana Steud.against Valsa mali Miyabe et Yamada.Journal of Shanxi Agricultural University(Natural Science Edition),29(6):559-561(in Chinese)[李少华,曹挥,刘素琪.2009.大戟狼毒石油醚提取物对苹果树腐烂病抑菌活性初步研究.山西农业大学学报(自然科学版),29(6):559-561]
Shao RJ,Shao SH.2015.Progress in study on medicinal value of medicinal parts of Kochia scoparia.Chinese Traditional and Herbal Drugs,46(23):3605-3610(in Chinese)[邵荣杰,邵世宏.2015.地肤的各药用部位药用价值研究进展.中草药,46(23):3605-3610]
Shi XJ,Yang QQ,Ren L,Han JC.2017.Variation of cell membrane permeability of early blight pathogen Alternaria solani resistant to boscalid.Journal of Plant Protection,44(2):240-245(in Chinese)[史晓晶,杨琴琴,任璐,韩巨才.2017.番茄早疫病菌抗啶酰菌胺菌株细胞膜透性变化.植物保护学报,44(2):240-245]
Wei Y,Zhang XH,Liu ZD,Hao SH.2009.Pesticidal activity of extracts of Kochia scoparia.Journal of Northwest Forestry University,24(6):129-132(in Chinese)[魏艳,张晓华,刘正东,郝双红.2009.地肤农用生物活性研究.西北林学院学报,24(6):129-132]
Wu J,Shi GL,Su XY,Wen ZY,Wang YN.2008.A preliminary study on bioactivity of extracts from fructus kochiae against several phytopathogens.Journal of Beijing University of Agriculture,23(3):36-39(in Chinese)[吴静,师光禄,苏学友,温哲屹,王有年.2008.地肤子提取物对几种植物病原菌生物活性的影响.北京农学院学报,23(3):36-39]
Xi ZM,Wang ZZ,Hu Y,Deng MM,Zhang ZW.2013.Effects of 24-epibrassinolide on the antioxidant system and osmotic adjustment substance in grape seedlings(V.vinifera L.)under chilling stress.Scientia Agricultura Sinica,46(5):1005-1013(in Chinese)[惠竹梅,王智真,胡勇,邓敏敏,张振文.2013.24-表油菜素内酯对低温胁迫下葡萄幼苗抗氧化系统及渗透调节物质的影响.中国农业科学,46(5):1005-1013]
Xu T,Hu TL,Wang YN,Wang ST,Cao KQ.2012.Isolation of endophytic fungi from apple bark and their potential for biological control of Valsa ceratosperma.Journal of Plant Protection,39(4):327-333(in Chinese)[徐涛,胡同乐,王亚南,王树桐,曹克强.2012.苹果树皮内生真菌的分离及其对腐烂病菌的生物防治潜力.植物保护学报,39(4):327-333]
Yan HD,Wang YN,Tang XM,Hu TL,Wang ST,Cui Y,Cao KQ.2014.Antifungal activity of extracts from 5 species of plants against 3phytopathogens of apple trees.China Fruits,(2):22-26(in Chinese)[闫红豆,王亚南,唐兴敏,胡同乐,王树桐,崔彦,曹克强.2014.5种植物提取物抑制苹果树3种病原菌活性的研究.中国果树,(2):22-26]
Yang SZ,Peng LT,Cheng YJ,Chen F,Pan SY.2010.Control of citrus green and blue molds by Chinese propolis.Food Science and Biotechnology,19(5):1303-1308
Yuan ZL,Luo L,Meng ZL.2006.Control effect of artificial synthesized fungicide Yintai on 2 kinds of apple diseases.Journal of Plant Protection,33(2):223-224(in Chinese)[袁忠林,罗兰,孟昭礼.2006.仿生农用杀菌剂银泰对苹果树两种主要病害的防治效果.植物保护学报,33(2):223-224]
Zhang X,Ma ZQ,Feng JT,Wu H,Han LR.2015.Review on research and development of botanical pesticides.Chinese Journal of Biological Control,31(5):685-698(in Chinese)[张兴,马志卿,冯俊涛,吴华,韩立荣.2015.植物源农药研究进展.中国生物防治学报,31(5):685-698]
Zhang ZL,Qu WJ,Li XF.2009.Guide of plant physiology experiment(4th edition).Beijing:Higher Education Press,pp.103-104(in Chinese)[张志良,瞿伟菁,李小方.2009.植物生理学实验指导(第4版).北京:高等教育出版社,pp.103-104]
Zhao L,Du J,Liu SQ,Cao H.2008.Antifeeding effects of Plutella xylostella L.by extracts of the seed of Kochia scoparia(L.)Schrad.Journal of Shanxi Agricultural Sciences,36(10):38-39(in Chinese)[赵磊,杜娟,刘素琪,曹挥.2008.地肤子粗提物对小菜蛾的拒食作用.山西农业科学,36(10):38-39]
Zhou MJ,Wan CP,Chen JY.2014.Screecing and antifungal mechanism of Chinese herb extracts against green mold of citrus.Modern Food Science and Technology,30(3):144-149(in Chinese)[周梦娇,万春鹏,陈金印.2014.柑橘绿霉病中草药高效抑菌剂的筛选及抑菌机理研究.现代食品科技,30(3):144-149]
Cantrell CL,Dayan FE,Duke SO.2012.Natural products as sources for new pesticides.Journal of Natural Products,75(6):1231-1242
Cao H,Wang YN,Zhang TQ,Li YS,Liu SQ,Li XH,Shi GL.2007.Acarocidal activity of different parts of Kochia scoparia against Tetranychus viennensis.Scientia Silvae Sinicae,43(9):27-30(in Chinese)[曹挥,王有年,张铁强,李奕松,刘素琪,李向花,师光禄.2007.地肤不同部位提取物对山楂叶螨活性的研究.林业科学,43(9):27-30]
Cao KQ,Guo LY,Li BH,Sun GY,Chen HJ.2009.Investigations on the occurrence and control of apple canker in China.Plant Protection,35(2):114-116(in Chinese)[曹克强,国立耘,李保华,孙广宇,陈汉杰.2009.中国苹果树腐烂病发生和防治情况调查.植物保护,35(2):114-116]
Gao KX,Liu XG.1995.Review of researches on the Valsa canker of apple trees.Journal of Hebei Forestry College,10(1):87-91(in Chinese)[高克祥,刘晓光.1995.苹果树腐烂病研究概况.河北林学院学报,10(1):87-91]
Gao ZP,Ke XW,Wei JL,Chen YC,Kang ZS,Huang LL.2009.Biocontrol efficacy of apple tree Valsa canker by endophytic actinomycetes.Journal of Plant Protection,36(5):410-416(in Chinese)[郜佐鹏,柯希望,韦洁玲,陈银潮,康振生,黄丽丽.2009.七株植物内生放线菌对苹果树腐烂病菌的防治作用.植物保护学报,36(5):410-416]
Lee HJ,Choi GJ,Cho KY.1998.Correlation of lipid peroxidation in Botrytis cinerea caused by dicarboximide fungicides with their fungicidal activity.Journal of Agricultural and Food Chemistry,46(2):737-741
Li SH,Cao H,Liu SQ.2009.Study on antifungal activities of the petroleum ether extract from Euphorbia fischeriana Steud.against Valsa mali Miyabe et Yamada.Journal of Shanxi Agricultural University(Natural Science Edition),29(6):559-561(in Chinese)[李少华,曹挥,刘素琪.2009.大戟狼毒石油醚提取物对苹果树腐烂病抑菌活性初步研究.山西农业大学学报(自然科学版),29(6):559-561]
Shao RJ,Shao SH.2015.Progress in study on medicinal value of medicinal parts of Kochia scoparia.Chinese Traditional and Herbal Drugs,46(23):3605-3610(in Chinese)[邵荣杰,邵世宏.2015.地肤的各药用部位药用价值研究进展.中草药,46(23):3605-3610]
Shi XJ,Yang QQ,Ren L,Han JC.2017.Variation of cell membrane permeability of early blight pathogen Alternaria solani resistant to boscalid.Journal of Plant Protection,44(2):240-245(in Chinese)[史晓晶,杨琴琴,任璐,韩巨才.2017.番茄早疫病菌抗啶酰菌胺菌株细胞膜透性变化.植物保护学报,44(2):240-245]
Wei Y,Zhang XH,Liu ZD,Hao SH.2009.Pesticidal activity of extracts of Kochia scoparia.Journal of Northwest Forestry University,24(6):129-132(in Chinese)[魏艳,张晓华,刘正东,郝双红.2009.地肤农用生物活性研究.西北林学院学报,24(6):129-132]
Wu J,Shi GL,Su XY,Wen ZY,Wang YN.2008.A preliminary study on bioactivity of extracts from fructus kochiae against several phytopathogens.Journal of Beijing University of Agriculture,23(3):36-39(in Chinese)[吴静,师光禄,苏学友,温哲屹,王有年.2008.地肤子提取物对几种植物病原菌生物活性的影响.北京农学院学报,23(3):36-39]
Xi ZM,Wang ZZ,Hu Y,Deng MM,Zhang ZW.2013.Effects of 24-epibrassinolide on the antioxidant system and osmotic adjustment substance in grape seedlings(V.vinifera L.)under chilling stress.Scientia Agricultura Sinica,46(5):1005-1013(in Chinese)[惠竹梅,王智真,胡勇,邓敏敏,张振文.2013.24-表油菜素内酯对低温胁迫下葡萄幼苗抗氧化系统及渗透调节物质的影响.中国农业科学,46(5):1005-1013]
Xu T,Hu TL,Wang YN,Wang ST,Cao KQ.2012.Isolation of endophytic fungi from apple bark and their potential for biological control of Valsa ceratosperma.Journal of Plant Protection,39(4):327-333(in Chinese)[徐涛,胡同乐,王亚南,王树桐,曹克强.2012.苹果树皮内生真菌的分离及其对腐烂病菌的生物防治潜力.植物保护学报,39(4):327-333]
Yan HD,Wang YN,Tang XM,Hu TL,Wang ST,Cui Y,Cao KQ.2014.Antifungal activity of extracts from 5 species of plants against 3phytopathogens of apple trees.China Fruits,(2):22-26(in Chinese)[闫红豆,王亚南,唐兴敏,胡同乐,王树桐,崔彦,曹克强.2014.5种植物提取物抑制苹果树3种病原菌活性的研究.中国果树,(2):22-26]
Yang SZ,Peng LT,Cheng YJ,Chen F,Pan SY.2010.Control of citrus green and blue molds by Chinese propolis.Food Science and Biotechnology,19(5):1303-1308
Yuan ZL,Luo L,Meng ZL.2006.Control effect of artificial synthesized fungicide Yintai on 2 kinds of apple diseases.Journal of Plant Protection,33(2):223-224(in Chinese)[袁忠林,罗兰,孟昭礼.2006.仿生农用杀菌剂银泰对苹果树两种主要病害的防治效果.植物保护学报,33(2):223-224]
Zhang X,Ma ZQ,Feng JT,Wu H,Han LR.2015.Review on research and development of botanical pesticides.Chinese Journal of Biological Control,31(5):685-698(in Chinese)[张兴,马志卿,冯俊涛,吴华,韩立荣.2015.植物源农药研究进展.中国生物防治学报,31(5):685-698]
Zhang ZL,Qu WJ,Li XF.2009.Guide of plant physiology experiment(4th edition).Beijing:Higher Education Press,pp.103-104(in Chinese)[张志良,瞿伟菁,李小方.2009.植物生理学实验指导(第4版).北京:高等教育出版社,pp.103-104]
Zhao L,Du J,Liu SQ,Cao H.2008.Antifeeding effects of Plutella xylostella L.by extracts of the seed of Kochia scoparia(L.)Schrad.Journal of Shanxi Agricultural Sciences,36(10):38-39(in Chinese)[赵磊,杜娟,刘素琪,曹挥.2008.地肤子粗提物对小菜蛾的拒食作用.山西农业科学,36(10):38-39]
Zhou MJ,Wan CP,Chen JY.2014.Screecing and antifungal mechanism of Chinese herb extracts against green mold of citrus.Modern Food Science and Technology,30(3):144-149(in Chinese)[周梦娇,万春鹏,陈金印.2014.柑橘绿霉病中草药高效抑菌剂的筛选及抑菌机理研究.现代食品科技,30(3):144-149]