反式肉桂醛对稻绿核菌细胞超微结构及3种呼吸酶活力的影响
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摘要
为初步探究反式肉桂醛(TC)对稻绿核菌的抑菌机制,采用透射电子显微镜观察了TC处理后稻绿核菌菌丝细胞的超微结构,并测定了2、4、8、15及30μg/mL系列质量浓度TC对稻绿核菌细胞壁完整性的影响,以及其对烟酰胺腺嘌呤二核苷酸-苹果酸脱氢酶(NADMDHase)、琥珀酸脱氢酶(SDHase)和三磷酸腺苷酶(ATPase)活力的影响。结果显示:经4μg/mL的TC处理后,菌丝细胞内脂质体显著增多,线粒体结构变模糊。当TC质量浓度升高至30μg/mL时,可破坏菌丝细胞壁的完整性,且对NAD-MDHase活力的相对抑制率为44.3%,对SDHase活力的相对抑制率为76.7%;而Na~+-K~+-ATPase、Ca~(2+)-Mg~(2+)-ATPase、Ca~(2+)-ATPase及Mg~(2+)-ATPase的活力随TC质量浓度升高呈U型变化,其中,4μg/mL的TC对ATPase活力的抑制效果最强,相对抑制率达78.4%以上。研究表明,细胞壁及线粒体可能是TC抑制稻绿核菌菌丝生长的作用靶点,具有进一步研究的价值。
The antifungal mechanism of trans-cinnamaldehyde(TC) against Villosiclava virens was investigated. The alteration of cell ultrastructure was observed using transmission electron microscope.The integrity of cell wall and activities of nicotinamide adenine dinucleotide-malic dehydrogenase(NAD-MDHase), succinate dehydrogenase(SDHase) and adenosine triphosphatase(ATPase) of V.virens were determined after treated with TC at the mass concentration of 2, 4, 8, 15 and 30 μg/mL. The results showed that the contents of lipidosome increased significantly and the structure of mitochondrion was indiscernible after treatment with TC at the mass concentration of 4 μg/mL. With the mass concentration of TC increasing to 30 μg/mL, the integrity of cell wall in V. virens was destroyed and the relative inhibition of NAD-MDHase and SDHase were reduced by 44.3% and 76.7%,respectively. Differently, the activities of Na~+-K~+-ATPase, Ca~(2+)-Mg~(2+)-ATPase, Ca~(2+)-ATPase and Mg~(2+)-ATPase fluctuated as U-curve with the concentration increase of TC. When the mass concentration of TC was 4 μg/mL, the highest inhibitive activity of TC on ATPase was the achieved among all the tested concentrations and the relative inhibition of TC on ATPase was higher than 78.4%. These results suggested that the cell wall and mitochondrion were the possible action targets for TC to inhibit the mycelial growth.
The antifungal mechanism of trans-cinnamaldehyde(TC) against Villosiclava virens was investigated. The alteration of cell ultrastructure was observed using transmission electron microscope.The integrity of cell wall and activities of nicotinamide adenine dinucleotide-malic dehydrogenase(NAD-MDHase), succinate dehydrogenase(SDHase) and adenosine triphosphatase(ATPase) of V.virens were determined after treated with TC at the mass concentration of 2, 4, 8, 15 and 30 μg/mL. The results showed that the contents of lipidosome increased significantly and the structure of mitochondrion was indiscernible after treatment with TC at the mass concentration of 4 μg/mL. With the mass concentration of TC increasing to 30 μg/mL, the integrity of cell wall in V. virens was destroyed and the relative inhibition of NAD-MDHase and SDHase were reduced by 44.3% and 76.7%,respectively. Differently, the activities of Na~+-K~+-ATPase, Ca~(2+)-Mg~(2+)-ATPase, Ca~(2+)-ATPase and Mg~(2+)-ATPase fluctuated as U-curve with the concentration increase of TC. When the mass concentration of TC was 4 μg/mL, the highest inhibitive activity of TC on ATPase was the achieved among all the tested concentrations and the relative inhibition of TC on ATPase was higher than 78.4%. These results suggested that the cell wall and mitochondrion were the possible action targets for TC to inhibit the mycelial growth.
引文
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[22]BANG K H,LEE D W,PARK H M,et al.Inhibition of fungal cell wall synthesizing enzymes by trans-cinnamaldehyde[J].Biosci Biotechnol Biochem,2000,64(5):1061-1063.
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[28]FERREIRA M D O P,CARDOSO M F,DA SILVA F D E C,et al.Antifungal activity of synthetic naphthoquinones against dermatophytes and opportunistic fungi:preliminary mechanism-ofaction tests[J].Ann Clin Microbiol Antimicrob,2014,13(1):26-31.
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[30]何玲,袁会珠,唐剑锋,等.新型杀菌剂氟醚菌酰胺对辣椒疫霉的作用机制初探[J].农药学学报,2016,18(2):185-193.HE L,YUAN H Z,TANG J F,et al.Preliminary studies on the action mechanism of the novel fungicide LH-2010A against Phytophthora capsici[J].Chin J Pestic Sci,2016,18(2):185-193.
[31]汪琨,徐峥,汪倩雯,等.肉桂醛特异性抑制酵母细胞壁合成的作用机理[J].食品与发酵工业,2012,38(3):68-72.WANG K,XU Z,WANG Q W,et al.Mechanism of yeast cell wall synthesis specifically inhibited by cinnamaldehyde[J].Food Ferment Ind,2012,38(3):68-72.
[32]曾红.莳萝子挥发油抗真菌活性及其作用机制研究[D].武汉:华中农业大学,2011.ZENG H.Antifungal activity and mechanism of essential oil from the seeds of Anethum graveolens L.[D].Wuhan:Huazhong Agricultural University,2011.
[33]CHEN C,LONG L,ZHANG F,et al.Antifungal activity,main active components and mechanism of Curcuma longa extract against Fusarium graminearum[J].PloS One,2018,13(3):e0194284.
[34]周智兴.黄岑苷、IgY抗白色念珠菌作用机制研究[D].南昌:南昌大学,2006.ZHOU Z X.Study of action mechanism of baicalin and IgY against Candida albicans[D].Nanchang:Nanchang University,2006.
[2]黄珊.水稻稻曲病研究进展[J].福建农业学报,2012,27(4):452-456.HUANG S.Advances on study of rice false smut(Ustilaginoidea virens)[J].Fujian J Agric Sci,2012,27(4):452-456.
[3]陆明红,刘万才,朱凤.稻曲病近年流行规律及治理对策探讨[J].中国植保导航,2018,38(5):44-47.LU M H,LIU W C,ZHU F.Discussion on epidemic rule of rice false smut in recent years and its controlling strategy[J].China Plant Prot,2018,38(5):44-47.
[4]KOISO Y,LI Y,IWASAKI S,et al.Ustiloxins,antimitotic cyclic peptides from false smut balls on rice panicles caused by Ustilaginoidea virens[J].J Antibiot,1994,47(7):765-773.
[5]SUN W B,WANG A L,XU D,et al.New ustilaginoidins from rice false smut balls caused by Villosiclava virens and their phytotoxic and cytotoxic activities[J].J Agric Food Chem,2017,65(25):5151-5160.
[6]王菲.水稻稻曲病菌遗传多样性及其抗DMI类杀菌剂分子机理的研究[D].武汉:华中农业大学,2015.WANG F.Study on genetic diversity and DMI fungicide resistance molecular mechanism of Villosiclava virens[D].Wuhan:Huazhong Agric Univ,2015.
[7]李晓菲,徐政.植物源杀菌剂研究进展[J].南方农业,2018,12(13):40-45.LI X F,XU Z.Progress in research on botanical fungicides[J].Southern Agric,2018,12(13):40-45.
[8]宋宗辉,张艺雯,王玲洁,等.肉桂醛的药理活性及其研究进展[J].解放军药学学报,2018,34(6):550-554.SONG Z H,ZHANG Y W,WANG L J,et al.Pharmacological activity of cinnamaldehyde and its research progress[J].Pharm J Chin PLA,2018,34(6):550-554.
[9]JAKHETIA V,PATEL R,KHATRI P,et al.Cinnamon:a pharmacological review[J].J Adv Sci Res,2010,1(2):19-23.
[10]农药登记毒理学试验方法:GB/T 15670.4-2017[S].北京:中国标准出版社,2017.Toxicological test methods of pesticides for registration:GB/T 15670.4-2017[S].Beijing:China Standards Press,2017.
[11]申素霞.植物源食品防腐剂的筛选及肉桂醛的抑菌机制研究[D].长春:吉林大学,2015.SHEN S X.The screening of natural plant source food preservatives and the research of antimicrobial mechanism of cinnamaldehyde[D].Changchun:Jilin University,2015.
[12]MAREI G I K,ABDELGALEIL S A M.Antifungal potential and biochemical effects of monoterpenes and phenylpropenes on plant[J].Plant Prot Sci,2017,54(1):9-16.
[13]SCHL?SSER I,PRANGE A.Antifungal activity of selected natural preservatives against the foodborne molds Penicillium verrucosum and Aspergillus westerdijkiae[J].FEMS Microbiol Lett,2018,365(13):1-8.
[14]XU L C,TAO N G,YANG W H,et al.Cinnamaldehyde damaged the cell membrane of Alternaria alternata and induced the degradation of mycotoxins in vivo[J].Ind Crop Prod,2018,112:427-433.
[15]WANG H W,YANG Z X,YING G Y,et al.Antifungal evaluation of plant essential oils and their major components against toxigenic fungi[J].Ind Crop Prod,2018,120:180-186.
[16]XIE Y J,HUANG Q Q,WANG Z J,et al.Structure-activity relationships of cinnamaldehyde and eugenol derivatives against plant pathogenic fungi[J].Ind Crop Prod,2017,97:388-394.
[17]FRIEDMAN M.Chemistry,antimicrobial mechanisms,and antibiotic activities of cinnamaldehyde against pathogenic bacteria in animal feeds and human foods[J].J Agric Food Chem,2017,65(48):10406-10423.
[18]SERRA E,HIDALGO-BASTIDA L A,VERRAN J,et al.Antifungal activity of commercial essential oils and biocides against Candida Albicans[J].Pathogens,2018,7(1):15.
[19]张文娟.肉桂醛抗黄曲霉作用及超微结构观察的研究[J].北京医科大学学报,1995,27(5):374.ZHANG W J.Sthudy on the antifungal effect of cinnamaldehyde against Aspergillus flavus and ultrastructure observation[J].J Peking Univ(Health Sci),1995,27(5):374.
[20]TANG X,SHAO Y L,TANG Y J,et al.Antifungal activity of essential oil compounds(geraniol and citral)and inhibitory mechanisms on grain pathogens(Aspergillus flavus and Aspergillus ochraceus)[J].Molecules,2018,23(9):2108-2125.
[21]PEREIRA F D E O,MENDES J M,LIMA I O,et al.Antifungal activity of geraniol and citronellol,two monoterpenes alcohols,against Trichophyton rubrum involves inhibition of ergosterol biosynthesis[J].Pharm Biol,2015,53(2):228-234.
[22]BANG K H,LEE D W,PARK H M,et al.Inhibition of fungal cell wall synthesizing enzymes by trans-cinnamaldehyde[J].Biosci Biotechnol Biochem,2000,64(5):1061-1063.
[23]SHREAZ S,BHATIA R,KHAN N,et al.Influences of cinnamic aldehydes on H+extrusion activity and ultrastructure of Candida[J].JMed Microbiol,2013,62(Pt 2):232-240.
[24]USTA J,KREYDIYYEH S,BARNABE P,et al.Comparative study on the effect of cinnamon and clove extracts and their main components on different types of ATPases[J].Hum Exp Toxicol,2003,22(7):355-362.
[25]ZENG H,CHEN X P,LIANG J N.In vitro antifungal activity and mechanism of essential oil from fennel(Foeniculum vulgare L.)on dermatophyte species[J].J Med Microbiol,2015,64(Pt 1):93-103.
[26]TIAN J,ZENG X B,FENG Z Z,et al.Zanthoxylum molle Rehd.essential oil as a potential natural preservative in management of Aspergillus flavus[J].Ind Crop Prod,2014,61(1):151-159.
[27]MA M M,WEN X F,XIE Y T,et al.Antifungal activity and mechanism of monocaprin against food spoilage fungi[J].Food Control,2018,84:561-568.
[28]FERREIRA M D O P,CARDOSO M F,DA SILVA F D E C,et al.Antifungal activity of synthetic naphthoquinones against dermatophytes and opportunistic fungi:preliminary mechanism-ofaction tests[J].Ann Clin Microbiol Antimicrob,2014,13(1):26-31.
[29]邢来君,李明春,魏东盛.普通真菌学[M].北京:高等教育出版社,2010:114-117.XING L J,LI M C,WEI D S.General mycology[M].Advanced Education Press,2010:114-117.
[30]何玲,袁会珠,唐剑锋,等.新型杀菌剂氟醚菌酰胺对辣椒疫霉的作用机制初探[J].农药学学报,2016,18(2):185-193.HE L,YUAN H Z,TANG J F,et al.Preliminary studies on the action mechanism of the novel fungicide LH-2010A against Phytophthora capsici[J].Chin J Pestic Sci,2016,18(2):185-193.
[31]汪琨,徐峥,汪倩雯,等.肉桂醛特异性抑制酵母细胞壁合成的作用机理[J].食品与发酵工业,2012,38(3):68-72.WANG K,XU Z,WANG Q W,et al.Mechanism of yeast cell wall synthesis specifically inhibited by cinnamaldehyde[J].Food Ferment Ind,2012,38(3):68-72.
[32]曾红.莳萝子挥发油抗真菌活性及其作用机制研究[D].武汉:华中农业大学,2011.ZENG H.Antifungal activity and mechanism of essential oil from the seeds of Anethum graveolens L.[D].Wuhan:Huazhong Agricultural University,2011.
[33]CHEN C,LONG L,ZHANG F,et al.Antifungal activity,main active components and mechanism of Curcuma longa extract against Fusarium graminearum[J].PloS One,2018,13(3):e0194284.
[34]周智兴.黄岑苷、IgY抗白色念珠菌作用机制研究[D].南昌:南昌大学,2006.ZHOU Z X.Study of action mechanism of baicalin and IgY against Candida albicans[D].Nanchang:Nanchang University,2006.