脂肽对蜜蜂球囊菌的抑制作用研究

doi:10.16431/j.cnki.1671-7236.2025.09.037

摘要/Abstract

摘要： 【目的】探究脂肽对蜜蜂球囊菌的抑制作用和机制，以及其体外抑菌和杀菌活性。【方法】通过形态学和分子生物学等方法鉴定蜜蜂球囊菌，采用抑菌圈试验法验证脂肽能否抑制蜜蜂球囊菌生长，采用倍比稀释法测定脂肽对蜜蜂球囊菌的最小抑菌浓度(MIC)和最小杀真菌浓度(MFC)。采用血球计数板观察脂肽对蜜蜂球囊菌孢子萌发的影响；高效液相色谱法测定蜜蜂球囊菌细胞膜中麦角甾醇和细胞壁中几丁质含量，确定脂肽Iturin对蜜蜂球囊菌是否存在抑制作用。【结果】显微镜观察发现，分离菌菌丝呈有隔膜的分枝状和具有球形的孢子囊、孢子等形态。18S rDNA测序结果显示,分离菌与蜜蜂球囊菌(GenBank登录号：GQ867785.1)序列相似性为100%，确定获得的菌株为蜜蜂球囊菌。脂肽伊枯草菌素(Iturin)对蜜蜂球囊菌的抑制作用最好，抑菌圈半径达8.53 mm,丰原素(Fengycin)抑菌圈半径为5.84 mm，表面活性素(Surfactin)无抑制作用。Iturin对蜜蜂球囊菌的MIC为6.25～25 μg/mL，MIC₅₀、MIC₉₀和MFC分别为6.25、25和50 μg/mL。与0 μg/mL Iturin处理组相比，6.25、12.5、25和50 μg/mL Iturin处理组蜜蜂球囊菌孢子萌发率均显著下降(P＜0.05)。麦角甾醇含量测定结果显示，与0 μg/mL Iturin处理组相比，12.5、25和50 μg/mL Iturin处理组蜜蜂球囊菌细胞膜麦角甾醇含量均显著下降(P＜0.05)。几丁质含量测定结果显示，与0 μg/mL Iturin处理组相比，25和50 μg/mL Iturin处理组蜜蜂球囊菌细胞壁几丁质含量显著下降(P＜0.05)。【结论】脂肽Iturin通过减少孢子萌发，破坏细胞膜中麦角甾醇的生物合成及损害细胞壁中几丁质的合成来有效抑制蜜蜂球囊菌。

关键词: 脂肽; 蜜蜂球囊菌; 抑制作用; 麦角甾醇; 几丁质

Abstract: 【Objective】 This study aimed to explore the inhibitory effect and mechanism of lipopeptides on Ascosphaera apis,as well as its antibacterial and bactericidal activities in vitro. 【Method】 Ascosphaera apis was identified by morphological and molecular biological methods.The inhibition zone test method was used to verify whether lipopeptides could inhibit the growth of Ascosphaera apis.The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of lipopeptides against Ascosphaera apis were determined by the multiple dilution method.The effect of lipopeptides on the spore germination of Ascosphaera apis was observed using a hemocytometer.The contents of ergosterol in the cell membrane and chitin in the cell wall of Ascosphaera apis were determined by high performance liquid chromatography to determine whether the lipopeptide Iturin had an inhibitory effect on Ascosphaera apis. 【Result】 Microscopic observation revealed that the isolated fungus exhibited septate,branched hyphae along with spherical sporangia and spores.18S rDNA sequencing results showed that the isolated strain had 100% sequence similarity with Ascosphaera apis (GenBank accession No.:GQ867785.1),confirming it was Ascosphaera apis.Among the tested lipopeptides,Iturin demonstrated the strongest inhibitory effect against Ascosphaera apis,with an inhibition zone radius of 8.53 mm,while Fengycin showed a smaller inhibition zone radius (5.84 mm),and Surfactin exhibited no inhibitory activity.The MIC of Iturin against Ascosphaera apis ranged from 6.25 to 25 μg/mL,with MIC₅₀,MIC₉₀ and MFC values of 6.25,25 and 50 μg/mL,respectively.Compared with 0 μg/mL Iturin group,the spore germination rates of Ascosphaera apis were significantly decreased (P＜0.05) in all Iturin-treated groups (6.25,12.5,25 and 50 μg/mL).Ergosterol content analysis revealed that compared with 0 μg/mL Iturin group,the ergosterol contents in the cell membrane of Ascosphaera apis were significantly decreased (P＜0.05) in 12.5,25 and 50 μg/mL Iturin-treated groups.Similarly,chitin content measurements showed that the chitin contents in the fungal cell wall were significantly decreased (P＜0.05) in 25 and 50 μg/mL Iturin-treated groups compared with 0 μg/mL Iturin group. 【Conclusion】 These results demonstrated that Iturin effectively inhibited Ascosphaera apis by reducing spore germination,disrupting ergosterol biosynthesis in the cell membrane,and impairing chitin synthesis in the cell wall.

Key words: lipopeptide; Ascosphaera apis; inhibitory effect; ergosterol; chitin

中图分类号:

S859.79

高杨, 常硕, 李志国. 脂肽对蜜蜂球囊菌的抑制作用研究[J]. 中国畜牧兽医, 2025, 52(9): 4416-4426.

GAO Yang, CHANG Shuo, LI Zhiguo. Study on the Inhibitory Effect of Lipopeptide on Ascosphaera apis[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(9): 4416-4426.

参考文献

[1] 黄文忠，张学锋，罗岳雄，等.蜜蜂白垩病的研究进展[J].黑龙江畜牧兽医，2017，15:69-71. HUANG W Z，ZHANG X F,LUO Y X,et al.Study on bee chalk-brood disease[J].Heilongjiang Animal Science and Veterinary Medicine,2017,15:69-71.(in Chinese)
[2] BOONMEE T，SINPOO C，THAYATHAM K，et al.Atmospheric non-thermal plasma inactivation of Ascosphaera apis,the causative agent of chalkbrood disease in honeybee[J].Scientific Reports,2024,14(1):1831.
[3] 李江红，郑志阳，洪双燕，等.蜜蜂患白垩病虫体内一株球囊菌拮抗细菌的分离与鉴定[J].中国农业科学，2012，45(5):973-980. LI J H,ZHENG Z Y,HONG S Y,et al.Isolation and identification of an antagonistic bacterial strain against Ascosphaera apis from honeybee larvae infected with chalkbrood disease[J].Scientia Agricultura Sinica,2012,45(5):973-980.(in Chinese)
[4] YODER J A,JAJACK A J,CORNACCHIONE W S,et al. In vitro evaluation of sugar syrups,antibiotics,and miticides on growth of honey bee pathogen,Ascosphaera apis:Emphasis for chalkbrood prevention is on keeping bees healthy[J].Apidologie,2014,45(5):568-578.
[5] 李江红，王海，庄建斌，等.蜜蜂对接种球囊菌孢子幼虫清除行为的研究[J].中国蜂业，2010，61(12):5-8. LI J H,WANG H,ZHUANG J B,et al.Removing behavior of adult honeybee to larvea innoculated by the spores of Ascospheara apis[J].Apiculture of China,2010,61(12):5-8.(in Chinese)
[6] MRAZ P，ZABKA M，HOSTICKOVA I，et al.Effect of selected botanical compounds on Ascosphaera apis and Apis mellifera[J].Industrial Crops & Products,2023,197:116649.
[7] 王文迪，赵婷婷，马红霞，等.解淀粉芽胞杆菌抗菌机制的研究进展[J].中国兽医杂志，2023，59(8):88-92. WANG W D,ZHAO T T,MA H X,et al.Research progress on antibacterial mechanism of Bacillus amyloliquefaciens[J].Chinese Journal of Veterinary Medicine,2023,59(8):88-92.(in Chinese)
[8] INES M,DHOUHA G.Lipopeptide surfactants:Production,recovery and pore forming capacity[J].Peptides,2015,71:100-112.
[9] JAHAN R,BODRATTI A M,TSIANOU M,et al.Biosurfactants,natural alternatives to synthetic surfactants:Physicochemical properties and applications[J].Advances in Colloid and Interface Science，2020,275:102061.
[10] MEENA K R,KANWAR S S.Lipopeptides as the antifungal and antibacterial agents:Applications in food safety and therapeutics[J].BioMed Research International,2015,2015:473050.
[11] DENG Y J,CHEN Z,CHEN Y P，et al.Lipopeptide C17 fengycin B exhibits a novel antifungal mechanism by triggering metacaspase-dependent apoptosis in Fusarium oxysporum[J].Journal of Agricultural and Food Chemistry,2024,72(14):7943-7953.
[12] 陈尚里，于福田，沈圆圆，等.高产抗菌脂肽Fengycin芽孢杆菌的诱变育种和发酵条件优化[J].食品工业科技，2023，44(23):134-143. CHEN S L,YU F T,SHEN Y Y,et al.Mutation breeding and optimization of fermentation conditions of Bacillus highly producing antimicrobial lipopeptide fengycin[J].Science and Technology of Food Industry,2023,44(23):134-143.(in Chinese)
[13] 胡仿香,李霜.生物表面活性剂Surfactin生产菌株的定向改造策略[J].微生物学报，2018，58(10):1711-1721. HU F X,LI S.Directed modification of strains for surfactin production[J].Acta Microbiologica Sinica,2018,58(10):1711-1721.(in Chinese)
[14] 孟勇，李侠清，张星，等.脂肽类表面活性剂及其应用研究进展(Ⅰ)——结构、性能及生物合成[J].精细石油化工，2023，40(3):69-73. MENG Y,LI X Q,ZHANG X,et al.Progress in the study of lipopeptide surfactants and their applications (Ⅰ)—Structure,properties and biosynthesis[J].Speciality Petrochemicals, 2023,40(3):69-73.(in Chinese)
[15] 李道明，王瑛，陈超，等.芽孢杆菌几种重要抗菌脂肽研究进展[J].生物工程学报，2022，38(5):1768-1783. LI D M,WANG Y,CHEN C,et al.Advances in several important antimicrobial lipopeptids from Bacillus spp.[J].Chinese Journal of Biotechnology,2022,38(5):1768-1783.(in Chinese)
[16] JEMIL N，BESBES I，GHARBI Y，et al.Bacillus methylotrophicus DCS1:Production of different lipopeptide families,in vitro antifungal activity and suppression of fusarium wilt in tomato plants[J].Current Microbiology,2024,81 (6):142.
[17] CAULIER S，NANNAN C，GILLIS A，et al.Overview of the antimicrobial compounds produced by members of the Bacillus subtilis group[J].Frontiers in Microbiology,2019,10:302.
[18] LI T X，LI L L，DU F Y，et al.Activity and mechanism of action of antifungal peptides from microorganisms:A review[J].Molecules,2021,26(11):3438.
[19] ZHAO H B，SHAO D Y，JIANG C，et al.Biological activity of lipopeptides from Bacillus[J].Applied Microbiology and Biotechnology,2017,101(15):5951-5960.
[20] 陈旭波.饲料中添加抗菌脂肽Surfactin对断奶仔猪生长性能及血液生化指标的影响[D].厦门：集美大学，2024. CHEN X B.Effects of dietary surfactin supplementation on growth performance and serum biochemical indexes of weanling piglets[D].Xiamen:Jimei University,2024.(in Chinese)
[21] 米婧璇,张飞燕,孟祥荷,等.伊枯草菌素抑菌稳定性及对雪梨采后保鲜的影响[J].河北省科学院学报,2024,41(1):65-70. MI J X,ZHANG F Y,MENG X H,et al.Antibacterial stability analysis of Iturin and its effect on postharvest preservation of snow pears[J].Journal of the Hebei Academy of Sciences,2024,41(1):65-70.
[22] JI Y Y,SUN C M,WU S M.Transcriptomic and biochemical analysis of the antimicrobial mechanism of lipopeptide Iturin W against Staphylococcus aureus[J].International Journal of Molecular Sciences,2024,25(18):9949.
[23] WEI X Y,WANG H F,LIU S J,et al.Surfactin’s impact on gut microbiota and intestinal tumor cells[J].International Journal of Biological Macromolecules,2024,287:138607.
[24] HAN D,JI Y L,YANG S X,et al.Therapeutic effect of iturin A on Candida albicans oral infection and its pathogenic factors[J].Antimicrobial Agents and Chemotherapy,2023,68(1):e0094823.
[25] 罗晓娇,孙静,陆颖健.解淀粉芽孢杆菌中脂肽的生物合成、抑菌机理及应用的研究进展[J].食品工业科技,2022,43(19):462-470. LUO X J,SUN J,LU Y J.Research progress in the biosynthesis,antimicrobial mechanism,and application of lipopeptides in Bacillus amyloliquefaciens[J].Science and Technology of Food Industry,2022,43(19):462-470.(in Chinese)
[26] 陈萍,冯芬,杨恬然,等.脂肽类抗生素及其作用机制[J].微生物学杂志,2015,35(5):89-93. CHEN P,FENG F,YANG T R,et al.Lipopeptide antibiotics and its action mechanism[J].Journal of Microbiology,2015,35(5):89-93.(in Chinese)
[27] 熊毅,段燕文,朱湘成.表面活性素在药物开发中应用的研究进展[J].中国医药工业杂志,2022,53(1):56-64. XIONG Y,DUAN Y W,ZHU X C.Research progress on the application of surfactin in drug development[J].Chinese Journal of Pharmaceuticals,2022,53(1):56-64.(in Chinese)
[28] NAUGHTON P J,MARCHANT R,NAUGHTON V,et al.Microbial biosurfactants:Current trends and applications in agricultural and biomedical industries[J].Journal of Applied Microbiology,2019,127(1):12-28.
[29] 戚伟，刘小兰，单心怡，等.蜜蜂球囊菌的分离鉴定及体外抑菌试验[J].江西畜牧兽医杂志，2019，6:12-17. QI W,LIU X L,SHAN X Y,et al.Isolation,identification and in vitro bacteriostatic assay of Ascospheara apis[J].Jiangxi Journal of Animal Husbandry & Veterinary Medicne,2019,6:12-17.(in Chinese)
[30] 候梦赏，赵必安，邱园妹，等.球囊菌侵染对意大利蜜蜂幼虫肠道免疫与解毒相关基因表达及蛋白质、脂质和糖类含量的影响[J].昆虫学报，2021，64(5):574-584. HOU M S,ZHAO B A,QIU Y M,et al.Effects of Ascosphaera apis infection on the expression of immune-and detoxification-related genes and contents of proteins,lipids and glucoses in the gut of Apis mellifera ligustica larvae[J].Acta Entomologica Sinica,2021,64(5):574-584.(in Chinese)
[31] 常志光，牛庆生，刘楠楠，等.大蒜素对蜜蜂球囊菌的抑菌和杀菌活性研究[J].特产研究，2018，40(4):35-38. CHANG Z G，NIU Q S,LIU N N,et al.The antibacterial and bactericidal activities of allicin against the pathogenic bacteria of Ascosphaera apis[J].Special Wild Economic Animal and Plant Research,2018,40(4):35-38.(in Chinese)
[32] 张岱.枯草芽胞杆菌ZD01次级代谢产物对马铃薯早疫病菌的抑制机理[D].保定：河北农业大学，2023. ZHANG D.The antifungal mechanism of secondary metabolites produced by Bacillus subtilis ZD01 against Alternaria solani[D].Baoding:Hebei Agriculture University，2023.(in Chinese)
[33] 朱晓兰,夏文水.甲壳素纯度的高效液相色谱法测定[J].分析测试学报，2003，22(4):9-13. ZHU X L,XIA W S.Determination of chitin purity by high performance liquid chromatography[J].Journal of Instrumental Analysis,2003,22(4):9-13.(in Chinese)
[34] 余林生,孟祥金.蜜蜂白垩病的病原和防治技术研究[J].中国兽医科技,1999,12:6-8. YU L S,MENG X J.Study on the pathogen and control technique of bee chalkbrood[J].Chinese Journal of Veterinary Science and Technology,1999,12:6-8.(in Chinese)
[35] 王晓琼，毕秀芳，谢晓凤，等.伊枯草菌素A对草莓腐败菌的抑制效果研究[J].天然产物研究与开发，2020，32(11):1889-1895. WANG X Q,BI X F,XIE X F,et al.Antibacterial effect of iturin A on strawberry spoilage mold[J].Natural Product Research and Development,2020,32(11):1889-1895.(in Chinese)
[36] WANG S Y,XU M,HAN Y,et al.Exploring mechanisms of antifungal lipopeptide Iturin A from Bacillus against Aspergillus niger[J].Journal of Fungi,2024,10(3):172.
[37] YANG S X,JI Y L,XUE P Y,et al.Insights into the antifungal mechanism of Bacillus subtilis cyclic lipopeptide iturin A mediated by potassium ion channel[J].International Journal of Biological Macromolecules,2024,277:134306.
[38] RODRIGUES M L,The multifunctional fungal ergosterol[J].mBio,2018,9(5):e01755-18.
[39] SHAW J A,MOL P C,BOWERS B,et al.The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle[J].Journal of Cell Biology,1991,114(1):111-123.
[40] 代富英，周金燕,谭红.捷安肽素抗真菌作用机理研究[J].成都医学院学报，2006，2:86-90. DAI F Y,ZHOU J Y,TAN H.A research on mechanism of antifungal polypeptide Jiean-peptide[J].Journal of Chengdu Medical College，2006,2:86-90.(in Chinese)
[41] MARTINEZ S D,RUIZ H J.Functional analysis of the MAPK pathways in fungi[J].Revista Iberoamericana de Micologia,2017,34(4):192-202.