抗花生青枯菌的穿心莲内生真菌筛选及抑菌成分研究

doi:10.16409/j.cnki.2095-039x.2025.02.028

摘要/Abstract

摘要： 为获得对花生青枯病具有生防潜力的菌株，本研究采用牛津杯抑菌法，从穿心莲叶片内生真菌中筛选对花生青枯菌Ralstonia solanacearum有较强抑制作用的菌株，基于形态学和16S rDNA系统发育分析对其进行鉴定，通过共培养试验探究其抑菌机理，采用LC-MS代谢组学分析该菌株发酵提取液中的抑菌成分并验证部分代谢物的抗花生青枯菌活性。结果表明，穿心莲内生真菌CXL-11发酵提取液的抑菌圈直径为19.08 mm，MIC值为0.625 mg/mL，该菌株为刺盘孢属菌Colletotrichum sp.；其发酵提取液可破坏花生青枯菌膜完整性、使菌体表面皱缩扭曲、并诱导活性氧积累造成氧化胁迫；抑菌活性物质以聚酮类、香豆素类为主，其中异龙胆黄素、佛手柑内酯、东莨菪素、东莨菪苷和七叶亭，均对花生青枯菌有一定抑菌作用，辅因子的生物合成途径是菌株CXL-11发挥抗青枯菌作用的关键途径。因此，穿心莲内生真菌CXL-11作为花生青枯病生防菌株具有较好的应用潜力，为天然抗菌剂的开发提供理论支持。

关键词: 花生青枯菌, 内生真菌, 抑菌活性成分, 生物防治

Abstract: To identify potential biocontrol strains against peanut bacterial wilt caused by Ralstonia solanacearum, we screened endophytic fungi of Andrographis paniculata leaves using the Oxford cup inhibition method. A strain with strong inhibitory activity against R. solanacearum was isolated and identified as Colletotrichum sp. CXL-11 based on morphological characteristics and 16S rDNA phylogenetic analysis. We further investigated its antibacterial mechanism through co-culture experiments and analyzed the antifungal components in its fermentation extract using LC-MS metabolomics. The bioactivity of selected metabolites against R. solanacearum was also validated. The results showed that the fermentation extract of CXL-11 produced an inhibition zone of 19.08 mm against R. solanacearum, with a minimum inhibitory concentration (MIC) of 0.625 mg/mL. The extract disrupted the membrane integrity of R. solanacearum, causing surface shrinkage and deformation of bacterial cells, and induced oxidative stress through the accumulation of reactive oxygen species. Metabolomic analysis revealed that the primary antifungal components were polyketides and coumarins, including isogentisin, bergapten, scopoletin, scopolin, and esculetin, all of which exhibited inhibitory effects against R. solanacearum. The biosynthesis pathway of cofactors was identified as a key mechanism underlying the antibacterial activity of CXL-11. In conclusion, the endophytic fungus Colletotrichum sp. CXL-11 demonstrates significant potential as a biocontrol agent for peanut bacterial wilt. This study provides theoretical support for the development of natural antimicrobial agents and highlights the promising application of CXL-11 in sustainable disease management.

Key words: Ralstonia solanacearum infecting peanuts, endophytic fungi, inhibition active substances, biological control

中图分类号:

S476

黄瑾, 胡静雯, 丁妹, 杨帆, 罗文荣, 杜勤. 抗花生青枯菌的穿心莲内生真菌筛选及抑菌成分研究[J]. 中国生物防治学报, 2025, 41(2): 396-410.

HUANG Jin, HU Jinwen, DING Mei, YANG Fan, LUO Wenrong, DU Qin. Screening of Endophytic Fungi of Andrographis paniculata Resistant to Ralstonia solanacearum Infecting Peanuts and Study of Their Inhibitory Components[J]. Chinese Journal of Biological Control, 2025, 41(2): 396-410.

参考文献

[1] 国家药典委员会. 中华人民共和国药典: 一部[M]. 北京: 中国医药科技出版社, 2020, 280.
[2] 母育成, 邢相宜, 晏子俊, 等. 穿心莲内酯的抗菌作用研究进展[J]. 贵州中医药大学学报, 2021, 43(1): 85-89.
[3] 卜宣尹, 杨卫丽. 植物内生菌抑菌机制和抑菌次生代谢产物的研究进展[J]. 现代药物与临床, 2021, 36(10): 2200-2206.
[4] 刘思成, 许言超, 吴耽, 等. 鱼腥草内生菌Aspergillus sp. GZWMJZ-636次级代谢产物研究[J]. 中草药, 2021, 52(10): 2863-2868.
[5] Cao L L, Zhang Y Y, Liu Y J, et al. Anti-phytopathogenic activity of sporothriolide, a metabolite from endophyte Nodulisporium sp. A21 in Ginkgo biloba[J]. Pesticide Biochemistry And Physiology, 2016, 129: 7-13.
[6] 邓振山, 高飞, 刘玉珍, 等. 一株酸枣内生菌的鉴定及其抗菌活性物质的初步分离[J]. 广西植物, 2018, 38(11): 1486-1492.
[7] 金玉, 任梦楠, 杨红澎, 等. 短柄龙胆内生菌的分离鉴定及抗菌活性分析[J]. 北方园艺, 2018(1): 135-139.
[8] Mansfield J, Genin S, Magori S, et al. Top 10 plant pathogenic bacteria in molecular plant pathology[J]. Molecular Plant Pathology, 2012, 13(6): 614-629.
[9] Cai L, Chen J, Liu Z, et al. Magnesium oxide nanoparticles: effective agricultural antibacterial agent against Ralstonia solanacearum[J]. Frontiers in Microbiology, 2018, 9: 790.
[10] 范会云, 陈卫明, 邓乔华, 等. 广藿香青枯病研究进展[J]. 中药材, 2021, 44(1): 229-232.
[11] 杨威. 姜青枯病的生物防治及生防菌株对土壤环境的影响研究[D]. 南京: 南京农业大学, 2011.
[12] 高欢乐. 论青枯菌病害的研究进展[J]. 法制与社会, 2009(12): 354.
[13] 吴思炫, 高复云, 张锐澎, 等. 番茄青枯病生物防治的研究进展[J]. 应用生态学报, 2023, 34(9): 2585-2592.
[14] 黄丽丹, 陈玉惠. 生防菌及相关生物技术在植物病害防治中的应用[J]. 西南林学院学报, 2006(1): 85-89.
[15] 宋薇薇, 朱辉, 余凤玉, 等. 植物内生菌及其对植物病害的防治作用综述[J]. 江苏农业科学, 2018, 46(6): 12-16.
[16] 卯婷婷, 陶刚, 赵兴丽, 等. 4种微生物菌剂对辣椒主要病害的生物防治作用[J]. 中国生物防治学报, 2020, 36(2): 258-264.
[17] 张祖姣, 袁志辉, 赵子豪, 等. 抗姜青枯菌银杏内生真菌的分离及抑菌特性[J]. 中国生物防治学报, 2018, 34(6): 890-896.
[18] 程小卿, 江浩铭, 崔业旋, 等. 内生真菌Alternaria sp. GHX-P17代谢产物防治广藿香青枯病及保护酶的变化[J]. 广西植物, 2023, 43(7): 1244-1251.
[19] Li N, Xu D, Huang R H, et al. A new source of diterpene lactones from Andrographis paniculata (Burm.f.) Nees-two endophytic fungi of Colletotrichum sp. with antibacterial and antioxidant activities[J]. Frontiers in Microbiology, 2022, 13: 819770.
[20] 赵燕妮, 余瑞, 刘欢, 等. 功能代谢组学技术在微生物研究中的应用[J]. 微生物学报, 2023, 63(8): 3009-3025.
[21] 朱君, 张鑫悦, 崔敏, 等. 4种中草药提取液的工艺优化及抗菌效果对比研究[J]. 常熟理工学院学报, 2023, 37(2): 65-71.
[22] 王星星, 潘旻, 蒋慧, 等. 马尾藻多酚对大肠杆菌的抑制效果研究[J]. 饲料工业, 2024, 45(13): 77-82.
[23] Xiao L, Niu H J, Qu T L, et al. Streptomyces sp. FX13 inhibits fungicide-resistant Botrytis cinerea in vitro and in vivo by producing oligomycin A[J]. Pesticide Biochemistry and Physiology, 2021, 175: 104834.
[24] Ma D Y, Ji D C, Zhang Z Q, et al. Efficacy of rapamycin in modulating autophagic activity of Botrytis cinerea for controlling gray mold[J]. Postharvest Biology and Technology, 2019, 150: 158-165.
[25] Zhang W H, Xie W, Hou J B, et al. Analytical research on the separation and residue of chiral pesticide triadimenolin fruit and vegetable puree[J]. Journal of Separation Science, 2021, 44(18): 3516-3523.
[26] 秦璐. 木犀草苷对运动性疲劳大鼠的心肌氧化应激及抗氧化基因表达的影响[J]. 分子植物育种, 2023, 21(9): 2900-2907.
[27] 吴晓萍, 胡英杰, 卢元媛, 等. 小叶榕叶有效成分表阿夫儿茶精的分析方法研究[J]. 药物分析杂志, 2008, 28(6): 992-995.
[28] 路晓庆, 杨芮, 李炘正, 等. 黄酮类物质的生物功能及作用机制研究进展[J]. 中西医结合心脑血管病杂志, 2018, 16(22): 3283-3286.
[29] 李素云, 李峥, 王立芹, 等. 槲皮素在Caco-2单层细胞模型上的跨膜吸收和甲基化代谢[J]. 中国药理学与毒理学杂志, 2019, 33(4): 273-280.
[30] 赵新贝, 倪云霞, 刘新涛, 等. 基于非靶标代谢组学分析拜赖青霉菌株47M-1的抑菌活性成分[J]. 中国生物防治学报, 2023, 39(5): 1156-1171.
[31] 吴劲松. 植物对病原微生物的“化学防御”:植保素的生物合成及其分子调控机制[J]. 应用生态学报, 2020, 31(7): 2161-2167.
[32] 杨亮. 羟基香豆素类化合物对青枯雷尔氏菌致病特性的调控作用研究[D]. 重庆: 西南大学, 2020.
[33] Yin C, Xie L, Guo Y. Phytochemical analysis and antibacterial activity of Gentiana macrophylla extract against bacteria isolated from burn wound infections[J]. Microbial Pathogenesis, 2018, 114: 25-28.
[34] 张国荣, 翟丽霞, 王燕萍, 等. 药用植物内生菌次级代谢产物药理作用研究进展[J]. 中国药房, 2021, 32(7): 880-884.
[35] Mao Z, Zhang W, Wu C, et al. Diversity and antibacterial activity of fungal endophytes from Eucalyptus exserta[J]. BMC Microbiology, 2021, 21(1): 155.
[36] Dey P, Barman M, Mitra A, et al. Lipid-rich endo-metabolites from a vertically transmitted fungal endophyte Penicillium sp. PM031 attenuate virulence factors of phytopathogenic Ralstonia solanacearum[J]. Microbiological Research, 2022, 261: 127058.
[37] 姜雯婷, 戴好富, 刘寿柏, 等. 52种热带药用植物抗烟草青枯菌的体外活性筛选[J]. 微生物学杂志, 2011, 31(6): 15-18.
[38] 姜宁, 刘晓鹏, 滕建勋, 等. 紫油厚朴叶提取物对马铃薯青枯病菌的抑制作用及防病效果[J]. 植物保护, 2008(2): 58-60.
[39] 黄雯. 青枯菌LAMP检测方法的建立及植物精油抑菌活性评价[D]. 重庆: 西南大学, 2017.
[40] Kim J W, Shim S H. The fungus Colletotrichum as a source for bioactive secondary metabolites[J]. Archives of Pharmacal Research, 2019, 42(9): 735-753.
[41] Wang W X, Kusari S, Laatsch H, et al. Antibacterial azaphilones from an endophytic fungus, Colletotrichum sp. BS4[J]. Journal of Natural Products, 2016, 79(4): 704-710.
[42] Zhang W, Draeger S, Schulz B, et al. Ring B aromatic steroids from an endophytic fungus, Colletotrichum sp[J]. Natural Product Communications, 2009, 4(11): 1449-1454.
[43] 周连玉, 钟松, 朵红梅, 等. 中华羊茅内生真菌Epichloё sp.挥发性物质的抑菌活性及成分分析[J]. 天然产物研究与开发, 2019, 31(9): 1543-1551.
[44] 袁中伟, 谷可欣, 张天翼, 等. 松萝酸对耐甲氧西林金黄色葡萄球菌的抑菌作用机制研究[J]. 甘肃农业大学学报, 2019, 54(4): 22-29.
[45] Li L, Shi Y, Cheng X, et al. A cell-penetrating peptide analogue, P7, exerts antimicrobial activity against Escherichia coli ATCC25922 via penetrating cell membrane and targeting intracellular DNA[J]. Food Chemistry, 2015, 166: 231-239.
[46] 张倩. 马齿苋内生菌抗青枯病的研究[D]. 广州: 华南农业大学, 2019.
[47] 于艳梅. 几种植物源化合物对青枯菌的抑制作用及机理研究[D]. 重庆: 西南大学, 2015.
[48] Zhang J, Gao L, Lin H, et al. Discovery of antibacterial compounds against Xanthomonas citri subsp. citri from a marine fungus Aspergillus terreus SCSIO 41202 and the mode of action[J]. Journal of Agricultural and Food Chemistry, 2024, 72(22): 12596-12606.
[49] Shimizu B, Miyagawa H, Ueno T, et al. Morning glory systemically accumulates scopoletin and scopolin after interaction with Fusarium oxysporum[J]. Zeitschrift Fur Naturforschung Section C-a Journal of Biosciences, 2005, 60(1-2): 83-90.
[50] Ku J T, Chen A Y, Lan E I. Metabolic engineering design strategies for increasing acetyl-CoA flux[J]. Metabolites, 2020, 10(4): 166.
[51] Shuang L, Wenya H, Zhiwen W, et al. Production of riboflavin and related cofactors by biotechnological processes[J]. Microbial Cell Factories, 2020, 19(1): 31.
[52] Wang R, He F, Ning Y, et al. Fine-tuning of RBOH-mediated ROS signaling in plant immunity[J]. Trends in Plant Science, 2020, 25(11): 1060-1062.
[53] 夏清珊. 核黄素参与水稻非生物胁迫响应的机理研究[D]. 长沙: 湖南大学, 2023.