2025年04月23日 星期三

广西师范大学学报（自然科学版） ›› 2025, Vol. 43 ›› Issue (1): 39-47.doi: 10.16088/j.issn.1001-6600.2024040701

• “生态保护与资源可持续利用”专辑 • 上一篇    下一篇

柑橘溃疡病菌的分离鉴定及其微生物源抑制剂筛选

刘铭希1,2,3,4, 李奇聪1,2,3,4, 冉川1,2,3,4, 张丝1,2,3,4, 施慧露1,2,3,4, 邓志勇1,2,3,4, 骆海玉1,2,3,4*, 邓业成1,2,3,4*   

  1. 1.珍稀濒危动植物生态与环境保护教育部重点实验室(广西师范大学),广西 桂林 541006;
    2.广西漓江流域景观资源保育与可持续利用重点实验室(广西师范大学),广西 桂林 541006;
    3.广西师范大学 生命科学学院,广西 桂林 541006;
    4.广西师范大学 可持续发展创新研究院,广西 桂林 541006
  • 收稿日期:2024-04-07 修回日期:2024-06-21 出版日期:2025-01-05 发布日期:2025-02-07
  • 通讯作者: 邓业成(1965—),男,广西全州人,广西师范大学教授,博士。E-mail:dyecheng@163.com
    骆海玉(1985—),女,广西桂林人,广西师范大学副教授,博士。E-mail:luohaiyu69@163.com
  • 基金资助:
    广西自然科学基金(2021GXNSFAA196064);广西重点研发计划(桂科AB21196065,桂科AB22080071)

Isolation and Identification of Xanthomonas citri subsp. citri and Screening of Microbial Derived Inhibitors

LIU Mingxi1,2,3,4, LI Qicong1,2,3,4, RAN Chuan1,2,3,4, ZHANG Si1,2,3,4, SHI Huilu1,2,3,4, DENG Zhiyong1,2,3,4, LUO Haiyu1,2,3,4*, DENG Yecheng1,2,3,4*   

  1. 1. Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection (Guangxi Normal University), Ministry of Education, Guilin Guangxi 541006, China;
    2. Guangxi Key Laboratory of Landscape Resources Conservation and Sustainable Utilization in Lijiang River Basin (Guangxi Normal University), Guilin Guangxi 541006, China;
    3. College of Life Sciences, Guangxi Normal University, Guilin Guangxi 541006, China;
    4. Institute for Sustainable Development and Innovation, Guangxi Normal University, Guilin Guangxi 541006, China
  • Received:2024-04-07 Revised:2024-06-21 Online:2025-01-05 Published:2025-02-07

PDF (PC)

162

摘要： 本文对柑橘溃疡病菌进行分离鉴定,并研究血散薯Stephania dielsiana Y. C. Wu内生真菌提取物对柑橘溃疡病菌的抑制活性,为柑橘溃疡病微生物源防治剂的开发奠定基础。利用组织分离法和平板划线法从患柑橘溃疡病的沃柑叶中分离纯化得到菌株KY(F),其在NA培养基上菌落呈黄色、圆形、黏稠状、全缘、有光泽、表面微隆起;经染色和形态学观察确定其为革兰氏阴性菌,具有极生单鞭毛,有荚膜;PCR结果显示菌株KY(F)能扩增出符合预期大小的目的条带(413 bp),具有和柑橘溃疡病原菌相同且独有的保守蛋白基因;根据柯赫氏法则,将菌株KY(F)接种到健康沃柑离体叶片上能产生典型的柑橘溃疡病症状,确定菌株KY(F)为柑橘溃疡病菌。通过带毒平板法测定18种血散薯内生真菌乙酸乙酯提取物对该病原菌的抑制活性。结果表明,供试浓度为5 g/L时,18种提取物对KY(F)均能完全抑制,MIC值为0.312 5～5 g/L。其中6株内生真菌(Stdif6、叶-7、叶-9、J-10、J-29和Y-10)的提取物对柑橘溃疡病菌的抑制活性最高,MIC值为0.312 5～0.625 g/L,均小于1 g/L;其次为5株内生真菌(J-8、J-21、J-36、Y-64、和YB-5)的提取物,MIC值为1.25 g/L,均小于2 g/L;Stdif9和Y-66提取物对柑橘溃疡病菌也有较高的抑制活性,MIC值为2.5 g/L;叶-11、Y-51、Y-59、YB-25和YB-26这5种提取物对柑橘溃疡病菌的抑制活性较其他菌株提取物低,MIC值为5 g/L。研究结果为后期从血散薯内生真菌次生代谢产物中寻找柑橘溃疡病防治剂提供参考。

关键词: 柑橘溃疡病菌, 血散薯, 内生真菌, 抑菌活性, 活性物质, 生物防治剂

Abstract: In this paper, the citrus canker pathogen, was isolated and identified, and the inhibitory activity of endophytic fungi associated with Stephania dielsiana Y. C. Wu against the pathogen was studied, which will lay a foundation for the development of microbial control agents for citrus canker disease. The pathogenic strain KY(F) was isolated from the infected Orah citrus leaves with canker disease using tissue isolation and streak plate methods. The bacterial colonies on NA medium were yellow, round, viscous, full edge, glossy, and slightly raised on the surface. After staining and morphological observation, it was found that it is a gram-negative bacterium with polar single flagella and a capsule. The PCR results showed that strain KY(F) could amplify a target band of expected size (413 bp), with a unique conserved protein gene similar to that of citrus canker pathogen. According to the Koch’s rule, the pathogen was inoculated to healthy detached leaves of Orah citrus, and typical symptoms of citrus canker disease was formed. The results indicated that strain KY(F) was a citrus canker pathogen. The inhibitory activity of ethyl acetate extracts from 18 endophytic fungi isolated from S. dielsiana against this pathogen was determined using the toxin plate method. When the test concentration was 5 g/L, all 18 extracts could completely inhibit strain KY(F), with a MIC value ranging from 0.312 5 g/L to 5 g/L. The extracts of six endophytic fungi (Stdif6, leaf-7, leaf -9, J-10, J-29, and Y-10) showed the highest inhibitory activity against strain KY(F), with MICs ranging from 0.312 5 g/L to 0.625 g/L, all less than 1 g/L. Next were extracts of 5 endophytic fungi (J-8, J-21, J-36, Y-64, and YB-5), with MICs all at 1.25 g/L, all less than 2 g/L. Extracts of Stdif9 and Y-66 also had high inhibitory activity against citrus canker pathogenic strain KY(F), with MICs both at 2.5 g/L. The inhibitory activity of the five extracts, leaf-11, Y-51, Y-59, YB-25, and YB-26, against strain KY(F) is lower than that of other extracts, with MICs all at 5 g/L. The research results laid a foundation for the application of secondary metabolites of endophytic fungi associated with S. dielsiana in the prevention and control of citrus canker disease.

Key words: Xanthomonas citri subsp. citri, Stephania dielsiana Y. C. Wu, endophytic fungi, inhibitory activity, active substances, biological control agents

中图分类号:  S436.66

[1] 姚廷山, 周彦, 周常勇. 柑橘溃疡病菌分化及防治研究进展[J]. 园艺学报, 2015, 42(9): 1699-1706. DOI: 10.16420/j.issn.0513-353x.2015-0128.
[2] SAVIETTO A, POLAQUINI C R, KOPACZ M, et al. Antibacterial activity of monoacetylated alkyl gallates against Xanthomonas citri subsp citri[J]. Archives of Microbiology, 2018, 200(6): 929-937. DOI: 10.1007/s00203-018-1502-6.
[3] 李奇聪, 金秋珠, 骆海玉, 等. 柑橘溃疡病生防微生物研究进展[J]. 广西师范大学学报(自然科学版), 2022, 40(5): 388-397. DOI: 10.16088/j.issn.1001-6600.2022022801.
[4] GAO L L, HUANG M L, XIONG Q, et al. Antibacterial mechanism, control efficiency, and nontarget toxicity evaluation of actinomycin X2 against Xanthomonas citri subsp. citri[J]. Journal of Agricultural and Food Chemistry, 2024, 72(9): 4788-4800. DOI: 10.1021/acs.jafc.3c08600.
[5] 靳小青, 钮燕, 刘长翠, 等. 依地红皮肤消毒剂治疗足癣的疗效观察[J]. 海军医学杂志, 2003,24(1): 60-61. DOI: 10.3969/j.issn.1009-0754.2003.01.026.
[6] 钮燕, 靳小青, 沈越, 等. 一种中药皮肤消毒剂使用安全性评价[J]. 中国消毒学杂志, 2007,24(5): 473-474. DOI: 10.3969/j.issn.1001-7658.2007.05.035.
[7] 陈兴兴, 张宏伟, 晁志. 岭南瑶族民间常用草药种类与分析[J]. 海峡药学, 2014, 26(3): 37-40.
[8] 颜桢灵, 李国萍, 骆海玉, 等. 血散薯内生真菌的分离鉴定及其抗菌活性研究[J]. 河南农业科学, 2019, 48(10): 84-92. DOI: 10.15933/j.cnki.1004-3268.2019.10.013.
[9] 林伟, 骆海玉, 邓业成, 等. 血散薯块根内生真菌Periconia igniaria Stdif10发酵产物的生物活性研究[J]. 河南农业科学, 2020, 49(1): 75-81. DOI: 10.15933/j.cnki.1004-3268.2020.01.010.
[10] WU H Y, YAN Z L, DENG Y C, et al. Endophytic fungi from the root tubers of medicinal plant Stephania dielsiana and their antimicrobial activity[J]. Acta Ecologica Sinica, 2020, 40(5): 383-387. DOI: 10.1016/j.chnaes.2020.02.008.
[11] 董金皋, 康振生, 周雪平. 植物病理学[M]. 北京: 科学出版社, 2016.
[12] HABIBOLLAHI F, HOSSEINIPOUR A, LOHRASBI-NEJAD A. Antibacterial activity of the CAP18 peptide against Xanthomonas citri ssp. citri, the causative agent of citrus canker, as evaluated by in vitro and in silico studies[J]. Annals of Applied Biology, 2022, 181(1): 93-106. DOI: 10.1111/aab.12762.
[13] 刘丹丹, 王鹤, 高文静. 柑橘溃疡病防治药剂的联合施用及减量化研究[J]. 广西科学, 2022, 29(2): 360-367. DOI: 10.13656/j.cnki.gxkx.20220526.017.
[14] 沈萍, 陈向东. 微生物学实验[M]. 第5版. 北京: 高等教育出版社, 2018.
[15] 王中康, 孙宪昀, 夏玉先, 等. 柑桔溃疡病菌PCR快速检验检疫技术研究[J]. 植物病理学报, 2004, 34(1): 14-20. DOI: 10.13926/j.cnki.apps.2004.01.003.
[16] 王中康, 夏玉先, 孙宪昀, 等. PCR、DIA与致病性测定法检测柑桔溃疡病菌的比较[J]. 中国农业科学, 2004, 37(11): 1728-1732. DOI: 10.3321/j.issn:0578-1752.2004.11.026.
[17] PENG A H, XU L Z, HE Y R, et al. Efficient production of marker-free transgenic ‘Tarocco’ Blood Orange (Citrus sinensis Osbeck) with enhanced resistance to citrus canker using a Cre/loxP site-recombination system[J]. Plant Cell Tissue and Organ Culture (PCTOC), 2015, 123(1): 1-13. DOI: 10.1007/s11240-015-0799-y.
[18] PENG A H, CHEN S C, LEI T G, et al. Engineering canker-resistant plants through CRISPR/Cas9-targeted editing of the susceptibility gene CsLOB1 promoter in citrus[J]. Plant Biotechnology Journal, 2017, 15(12): 1509-1519. DOI: 10.1111/pbi.12733.
[19] 罗旭钊, 朱韬, 郝晨星, 等. 湖南柑橘溃疡病菌的多态性鉴定及致病力分析[J]. 湖南农业大学学报(自然科学版), 2022, 48(6): 691-698. DOI: 10.13331/j.cnki.jhau.2022.06.010.
[20] 颜桢灵, 陈洁萍, 农小霞, 等. 柑橘内生真菌的分离鉴定及其发酵产物对柑橘溃疡病菌的抑制活性[J]. 广西植物, 2021, 41(7): 1196-1208. DOI: 10.11931/guihaia.gxzw202002021.
[21] MIRZAEI-NAJAFGHOLI H, TARIGHI S, GOLMOHAMMADI M, et al. The effect of citrus essential oils and their constituents on growth of Xanthomonas citri subsp. citri[J]. Molecules, 2017, 22(4): 591. DOI: 10.3390/molecules22040591.
[22] VALLEJO C V, SOSA-MÁRMOL S M, SAGUIR F M, et al. Antibacterial activity of grape (Vitis vinifera) pomace fatty extracts against Xanthomonas citri subsp. citri in lemons[J]. Archives of Phytopathology and Plant Protection, 2019, 52(19/20): 1359-1368. DOI: 10.1080/03235408.2019.1691427.
[23] ZHANG J, GAO L L, LIN H T, 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. DOI: 10.1021/acs.jafc.4c02769.
[24] LING W, DAI T R, ZHANG J Y, et al. Evaluation of pomelo seed extracts as natural antioxidant, antibacterial, herbicidal agents, and their functional components[J]. Chemistry Biodiversity, 2021, 18(12):e2100679. DOI: 10.1002/cbdv.202100679.
[25] 廖石榴, 尹维, 廖晓兰, 等. 苋菜乙酸乙酯提取物对柑橘溃疡病菌的抑制及机理研究[J]. 湖南农业大学学报(自然科学版), 2017, 43(5): 544-550. DOI: 10.13331/j.cnki.jhau.2017.05.014.
[26] ZHANG J, ZHANG J Y, LIN H T, et al. Semiliquidambar chingii is a highly potent antibacterial plant resource against Xanthomonas citri subsp. citri: insights into the possible mechanisms of action, chemical basis, and synergistic effect of bioactive compounds[J]. Industrial Crops and Products, 2023, 202: 117020. DOI: 10.1016/j.indcrop.2023.117020.
[27] LIN H T, LIANG Y, KALIAPERUMAL K, et al. Linoleic acid from the endophytic fungus Diaporthe sp. HT-79 inhibits the growth of Xanthomonas citri subsp. citri by destructing the cell membrane and producing reactive oxygen species (ROS)[J]. Pesticide Biochemistry and Physiology, 2023, 192: 105423. DOI: 10.1016/j.pestbp.2023.105423.
[28] 郭明程, 李保同, 汤丽梅, 等. 爵床提取物的抑菌杀虫活性研究[J]. 中国生态农业学报, 2013, 21(2): 212-216. DOI: 10.3724/SP.J.1011.2013.00212.
[29] NORILER S A, SAVI D C, PONOMAREVA L V, et al. Vochysiamides A and B: Two new bioactive carboxamides produced by the new species Diaporthe vochysiae[J]. Fitoterapia, 2019, 138: 104273. DOI: 10.1016/j.fitote.2019.104273.
[30] TONG W Y, LEONG C R, TAN W N, et al. Endophytic Diaporthe sp. ED2 produces a novel anti-candidal ketone derivative[J]. Journal of Microbiology and Biotechnology, 2017, 27(6): 1065-1070. DOI: 10.4014/jmb.1612.12009.
[31] HUANG X S, ZHOU D X, LIANG Y, et al. Cytochalasins from endophytic Diaporthe sp. GDG-118[J]. Natural Product Research, 2021, 35(20): 3396-3403. DOI: 10.1080/14786419.2019.1700504.
[32] 姚裕群. 越南槐内生真菌多样性、抑菌活性及其次生代谢产物研究[D]. 南宁: 广西大学, 2017. DOI: 10.7666/d.Y3277003.
[33] SHAABAN M, SHAABAN K A, ABDEL-AZIZ M S. Seven naphtho-γ-pyrones from the marine-derived fungus Alternaria alternata: structure elucidation and biological properties[J]. Organic and Medicinal Chemistry Letters, 2012, 2: 6. DOI: 10.1186/2191-2858-2-6.
[34] YANG Z, DAN W J, LI Y X,et al. Antifungal metabolites from Alternaria atrans: an endophytic fungus in Psidium guajava[J]. Natural Product Communications, 2019, 14(5): 1-5. DOI: 10.1177/1934578X19844116.
[35] GUPTA S, KAUL S, SINGH B, et al. Production of gentisyl alcohol from Phoma herbarum Endophytic in Curcuma longa L. and its antagonistic activity towards leaf spot pathogen Colletotrichum gloeosporioides[J]. Applied Biochemistry and Biotechnology, 2016, 180(6): 1093-1109. DOI: 10.1007/s12010-016-2154-0.
[36] HUSSAIN H, KOCK I, AI-HARRASI A, et al. Antimicrobial chemical constituents from endophytic fungus Phoma sp.[J]. Asian Pacific Journal of Tropical Medicine, 2014, 7(9): 699-702. DOI: 10.1016/S1995-7645(14)60119-X.
[37] ZHAO S S, ZHANG Y Y, YAN W, et al. Chaetomium globosum CDW7, a potential biological control strain and its antifungal metabolites[J]. FEMS Microbiology Letters, 2017, 364(3): fnw287. DOI: 10.1093/femsle/fnw287.
[38] NONGTHOMBAM K S, MUTUM S S. Biological activities and metabolite profiling of Chaetomium sp. R1C1, an endophytic fungus from Chromolaena odorata of Manipur[J]. Biologia, 2024, 79(2): 643-656. DOI: 10.1007/s11756-023-01584-3.
[39] WANG H, LIU Z Y, DUAN F F, et al. Isolation, identification, and antibacterial evaluation of endophytic fungi from Gannan navel orange[J]. Frontiers in Microbiology, 2023, 14: 1172629. DOI: 10.3389/fmicb.2023.1172629.
[1] 施慧露, 莫燕华, 骆海玉, 马姜明. 檵木乙酸乙酯萃取物抑菌活性研究[J]. 广西师范大学学报（自然科学版）, 2025, 43(1): 1-8.
[2] 徐紫薇, 邓业成, 骆海玉, 邓志勇, 韦坚芬, 黄玲玉, 甘萍贵. 鸡屎藤内生真菌活性菌株筛选及其抗菌活性物质研究[J]. 广西师范大学学报（自然科学版）, 2025, 43(1): 31-38.
[3] 韦坚芬, 邓志勇, 邓业成, 骆海玉, 蒙思妤, 徐紫薇, 施瑶, 梁文垦. 草珊瑚内生真菌SgG4抗植物病原真菌的活性物质研究[J]. 广西师范大学学报（自然科学版）, 2024, 42(2): 175-182.
[4] 肖泽恩, 邹思华, 江娇, 罗海梅, 黄锡山, 刘永宏, 谭振. 木榄内生真菌Penicillium sp. GXIMD00006的次级代谢产物及其降血糖活性研究[J]. 广西师范大学学报（自然科学版）, 2023, 41(6): 150-157.
[5] 李奇聪, 陈洁萍, 欧艳绍, 杨树贤, 邓志勇, 骆海玉, 邓业成. 柑橘内生真菌LJZ-Y-11次生代谢产物抑菌活性研究[J]. 广西师范大学学报（自然科学版）, 2023, 41(1): 155-163.
[6] 杜丽波, 李金玉, 张晓, 李永红, 潘卫东. 毛红椿皮的化学成分及生物活性研究[J]. 广西师范大学学报（自然科学版）, 2022, 40(4): 162-172.
[7] 李本超, 梁艳, 覃小芽, 莫土香, 徐照隆, 李俊, 杨瑞云. 广豆根内生真菌GDG-178代谢产物研究[J]. 广西师范大学学报（自然科学版）, 2021, 39(2): 139-143.
[8] 覃玉月, 刘晓波, 徐照隆, 莫土香, 李俊, 杨瑞云. 广豆根内生真菌Xylaria sp. GDGJ-368代谢产物研究[J]. 广西师范大学学报（自然科学版）, 2020, 38(5): 71-77.
[9] 梁艳,周德雄,薛佳津,刘晓波,李俊,杨瑞云. 中药血散薯中非生物碱类化学成分研究[J]. 广西师范大学学报（自然科学版）, 2018, 36(1): 95-98.
[10] 徐伟锋,姚飞华,梁学锋,郑娜,杨瑞云,李俊. 广豆根内生真菌GDG-180代谢产物研究[J]. 广西师范大学学报（自然科学版）, 2017, 35(1): 58-61.
[11] 刘汉甫, 马献力, 陈素一, 沈雪松, 黄富平. 锌三元配合物的合成及其抑菌活性研究[J]. 广西师范大学学报（自然科学版）, 2015, 33(4): 103-107.
[12] 周德雄, 王歆竹, 徐伟峰, 杨瑞云, 邵长伦, 李俊. 短指软珊瑚Sinularia sp.内生真菌Ta31-2代谢产物研究(Ⅱ)[J]. 广西师范大学学报（自然科学版）, 2014, 32(4): 91-95.
[13] 邓业成, 宁蕾. 4种植物精油的抑菌活性及应用[J]. 广西师范大学学报（自然科学版）, 2012, 30(3): 288-294.
[14] 杨瑞云, 黄忠京, 邵长伦, 李俊, 佘志刚, 林永成. 南海马尾藻内生真菌No.ZZF36代谢产物研究[J]. 广西师范大学学报（自然科学版）, 2010, 28(2): 53-56.
Viewed
Full text
160
HTML PDF
Just accepted Online first Issue Just accepted Online first Issue
0 0 0 0 0 160

  From Others local
  Times 27 133
  Rate 17% 83%

Abstract
47
Just accepted Online first Issue
0 0 47
  From Others local
  Times 41 6
  Rate 87% 13%

Cited
Web of Science  Crossref   ScienceDirect  Search for Citations in Google Scholar >>
 
This page requires you have already subscribed to WoS.
  Shared   
  Discussed   
[1] 何静, 冯元柳, 邵靖雯. 基于CiteSpace的多源数据融合研究进展[J]. 广西师范大学学报（自然科学版）, 2024, 42(5): 13 -27 .
[2] 刘畅平, 宋树祥, 蒋品群, 岑明灿. 基于开关电容的差分无源N通道滤波器[J]. 广西师范大学学报（自然科学版）, 2024, 42(5): 52 -60 .
[3] 王淑颖, 卢宇翔, 董淑彤, 陈默, 康秉娅, 蒋长兰, 宿程远. 污水中抗生素抗性基因传播过程及控制技术研究进展[J]. 广西师范大学学报（自然科学版）, 2024, 42(6): 1 -15 .
[4] 钟俏, 陈生龙, 唐聪聪. 水凝胶技术在微藻采收中的应用:现状、挑战与发展分析[J]. 广西师范大学学报（自然科学版）, 2024, 42(6): 16 -29 .
[5] 翟思琪, 蔡文君, 朱苏, 李韩龙, 宋海亮, 杨小丽, 杨玉立. 汲取液溶质反向扩散与正渗透中膜污染的相互关系研究[J]. 广西师范大学学报（自然科学版）, 2024, 42(6): 30 -39 .
[6] 郑国权, 秦永丽, 汪晨祥, 葛仕佳, 闻倩敏, 蒋永荣. ABR硫酸盐还原体系分级沉淀酸性矿山废水中重金属及矿物形成[J]. 广西师范大学学报（自然科学版）, 2024, 42(6): 40 -52 .
[7] 刘洋, 张毅杰, 章延, 李玲, 孔祥铭, 李红. 饮用水处理中藻类混凝消除技术的现状与趋势——基于CiteSpace的可视化分析[J]. 广西师范大学学报（自然科学版）, 2024, 42(6): 53 -66 .
[8] 田晟, 陈东. 基于深度强化学习的网联燃料电池混合动力汽车生态驾驶联合优化方法[J]. 广西师范大学学报（自然科学版）, 2024, 42(6): 67 -80 .
[9] 陈秀锋, 王成鑫, 赵凤阳, 杨凯, 谷可鑫. 改进DQN算法的单点交叉口信号控制方法[J]. 广西师范大学学报（自然科学版）, 2024, 42(6): 81 -88 .
[10] 李欣, 宁静. 基于时空特征融合的电力系统暂态稳定评估[J]. 广西师范大学学报（自然科学版）, 2024, 42(6): 89 -100 .
版权所有 © 广西师范大学学报（自然科学版）编辑部
地址：广西桂林市三里店育才路15号　邮编：541004
电话：0773-5857325　E-mail: gxsdzkb@mailbox.gxnu.edu.cn
本系统由北京玛格泰克科技发展有限公司设计开发