Journal of Guangxi Normal University(Natural Science Edition) ›› 2025, Vol. 43 ›› Issue (1): 39-47.doi: 10.16088/j.issn.1001-6600.2024040701

Previous Articles     Next Articles

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)

673

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

CLC Number:  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] SHI Huilu, MO Yanhua, LUO Haiyu, MA Jiangming. Inhibitory Activity of Ethyl Acetate Extracts of Loropetalum chinense against Pathogens [J]. Journal of Guangxi Normal University(Natural Science Edition), 2025, 43(1): 1-8.
[2] XU Ziwei, DENG Yecheng, LUO Haiyu, DENG Zhiyong, WEI Jianfen, HUANG Lingyu, GAN Pinggui. Study on the Antifungal Activity of Secondary Metabolites from Active Endophytic Fungi Isolated from Paederia scandens (Lour.) Merr. [J]. Journal of Guangxi Normal University(Natural Science Edition), 2025, 43(1): 31-38.
[3] WEI Jianfen, DENG Zhiyong, DENG Yecheng, LUO Haiyu, MENG Siyu, XU Ziwei, SHI Yao, LIANG Wenken. Study on the Active Substances of Endophytic Fungus SgG4 from Sarcandra glabra Against Plant Pathogenic Fungi [J]. Journal of Guangxi Normal University(Natural Science Edition), 2024, 42(2): 175-182.
[4] LI Qicong, CHEN Jieping, OU Yanshao, YANG Shuxian, DENG Zhiyong, LUO Haiyu, DENG Yecheng. Antimicrobial Activity of the Secondary Metabolites from Citrus Endophytic Fungus Nemania sp. LJZ-Y-11 [J]. Journal of Guangxi Normal University(Natural Science Edition), 2023, 41(1): 155-163.
[5] LI Benchao, LIANG Yan, QIN Xiaoya, MO Tuxiang, XU Zhaolong, LI Jun, YANG Ruiyun. Secondary Metabolites of Endophytic Fungus GDG-178 from Sophora tonkinensis [J]. Journal of Guangxi Normal University(Natural Science Edition), 2021, 39(2): 139-143.
[6] QIN Yuyue, LIU Xiaobo, XU Zhaolong, MO Tuxiang, LI Jun, YANG Ruiyun. Secondary Metabolites of Endophytic Fungus Xylaria sp. GDGJ-368 from Sophora tonkinensis [J]. Journal of Guangxi Normal University(Natural Science Edition), 2020, 38(5): 71-77.
[7] LIANG Yan. Non-alkaloid Chemical Constituents from the Chinese Medical Plant Stephania dielsiana [J]. Journal of Guangxi Normal University(Natural Science Edition), 2018, 36(1): 95-98.
[8] XU Weifeng,YAO Feihua, LIANG Xuefeng, ZHENG Na, YANG Ruiyun,LI Jun. Secondary Metabolites of Endophytic Fungus GDG-180 from Sophora tonkinensis [J]. Journal of Guangxi Normal University(Natural Science Edition), 2017, 35(1): 58-61.
[9] ZHOU De-xiong, WANG Xin-zhu, XU Wei-feng, YANG Rui-yun, SHAO Chang-lun, LI Jun. Metabolites of Endophytic Fungus Ta31-2 from Soft Coral Sinularia sp. (Ⅱ) [J]. Journal of Guangxi Normal University(Natural Science Edition), 2014, 32(4): 91-95.
[10] DENG Ye-cheng, NING Lei. Inhibitory Activities and Application of Four Plant Essential Oils [J]. Journal of Guangxi Normal University(Natural Science Edition), 2012, 30(3): 288-294.
[11] YANG Rui-yun, HUANG Zhong-jing, SHAO Chang-lun, LI Jun, SHE Zhi-gang, LIN Yong-cheng. Metabolites of Brown Alga Endophytic Fungus (No.ZZF36) from the South China Sea [J]. Journal of Guangxi Normal University(Natural Science Edition), 2010, 28(2): 53-56.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
[1] HE Jing, FENG Yuanliu, SHAO Jingwen. Research Progress on Multi-source Data Fusion Based on CiteSpace[J]. Journal of Guangxi Normal University(Natural Science Edition), 2024, 42(5): 13 -27 .
[2] LIU Changping, SONG Shuxiang, JIANG Pinqun, CEN Mingcan. Differential Passive N-path Filter Based on Switched Capacitors[J]. Journal of Guangxi Normal University(Natural Science Edition), 2024, 42(5): 52 -60 .
[3] WANG Shuying, LU Yuxiang, DONG Shutong, CHEN Mo, KANG Bingya, JIANG Zhanglan, SU Chengyuan. Research Progress on the Propagation Process and Control Technology of ARGs in Wastewater[J]. Journal of Guangxi Normal University(Natural Science Edition), 2024, 42(6): 1 -15 .
[4] ZHONG Qiao, CHEN Shenglong, TANG Congcong. Hydrogel Technology for Microalgae Collection: Status Overview, Challenges and Development Analysis[J]. Journal of Guangxi Normal University(Natural Science Edition), 2024, 42(6): 16 -29 .
[5] ZHAI Siqi, CAI Wenjun, ZHU Su, LI Hanlong, SONG Hailiang, YANG Xiaoli, YANG Yuli. Dynamic Relationship Between Reverse Solute Flux and Membrane Fouling in Forward Osmosis[J]. Journal of Guangxi Normal University(Natural Science Edition), 2024, 42(6): 30 -39 .
[6] ZHENG Guoquan, QIN Yongli, WANG Chenxiang, GE Shijia, WEN Qianmin, JIANG Yongrong. Stepwise Precipitation of Heavy Metals from Acid Mine Drainage and Mineral Formation in Sulfate-Reducing Anaerobic Baffled Reactor System[J]. Journal of Guangxi Normal University(Natural Science Edition), 2024, 42(6): 40 -52 .
[7] LIU Yang, ZHANG Yijie, ZHANG Yan, LI Ling, KONG Xiangming, LI Hong. Current Status and Trends of Algal Coagulation Elimination Technology in Drinking Water Treatment: a Visual Analysis Based on CiteSpace[J]. Journal of Guangxi Normal University(Natural Science Edition), 2024, 42(6): 53 -66 .
[8] TIAN Sheng, CHEN Dong. A Joint Eco-driving Optimization Research for Connected Fuel Cell Hybrid Vehicle via Deep Reinforcement Learning[J]. Journal of Guangxi Normal University(Natural Science Edition), 2024, 42(6): 67 -80 .
[9] CHEN Xiufeng, WANG Chengxin, ZHAO Fengyang, YANG Kai, GU Kexin. A Single Intersection Signal Control Method Based on Improved DQN Algorithm[J]. Journal of Guangxi Normal University(Natural Science Edition), 2024, 42(6): 81 -88 .
[10] LI Xin, NING Jing. Online Assessment of Transient Stability in Power Systems Based on Spatiotemporal Feature Fusion[J]. Journal of Guangxi Normal University(Natural Science Edition), 2024, 42(6): 89 -100 .
Copyright © Journal of Guangxi Normal University(Natural Science Edition), All Rights Reserved.
Tel: 0773-5857325　E-mail: gxsdzkb@mailbox.gxnu.edu.cn
Powered by Beijing Magtech Co. Ltd