Screening and Identification of Endophytic Fungus HU0460 for Biocontrol of Potato (Solanum tuberosum) Blackleg and the Mechanism of Its Metabolites Against Pectobacterium atroseptica
HUANG Yu-Han, MAO Lu-Tian*, LU Zhen-Zhang, JI Xia, XU Liang-Xiong, ZENG Xue-Yun, YE Yu-Zhen, HUANG Yan-Fen
College of Life Science, Huizhou University, Huizhou 516007, China
摘要马铃薯黑胫病是由软腐果胶菌属马铃薯黑胫病菌(Pectobacterium atroseptica, Pat)引起的细菌性土传病害,该病严重影响马铃薯(Solanum tuberosum)产业的发展。本研究采用马铃薯琼脂培养基(PDA)从银叶树(Heritiera littoralis)的茎、叶组织分离出内生真菌 ,通过玉米培养基和大米培养基固体发酵得到真菌代谢产物,以滤纸片法筛选抗黑胫病菌的高活性代谢产物,采用 ITS 序列比对和形态观察进行菌种鉴定。代谢产物经乙醇浸提浓缩和正相硅胶柱层析获得活性组分,通过抑菌曲线和生长曲线测定、扫描电镜观察菌体形态及 DiBAC4(3)荧光探针检测膜电位等手段探究了代谢产物对 Pat 的作用及机制,采用薯块接菌法分析活性代谢产物对 Pat 的防控效果。结果发现,内生真菌 HU0460 的玉米发酵物对 Pat 有较好的抑菌活性;HU0460 经形态学和 ITS 序列鉴定为黑孢属真菌(Nigrospora sp.)。玉米发酵物经乙醇浸提和正相硅胶柱层析初步分离获得对 Pat 有明显抑菌效果的活性组分 ,其对 Pat 的最低抑菌浓度(minimum inhibitory concentration, MIC)和最低杀菌浓度(minimum bactericidal concentration, MBC)分别为2.44×10-3和3.91×10-2 μg/mL。扫描电镜观察发现,随着活性代谢产物浓度的上升,Pat 的形态变化程度加剧,其浓度达到 MBC 时,菌体出现破裂、细胞内容物流失等现象。DiBAC4(3)荧光探针检测结果显示,HU0460 活性代谢产物处理导致 Pat 的荧光强度上调,使 Pat 的膜电位发生去极化。进一步用薯块接菌实验证实,活性代谢产物可明显抑制 Pat 对薯块的侵染性。本研究分离筛选获得了抗马铃薯黑胫病菌的生防内生真菌—黑孢菌 HU0460,分离的活性代谢产物对 Pat 有较好的抗菌作用,为马铃薯黑胫病的生物防控及后续农用抗生素研发提供菌种和参考数据。
Abstract：Potato blackleg disease (PBD) is a bacterial soil-borne disease caused by the pathogen Pectobacterium atroseptica (Pat), which seriously obstructs the development of the potato (Solanum tuberosum) industry. In this study, endophytic fungi were isolated from the stem and leaf tissues of Heritiera littoralis using the patato dextrose agar medium (PDA). Fungal metabolites were obtained by solid fermentation of corn medium and rice medium, and the highly active metabolites with significant resistance to Pat were screened using the filter paper method. High activity strain HU0460 was identified by combining morphological observation and ITS sequence comparison. The active components in the metabolites were obtained by ethanol extraction, concentration, and normal silica gel column chromatography. The antibacterial mechanism of the active components on Pat was explored using determination of bacteriostatic and growth curve, observation of cell morphology by scanning electron microscope, and detection of membrane potential by DiBAC4 (3) fluorescence probe. Furthermore, the inhibitory effect of the active metabolites on Pat was analyzed through tuber inoculation. The results showed that the corn fermentation product of the endophytic fungus HU0460 had good antibacterial activity against Pat. HU0460 was identified as Nigrospora sp. by morphology and ITS sequence.The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the bacteriostatic components isolated from the HU0460 corn fermentation medium by ethanol extraction and normal silica gel column chromatography on Pat were 2.44×10-3 and 3.91×10-2 μg/mL, respectively. Results of scanning electron microscope (SEM) showed that the degree of morphological deformation of Pat increased with the increase of the concentration of active metabolites. When the concentration reached the MBC, the Pat cells broke and the content of the cells was lost. The DiBAC4(3) fluorescence probe detection showed that the treatment of HU0460 active metabolites resulted in the up-regulation of the fluorescence intensity of the bacteria and depolarization of the membrane potential of Pat fluorescenceintensity which led to the infectivity to the tube was further confirmed by the tuber inoculation experiment. In this study, the endophytic fungus Nigrospora sp. HU0460 against Pat was isolated and analyzed. The isolated bacteriostatic metabolites have a good antibacterial effect on Pat, providing strain and reference data for the biological control of the PBD and the subsequent agricultural antibiotic research and development.
黄钰涵, 毛露甜, 陆镇樟, 冀霞, 徐良雄, 曾雪云, 叶玉珍, 黄艳芬. 马铃薯黑胫病生防内生真菌 HU0460 的筛选鉴定及其代谢产物对黑胫病菌的作用机制[J]. 农业生物技术学报, 2023, 31(6): 1252-1261.
HUANG Yu-Han, MAO Lu-Tian, LU Zhen-Zhang, JI Xia, XU Liang-Xiong, ZENG Xue-Yun, YE Yu-Zhen, HUANG Yan-Fen. Screening and Identification of Endophytic Fungus HU0460 for Biocontrol of Potato (Solanum tuberosum) Blackleg and the Mechanism of Its Metabolites Against Pectobacterium atroseptica. 农业生物技术学报, 2023, 31(6): 1252-1261.
[1] 白明, 钟志娟, 李晓洁, 等. 2021. 水葫芦内生拟枝孢镰刀菌SC1608 的次生代谢产物[J]. 菌物学报 , 40(01): 232-239. (Bai M, Zhong Z J, Li X J, et al. 2021. Secondary metabolites of endophytic Fusarium oxysporum SC1608 in water hyacinth[J]. Acta Bacteriologica Sinica, 40(01): 232-239. ) [2] 曹瑶, 戚馨月, 汪海峰, 等. 2021. 花生壳乙酸乙酯萃取物对金黄色葡萄球菌抗菌作用及机理[J]. 中国粮油学报, 36(02): 138-144. (Cao Y, Qi X Y, Wang H F, et al. 2021. Antibacterial effect and mechanism of ethyl acetate extract of peanut shell on Staphylococcus aureus[J]. Chinese Journal of Grain and Oil, 36(02): 138-144. ) [3] 方园, 彭勇政, 廖长贵, 等. 2022. 一株具有防病促生功能的贝莱斯芽孢杆菌 SF327[J]. 微生物学报, 62(10): 4071-4088. (Fang Y, Peng Y Z, Liao C G, et al. 2022. Bacillus velezensis SF327, a potential biocontrol agent with the functions of preventing plant diseases and promoting plant growth)[J]. Acta Microbiologica Sinica, 62(10): 4071-4088. ) [4] 冯志文, 曹亚宁, 孙清华, 等. 2020. 10 种杀菌剂对马铃薯黑胫病主要致病菌的室内毒力[J]. 中国马铃薯, 34(05): 281-289. (Feng Z W, Cao Y N, Sun Q H, et al. 2020. Indoor virulence of 10 fungicides against main pathogens of potato black shank[J]. Chinese Potato, 34(05): 281-289. ) [5] 高玉林, 徐进, 刘宁, 等. 2019. 我国马铃薯病虫害发生现状与防控策略[J]. 植物保护. 45(05): 106-111. (Gao Y L, Xu J, Liu N, et al. 2019. Current status and management strategies for potato insect pests and diseases in China[J]. Plant Protection, 45(05): 106-111. ) [6] 贾睿, 蔡丹, 葛思彤, 等. 2021. 红豆皮多酚提取物对两种致病菌的抑菌活性及作用机理[J]. 食品科学, 42(23): 64-71. (Jia R, Cai D, Ge S T, et al. 2021. Antibacterial activity and mechanism of polyphenol extracts from adzuki bean seed coat against two pathogens[J]. Food Science, 42(23): 64-71. ) [7] 李华伟, 林志坚, 许国春, 等. 2020. 福建省霞浦县马铃薯黑胫病菌分离及田间药剂防治效果[J]. 中国蔬菜, 9: 57-63. (Li H W, Lin Z J, Xu G C, et al. 2020. Isolation of potato Phytophthora parasitica and field control effect in Xiapu county of Fujian province[J]. China Vegetables, 9: 57-63. ) [8] 李腊, 史洪丽, 余婷婷, 等. 2022. 番茄灰霉病生防内生细菌 BR-1 的筛选鉴定及其生防性能分析[J]. 农业生物技术学报, 30(08): 1594-1605. (Li L, Shi H L, Yu T T, et al. 2022. Screening and identification of endophytic bacteria BR-1 for the biocontrol of tomato (Solanum lycopersi-cum) gray mold and analysis of its biocontrol performance[J]. Journal of Agricultural Biotechnology, 30(08): 1594-1605. ) [9] 李露丹, 吴宣, 徐丹, 等. 2021. 珍稀药用石斛内生菌研究进展[J]. 生物资源 , 43(03): 246-256. (Li L D, Wu X, Xu D, et al. 2021. Research advances on endophytes of rare medicinal dendrobium[J]. Biotic Resources, 43(03): 246-256. ) [10] 林国盛. 2019. 基于NF-κB 和肠道菌群研究银叶树果对 DSS 所致炎症性肠病的作用[D]. 硕士学位论文, 广州中医药大学 , 导师 : 苏子仁. pp. 2-3. (Lin G S. 2019. The fruit of Heritiera littoralis Dryand. ameliorates dextran sulfate sodium-induced mice colitis by suppressing NF- κB pathway and regulating gut microbiota[D]. Thesis of M. S. , Guangzhou University of Traditional Chinese Medicine, Supervisor: Shu Z R. pp. 2-3. ) [11] 孟焕文, 赵远征, 梁东超, 等. 2020. 马铃薯黑胫病生防菌株的筛选与鉴定[J]. 中国植保导刊 , 40(08): 24-29. (Meng H W, Zhao Y Z, Liang D C, et al. 2020. Screening and identification of biocontrol strains of potato black shank[J]. China Plant Protection Guide, 40(08): 24-29. ) [12] 宋璐忆, 李佳辉, 王硕, 等. 2021. 原儿茶醛对福氏志贺菌的抑制作用[J]. 中国食品学报, 21(11): 62-70. (Song L Y, Li J H, Wang S. et al. 2021. Inhibitory effect of protocatechu‐icaldehyde against Shigella flexneri and its mechanism[J]. Chinese Journal of Food Science, 21(11): 62-70. ) [13] 汪晓辉, 郭溶, 聂晓彬, 等. 2021. 佛手抗菌活性及其药效成分橙皮苷对金黄色葡萄球菌的作用机制研究[J]. 中国抗生素杂志, 46(05): 437-441. (Wang X H, Guo R, Nie X B, et al. 2021. Study on the antibacterial activity of bergamot and the mechanism of action of hesperidin on Staphylococcus aureus[J]. Chinese Journal of Antibiotics, 46(05): 437-441. ) [14] 徐进, 朱杰华, 杨艳丽, 等. 2019. 中国马铃薯病虫害发生情况与农药使用现状[J]. 中国农业科学 , 52(16): 2800-2808. (Xu J, Zhu J H, Yang Y L, et al. 2019. Status of major diseases and insect pests of potato and pesticideusage in China[J]. Scientia Agricultura Sinica, 52(16): 2800-2808. ) [15] 于艳梅. 2015. 几种植物源化合物对青枯菌的抑制作用及机理研究[D]. 硕士学位论文, 西南大学, 导师: 丁伟. pp. 25-27. (Yu Y M. 2015. Study on the inhibitory effect and mechanism of several plant derived compounds on bacterial wilt[D]. Thesis of M. S. , Southwest University, Supervisor: Ding W. pp. 25-27. ) [16] 翟大才, 房震, 吕彩云, 等. 2019. 黄精内生菌枯草芽胞杆菌HJ-2 的抑菌活性研究[J]. 农业生物技术学报 , 27(9):1664-1672. (Zhai D C, Fang Z, Lv C Y, et al. 2019. Research on antibacterial activity of the endophytic Bacil- lus subtilis HJ-2 from Polygonatum cyrtonema[J]. Journal of Agricultural Biotechnology, 27(9): 1664-1672. ) [17] 张倩. 2019. 马齿苋内生菌抗青枯病的研究[D]. 硕士学位论文 , 华南农业大学 , 导师 : 颜健. pp. 51-52. (Zhang Q. 2019. Study on the endophytes in Portulaca oleracea L. against bacterial wilt[D]. Thesis of M. S. , South China Agricultural University, Supervisor: Yan J. pp. 51-52. ) [18] Gao D Y, Ji X, Wang J L, et al. 2018. Engineering protein-based nanoplatform as antibacterial agents for light activated dual-modal photothermal and photodynamic therapy of infection in both the NIR Ⅰ and Ⅱ windows[J]. Journal of Materials Chemistry B, 6(5): 732-739. [19] Fadhel A, Albaayit S, Maharjan R, et al. 2022. Evaluation of anti-methicillin-resistant Staphylococcus aureus property of zerumbone[J]. Journal of Applied Biomedicine, 20: 15-21. [20] Gautam V S, Nishad, J H, Kumari P, et al. 2022. Isolation and functional characterization of a fungal plant symbiont Nigrospora sphaerica, associated to Euphorbia hirta L. [J]. Indian Phytopathology, 75: 345-355. [21] Mao L T, Chen Z G, Xu L X, et al. 2019. Rhizosphere microbiota compositional changes reflect potato blackleg disease[J]. Applied Soil Ecology, 140: 11-17. [22] Mao L T, Huang Z C, Lu Z Z, et al. 2022. Multi-species bacillus inoculant and its effect on potato growth and control of patato (Solanum tuberosuml) blackleg disease[J]. Applied Ecology and Environmental Research, 20(2): 1571-1583. [23] Wang B, He B, Chen T, et al. 2022. Discovery of tropolone stipitaldehyde as a potential agent for controlling Phytophthora Blight and its action mechanism research[J]. Journal of Agricultural and Food Chemistry, 70(28): 8693-8703.
