老鼠簕真菌病病原的分离鉴定及生物学特性

马林; 袁宗胜; 郑瑜婷; 郑雄; 陈维峰; 王思凡; 谢宝贵; 刘芳

doi:10.19303/j.issn.1008-0384.2023.11.009

老鼠簕真菌病病原的分离鉴定及生物学特性

doi: 10.19303/j.issn.1008-0384.2023.11.009

马林^1,,
袁宗胜²,
郑瑜婷³,
郑雄¹,
陈维峰¹,
王思凡⁴,
谢宝贵¹,
刘芳^1, ,

1.
福建农林大学生命科学学院，福建　福州，350002
2.
闽江学院地理与海洋学院，福建　福州，350108
3.
古田县食用菌研发中心，福建　宁德，352200
4.
福建农林大学未来技术学院，福建　福州，350002

基金项目: 国家自然科学基金项目（32002108）；闽江学院引进人才预研项目（33004328）

详细信息

作者简介:
马林（1997 — ），男，硕士研究生，主要从事食用菌和农业微生物研究，E-mail：worldmalinmm@gmail.com

通讯作者:
刘芳（1980 — ），女，讲师，主要从事食用菌和农业微生物研究，E-mail：fjliufang@163.com

中图分类号: S436
计量
- 文章访问数: 209
- HTML全文浏览量: 125
- PDF下载量: 38
- 被引次数: 0
出版历程
- 收稿日期: 2023-07-22
- 修回日期: 2023-08-16
- 网络出版日期: 2023-12-21
- 刊出日期: 2023-11-28

Isolation, Identification, and Biological Characteristics of a Fungal Pathogen on Acanthus ilicifolius

1.
College of Life Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China
2.
College of Geography and Oceanography, Minjiang University, Fuzhou, Fujian　350108, China
3.
Research and Development Center of Edible Fungi of Gutian, Ningde, Fujian　352200, China
4.
College of Future Technology, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China

摘要

摘要: 目的明确老鼠簕（Acanthus ilicifolius）叶斑病的病原种类及其生物学特性。方法从福建省漳江口红树林国家级自然保护区采集病叶，采用离体组织分离法对病叶病原菌进行分离，采用形态学与分子生物学（ITS和EF1-α基因序列分析）的方法鉴定其病原菌种类，并对该病原菌的菌丝在不同温度、pH、碳源和氮源等条件下的生物学特性进行研究，以及对过氧化氢酶（CAT），超氧化物歧化酶（SOD）、过氧化物酶（POD）、漆酶、纤维素酶（CL）、β-葡萄糖苷酶（β-GC）进行活性检测。结果经鉴定该病原菌为真菌界子囊菌门镰刀菌科的轮枝镰刀菌（Fusarium verticillioides）。这是轮枝镰刀菌侵染老鼠簕引发叶斑病的首次报道。生物学特性研究显示该病原菌生长的最适温度25 ℃，最适pH为10，最佳碳源为淀粉，最佳氮源为酵母粉。3种氧化还原酶中，过氧化物酶（POD）活性最高；3种水解酶中，纤维素酶（CL）活性最高。结论分离鉴定了福建省漳江口老鼠簕叶斑病病原菌为轮枝镰刀菌（Fusarium verticillioides），明确了病原菌的生物学特性。
- 老鼠簕 /
- 叶斑病 /
- 鉴定 /
- 轮枝镰刀菌 /
- 生物学特性
Abstract: Objective Pathogen of the leaf spot disease on Acanthus ilicifolius was isolated and identified with its biological characteristics determined. Method Microbes were isolated from the diseased A. ilicifolius leaves collected from the Zhangjiangkou Estuary Mangrove National Nature Reserve in Fujian Province by the detached tissue method. Morphological and molecular biological techniques including ITS and EF1-α gene sequence analysis were employed to identify the pathogen. Optimal growth conditions of the identified fungus with respect to temperatures, pH, carbon sources, and nitrogen sources, in addition to its activities of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), laccase, cellulase (CL), andβ-glucosidase (β-GC) were determined. Result For the first time Fusarium verticillioides in the Ascomycota phylum was identified as the responsible pathogen of the disease. It thrived at 25°C and pH 10 on a medium using starch as carbon source and yeast powder as nitrogen source. POD was the oxidoreductase, and CL the hydrolase, that showed the highest activity in the fungus. Conclusion The pathogen that caused the leaf spot disease on bougainvillea in the Zhangjiang Estuary was identified as F. verticillioides. The biological characteristics of the strongly adaptable fungus were determined.
- Acanthus ilicifolius /
- leaf spot disease /
- identification /
- Fusarium verticillium /
- biological characteristics

HTML全文

图 1 老鼠簕叶斑病症状

A为自然发病叶片症状；B为室内离体接种后叶片正面症状；C为室内离体接种后叶片背面症状。

Figure 1. Symptoms on A. ilicifolius infected by leaf spot disease

A: Symptoms on diseased leaves in nature; B: symptoms shown on face of diseased leaf after in vitro inoculation; C: symptoms shown on back of diseased leaf after in vitro inoculation.

下载: 全尺寸图片幻灯片

图 2 老鼠簕叶斑病病原菌形态特征

A为菌落的正面形态；B为菌丝的圆形缠绕形态；C为孢子聚集结构；D为小分生孢子；E为大分生孢子；F为厚垣孢子。

Figure 2. Morphology of pathogen causing leaf spot disease on A. ilicifolius

A: Top view of fungal colony; B: circular winding mycelial growth; C: aggregated spores; D: microconidia; E: macroconidia; F: chlamydospore.

下载: 全尺寸图片幻灯片

图 3 基于EF-1α基因序列的系统发育树

Figure 3. Phylogenetic tree based on EF-1α gene sequences

下载: 全尺寸图片幻灯片

图 4 基于ITS基因序列的系统发育树

Figure 4. Phylogenetic tree based on ITS gene sequences

下载: 全尺寸图片幻灯片

图 5 病原菌在不同温度的生长情况

在显著性水平为0.05的情况下，不同小写字母表示显著差异。下同。

Figure 5. Growth of pathogen at different temperatures

Data with different lowercase letters indicate significant differences at level of 0.05. Same for below.

下载: 全尺寸图片幻灯片

图 6 病原菌在不同pH的生长情况

Figure 6. Growth of pathogen at different pH

下载: 全尺寸图片幻灯片

图 7 病原菌在不同碳源的生长情况

Figure 7. Growth of pathogen on different carbon sources

下载: 全尺寸图片幻灯片

图 8 病原菌在不同氮源的生长情况

Figure 8. Growth of pathogen on different nitrogen sources

下载: 全尺寸图片幻灯片

图 9 病原菌的氧化还原酶活性

Figure 9. Oxidoreductase activity of pathogen

下载: 全尺寸图片幻灯片

图 10 病原菌的水解酶活性

Figure 10. Hydrolase activity of pathogen

下载: 全尺寸图片幻灯片

参考文献(36)

[1]	黄源欣, 辛琨, 熊燕梅, 等. 基于多环境因子条件下老鼠簕的生长适应性 [J]. 中南林业科技大学学报, 2023, 43(5):86−94. HUANG Y X, XIN K, XIONG Y M, et al. Growth adaptability of Acanthus ilicifolius based on multiple environmental factors [J]. Journal of Central South University of Forestry & Technology, 2023, 43(5): 86−94.(in Chinese)
[2]	DAHDOUH-GUEBAS F. World atlas of mangroves: Mark spalding, mami kainuma and lorna collins (eds) [J]. Human Ecology, 2011, 39(1): 107−109. doi: 10.1007/s10745-010-9366-7
[3]	徐慧鹏, 刘涛, 张建兵. 红树林碳埋藏过程对海平面上升、气候变化和人类活动的响应 [J]. 广西科学, 2020, 27(1):84−90. XU H P, LIU T, ZHANG J B. Response of carbon sequestration in mangroves to sea level rise, climate change and human activities [J]. Guangxi Sciences, 2020, 27(1): 84−90.(in Chinese)
[4]	许方宏, 张进平, 张倩媚, 等. 广东湛江高桥三个天然红树林的土壤碳库 [J]. 价值工程, 2012, 31(15):5−6. XU F H, ZHANG J P, ZHANG Q M, et al. Carbon storage of three natural mangrove forests in Gaoqiao, Zhanjiang [J]. Value Engineering, 2012, 31(15): 5−6.(in Chinese)
[5]	但新球, 廖宝文, 吴照柏, 等. 中国红树林湿地资源、保护现状和主要威胁 [J]. 生态环境学报, 2016, 25(7):1237−1243. DAN X Q, LIAO B W, WU Z B, et al. Resources, conservation status and main threats of mangrove wetlands in China [J]. Ecology and Environmental Sciences, 2016, 25(7): 1237−1243.(in Chinese)
[6]	韦秀桂, 王红园, 黄秀昆, 等. 老鼠簕生物碱A对小鼠药物性肝损伤的保护作用 [J]. 中国实验方剂学杂志, 2019, 25(15):103−107. WEI X G, WANG H Y, HUANG X K, et al. Protective effect of alkaloid A from acanthi ilicifolii herba seu Radix on drug-induced liver injury in mice [J]. Chinese Journal of Experimental Traditional Medical Formulae, 2019, 25(15): 103−107.(in Chinese)
[7]	王宗英, 陈永好, 郑广进, 等. 老鼠簕生物碱A及其衍生物乙酰老鼠簕生物碱A的抗炎镇痛作用 [J]. 中国医院药学杂志, 2011, 31(10):807−810. WANG Z Y, CHEN Y H, ZHENG G J, et al. Study on the anti-inflammatory and analgesic effects of ilicifolius alkaloids A and its derivatives acetyl ilicifolius alkaloids A [J]. Chinese Journal of Hospital Pharmacy, 2011, 31(10): 807−810.(in Chinese)
[8]	SARDAR P K, DEV S, AL BARI M A, et al. Antiallergic, anthelmintic and cytotoxic potentials of dried aerial parts of Acanthus ilicifolius L [J]. Clinical Phytoscience, 2018, 4(1): 1−13. doi: 10.1186/s40816-018-0060-4
[9]	李培谦, 冯宝珍, 赵燕飞, 等. 芍药红斑病病原菌鉴定、生物学特性及有效药剂筛选 [J]. 核农学报, 2023, 37(8):1533−1541. LI P Q, FENG B Z, ZHAO Y F, et al. Identification, biological characteristics and screening of effective fungicides for red spot on peony [J]. Journal of Nuclear Agricultural Sciences, 2023, 37(8): 1533−1541.(in Chinese)
[10]	范绍斌, 苏济钰, 方甜, 等. 枫香叶斑病病原菌 [J]. 菌物学报, 2022, 41(3):420−434. FAN S B, SU J Y, FANG T, et al. Pathogen of Liquidambar formosana leaf spot [J]. Mycosystema, 2022, 41(3): 420−434.(in Chinese)
[11]	何苏琴, 文朝慧, 白滨, 等. 莴苣叶疫病和茎腐病的病原 [J]. 菌物学报, 2023, 42(2):468−483. HE S Q, WEN Z H, BAI B, et al. Leaf blight and stem rot caused by Pythium tracheiphilum on lettuces in Gansu and Qinghai Provinces, China [J]. Mycosystema, 2023, 42(2): 468−483.(in Chinese)
[12]	APPLEBY J M, NELSON G, MCPHERSON M J, et al. Polymerase chain reaction amplification of the RuBisCo small subunit genes and their novel application to plant tissue identification [J]. Heredity, 1997, 79(6): 557−563. doi: 10.1038/hdy.1997.199
[13]	谭秀梅, 阿地力·沙塔尔, 朴春根, 等. 无柄灵芝遗传多样性的SRAP、ITS、TEF1-α和LSU分析 [J]. 微生物学通报, 2016, 43(12):2667−2677. TAN X M, ADIL·SATTAR, PIAO C G, et al. Genetic diversity of Ganoderma resinaceum by SRAP, ITS, TEF1-α and LSU markers [J]. Microbiology China, 2016, 43(12): 2667−2677.(in Chinese)
[14]	方中达. 植病研究方法[M]. 3版. 北京: 中国农业出版社, 1998.
[15]	普梅英, 武自强, 张诗文, 等. 华东山茶花腐病病原菌分离鉴定 [J]. 浙江农业学报, 2023, 35(1):121−127. PU M Y, WU Z Q, ZHANG S W, et al. Isolation and identification of petal blight disease of Camellia japonica [J]. Acta Agriculturae Zhejiangensis, 2023, 35(1): 121−127.(in Chinese)
[16]	朱轶慧, 刘玉军, 毕飞虎, 等. 红叶石楠叶斑病病原鉴定及其生物学特性研究 [J]. 植物保护, 2021, 47(5):210−215,235. ZHU Y H, LIU Y J, BI F H, et al. Identification and biological characteristics of the pathogen causing leaf spot disease on Photinia × fraseri [J]. Plant Protection, 2021, 47(5): 210−215,235.(in Chinese)
[17]	JOHNSON-BUCK A, LI J M, TEWARI M, et al. A guide to nucleic acid detection by single-molecule kinetic fingerprinting [J]. Methods, 2019, 153: 3−12. doi: 10.1016/j.ymeth.2018.08.002
[18]	BONNY S Q, MOTALIB HOSSAIN M A, UDDIN S M K, et al. Current trends in polymerase chain reaction based detection of three major human pathogenic vibrios [J]. Critical Reviews in Food Science and Nutrition, 2022, 62(5): 1317−1335. doi: 10.1080/10408398.2020.1841728
[19]	POCZAI P, VARGA I, HYVÖNEN J. Internal transcribed spacer (ITS) Evolution in Populations Of The Hyperparasitic European Mistletoe Pathogen Fungus, Sphaeropsis Visci (Botryosphaeriaceae): The utility of ITS2 secondary structures [J]. Gene, 2015, 558(1): 54−64. doi: 10.1016/j.gene.2014.12.042
[20]	黎肇家, 李汶晋, 毛跃彦, 等. 栾树枝枯病病原菌鉴定及其生物学特性研究 [J]. 西北农林科技大学学报(自然科学版), 2020, 48(9):53−63. LI Z J, LI W J, MAO Y Y, et al. Identification and biological characteristics of branch withered disease causing pathogen to Koelreuteria paniculata Laxm [J]. Journal of Northwest A & F University (Natural Science Edition), 2020, 48(9): 53−63.(in Chinese)
[21]	WANG J, YU X P, JIA R, et al. An in vitro and in silico study to explore the response of catalase to 4-chlorophenol and their interacting mechanisms [J]. Journal of Molecular Liquids, 2021, 337: 116444. doi: 10.1016/j.molliq.2021.116444
[22]	WANG H, YANG H S, LIU J H, et al. Combined effects of temperature and copper ion concentration on the superoxide dismutase activity in Crassostrea ariakensis [J]. Acta Oceanologica Sinica, 2016, 35(4): 51−57. doi: 10.1007/s13131-016-0838-5
[23]	白一彤, 张应朝. 氧化石墨烯对辣根过氧化物酶酶活的影响 [J]. 广东化工, 2018, 45(4):20−21. BAI Y T, ZHANG Y C. Influence of graphene oxide on the activity of horseradish peroxidase [J]. Guangdong Chemical Industry, 2018, 45(4): 20−21.(in Chinese)
[24]	田晓丽, 姜文侠, 张笑然, 等. 基于恒温催化反应过程中吸光度实时测定的漆酶活性快速测定 [J]. 食品研究与开发, 2022, 43(16):139−145. TIAN X L, JIANG W X, ZHANG X R, et al. Rapid detection method for laccase activity based on real-time absorbance measurement at constant temperature [J]. Food Research and Development, 2022, 43(16): 139−145.(in Chinese)
[25]	CAI L N, XU S N, LU T, et al. Salt-tolerant mechanism of marine Aspergillus niger cellulase cocktail and improvement of its activity [J]. Chinese Journal of Chemical Engineering, 2020, 28(4): 1120−1128. doi: 10.1016/j.cjche.2019.11.012
[26]	王茜, 许光治. Leifsonia菌β-葡萄糖苷酶(Lf18920)的酶学性质研究及对藏红花素转化作用的初步研究 [J]. 中国食品添加剂, 2023, 34(2):11−18. WANG X, XU G Z. Enzymatic properties of Leifsonia β-glucosidase (Lf18920) and its degradation of crocin [J]. China Food Additives, 2023, 34(2): 11−18.(in Chinese)
[27]	黄立飞, 刘伟明, 刘也楠, 等. 甘薯茎基部腐烂病调查及病原鉴定 [J]. 中国农学通报, 2019, 35(18):135−141. HUANG L F, LIU W M, LIU Y N, et al. Investigation of stem base rot of sweetpotato and the pathogeny identification [J]. Chinese Agricultural Science Bulletin, 2019, 35(18): 135−141.(in Chinese)
[28]	陈宏州, 杨红福, 姚克兵, 等. 水稻恶苗病病原菌鉴定及室内药剂毒力测定 [J]. 植物保护学报, 2018, 45(6):1356−1366. CHEN H Z, YANG H F, YAO K B, et al. Identification of rice bakanae pathogens and indoor toxicity test of fungicides [J]. Journal of Plant Protection, 2018, 45(6): 1356−1366.(in Chinese)
[29]	刘丽萍, 方鹏鹏. 马铃薯干腐病镰刀菌的分离及生物学特性研究 [J]. 陇东学院学报, 2017, 28(5):62−66. LIU L P, FANG P P. The isolation and biological characteristic of Fusarium associated with potato dry rot [J]. Journal of Longdong University, 2017, 28(5): 62−66.(in Chinese)
[30]	柴海燕, 贾娇, 白雪, 等. 吉林省玉米穗腐病致病镰孢菌的鉴定与部分菌株对杀菌剂的敏感性 [J]. 中国农业科学, 2023, 56(1):64−78. CHAI H Y, JIA J, BAI X, et al. Identification of pathogenic Fusarium spp. causing maize ear rot and susceptibility of some strains to fungicides in Jilin Province [J]. Scientia Agricultura Sinica, 2023, 56(1): 64−78.(in Chinese)
[31]	李孟滕, 王芯, 马晓冉, 等. 紫花苜蓿根腐病病原菌分离鉴定与致病性 [J]. 中国草地学报, 2022, 44(11):84−91. LI M T, WANG X, MA X R, et al. Isolation, identification and pathogenicity of pathogens causing alfalfa root rot [J]. Chinese Journal of Grassland, 2022, 44(11): 84−91.(in Chinese)
[32]	郭强, 马文清, 陈海生, 等. 广西崇左市甘蔗梢腐病病原菌的分离鉴定及生物学特性测定 [J]. 南方农业学报, 2019, 50(8):1728−1734. GUO Q, MA W Q, CHEN H S, et al. Isolation identification and biological characteristics of the pathogen causing sugarcane pokkah boeng in Chongzuo, Guangxi [J]. Journal of Southern Agriculture, 2019, 50(8): 1728−1734.(in Chinese)
[33]	高婧, 张园园, 王凯, 等. 向日葵枯萎病菌的分离鉴定及其生物学特性 [J]. 中国油料作物学报, 2016, 38(2):214−222. GAO J, ZHANG Y Y, WANG K, et al. Identification of sunflower wilt pathogen and its biological characteristics [J]. Chinese Journal of Oil Crop Sciences, 2016, 38(2): 214−222.(in Chinese)
[34]	郑童童, 王宁, 李进, 等. 小麦镰刀菌叶枯病病原鉴定及生物学特性研究 [J]. 中国农学通报, 2023, 39(8):90−98. ZHENG T T, WANG N, LI J, et al. Pathogen identification and biological characteristics of wheat Fusarium leaf blight [J]. Chinese Agricultural Science Bulletin, 2023, 39(8): 90−98.(in Chinese)
[35]	覃茜, 陆祖正, 丁丽琼, 等. 广西兰花镰刀菌叶斑病病原菌鉴定及生物学特性 [J]. 北方园艺, 2019(18):40−46. QIN Q, LU Z Z, DING L Q, et al. Identification and biological characteristics of the pathogen causing orchid Fusarium leaf spot in Guangxi [J]. Northern Horticulture, 2019(18): 40−46.(in Chinese)
[36]	LI L J, LIU X, SHI Y X, et al. Synthesis of Fe³⁺-doped aminated lignin as A peroxidase mimic for colorimetric detection of H₂O₂ and glucose [J]. Paper and Biomaterials, 2021, 6(4): 1−11.