基于环介导等温扩增技术检测Colletotrichum camelliae

涂一怡; 张幼; 徐婷; 陈俊杰; 王玉春; 吕务云

doi:10.13305/j.cnki.jts.2025.05.007

茶叶科学 >

2025 , Vol. 45 >Issue 5: 770 - 782

DOI: https://doi.org/10.13305/j.cnki.jts.2025.05.007

研究报告

基于环介导等温扩增技术检测Colletotrichum camelliae

涂一怡 ,
张幼 ,
徐婷 ,
陈俊杰 ,
王玉春 ,
吕务云

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浙江农林大学茶学与茶文化学院,浙江杭州 311300

涂一怡,女,硕士研究生,主要从事茶树抗病机理方面的研究。

收稿日期: 2025-02-25

修回日期: 2025-04-11

网络出版日期: 2025-10-17

基金资助

浙江省农业新品种选育重大科技专项（2021C02067-7）、浙江省大学生科技创新活动计划（新苗人才计划）项目（2024R412B052）

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Loop-mediated Isothermal Amplification-based Detection of Colletotrichum camelliae

TU Yiyi ,
ZHANG You ,
XU Ting ,
CHEN Junjie ,
WANG Yuchun ,
LÜ Wuyun

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College of Tea Science and Tea Culture, Zhejiang A & F University, Hangzhou 311300, China

Received date: 2025-02-25

Revised date: 2025-04-11

Online published: 2025-10-17

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摘要

山茶炭疽菌Colletotrichum camelliae是引起茶炭疽病的优势病原菌,该病害可致茶树大量发病叶片脱落,严重影响茶叶产量,造成较大的经济损失。因此,早期及时检测茶树叶片中有无C. camelliae侵染,对生产上及时防治茶炭疽病有重要意义。基于环介导等温扩增（Loop-mediated isothermal amplification,LAMP）技术,以C. camelliae中1个编码LEA（Late embryogenesis abundant）结构域蛋白的特异基因作为靶标,设计并筛选了3对特异性LAMP引物,包括1对外引物、1对内引物和1对环引物。随后通过特异性和灵敏度检测,以及室内接种和田间发病组织检测试验,建立了C. camelliae的LAMP检测方法。该方法在65 ℃下反应30 min效果最佳,检测结果可通过2%琼脂糖凝胶电泳验证和SYBR Green Ⅰ显色判断。在特异性检测试验中,仅C. camelliae扩增后呈阳性反应;灵敏度检测试验中,该方法对C. camelliae基因组DNA模板的最低检测质量浓度为100 ag·µL^-1;对室内接种C. camelliae后的病叶和田间18个茶树品系的炭疽病病叶的检测结果显示,该LAMP方法能准确地检测出C. camelliae。综上,本研究建立了1种C. camelliae的LAMP检测方法,具有快速简便、灵敏度高等优点,可实现C. camelliae的特异性检测及茶炭疽病的快速诊断。

关键词： 病害检测; 茶炭疽病; 环介导等温扩增技术; 山茶炭疽菌; 特异基因

本文引用格式

涂一怡 , 张幼 , 徐婷 , 陈俊杰 , 王玉春 , 吕务云 . 基于环介导等温扩增技术检测Colletotrichum camelliae[J]. 茶叶科学, 2025 , 45(5) : 770 -782 . DOI: 10.13305/j.cnki.jts.2025.05.007

Abstract

Colletotrichum camelliae is the dominant pathogen causing tea anthracnose. This disease causes significant defoliation, which has a major impact on the following year's yield and results in great economic losses. Therefore, the early and timely detection of C. camelliae in tea leaves is of great significance for effective control of anthracnose in production. In this study, based on a loop-mediated isothermal amplification (LAMP) assay, a specific gene encoding a late embryogenesis abundant (LEA) domain protein from C. camelliae was selected for the design of LAMP primers. Three pairs of specific LAMP primers, including two outer primers, two inner primers, and two loop primers, were designed and selected. Subsequently, a LAMP assay for C. camelliae was established through specificity and sensitivity tests, and by detecting C. camelliae in artificially inoculated and infected tea leaves. The optimal reaction condition was determined to be at 65 ℃ for 30 min. The results were verified by 2% agarose gel electrophoresis with SYBR Green Ⅰ added. Specificity tests reveal that, only DNA extracted from C. camelliae isolates exhibited a positive amplification reaction. In sensitivity tests, the assay could detect genomic DNA templates of C. camelliae at a minimum concentration of 100 ag·µL^-1. The LAMP assay accurately detected C. camelliae in artificially inoculated diseased leaves and anthracnose-affected leaves of 18 tea germplasms in the field. In summary, the LAMP assay for detecting C. camelliae established in this study has the advantages of rapid, simple and high sensitivity, enabling the specific detection of C. camelliae and the quick diagnosis of tea anthracnose.

Key words： Colletotrichum camelliae; disease detection; loop-mediated isothermal amplification; specific gene; tea anthracnose

参考文献

[1] Manawasinghe I S, Jayawardena R S, Li H L, et al.Microfungi associated with Camellia sinensis: a case study of leaf and shoot necrosis on tea in Fujian, China[J]. Mycosphere, 2021, 12(1): 430-518.
[2] 孙钦玉, 张家侠, 夏先江, 等. 茶炭疽病生物防治研究现状与展望[J]. 茶业通报, 2017, 39(2): 69-72.
Sun Q Y, Zhang J X, Xia X J, et al.Research status and prospect of biological control of tea anthracnose[J]. Journal of Tea Business, 2017, 39(2): 69-72.
[3] Guo M, Pan Y M, Dai Y L, et al.First report of brown blight disease caused by Colletotrichum gloeosporioides on Camellia sinensis in Anhui Province, China[J]. Plant Disease, 2014, 98(2): 284. doi: 10.1094/PDIS-08-13-0896-PDN.
[4] Xiong F, Wang Y C, Lu Q H, et al.Lifestyle characteristics and gene expression analysis of Colletotrichum camelliae isolated from tea plant [Camellia sinensis (L.) O. Kuntze] based on transcriptome[J]. Biomolecules, 2020, 10(5): 782. doi: 10.3390/biom10050782.
[5] Hyde K D, Nooedeloos M E, Savchenko K G, et al.The 2024 outline of fungi and fungus-like taxa[J]. Mycosphere, 2024, 15(1): 5146-6239.
[6] 陈宗懋, 罗逢健, 周利, 等. 茶叶中农药残留和污染物管控技术体系创建及应用[J]. 茶叶科学, 2021, 41(1): 1-6.
Chen Z M, Luo F J, Zhou L, et al.Innovation and application of control system for pesticide residues and contaminants in tea[J]. Journal of Tea Science, 2021, 41(1): 1-6.
[7] 李红莉, 周铁峰, 毛宇骁, 等. 西湖龙井产区茶树炭疽菌分离鉴定及其植物源抑菌剂筛选[J]. 茶叶科学, 2023, 43(2): 194-204.
Li H L, Zhou T F, Mao Y X, et al.Isolation and identification of anthracnose pathogen from Xihu Longjing plantation and screening of its plant-derived fungicides[J]. Journal of Tea Science, 2023, 43(2): 194-204.
[8] Li Q S, Zhu J Y, Ren N, et al.Characteristics and pathogenicity of Discula theae-sinensis isolated from tea plant (Camellia sinensis) and interaction with Colletotrichum spp.[J]. Plants, 2023, 12(19): 3427. doi: 10.3390/plants12193427.
[9] 张淑梅, 李晶, 王玉霞, 等. 植物病原真菌分子诊断技术研究进展[J]. 安徽农业科学, 2010, 38(2): 597-599.
Zhang S M, Li J, Wang Y X, et al.Advances in molecular diagnosis of plant fungal pathogens[J]. Journal of Anhui Agricultural Sciences, 2010, 38(2): 597-599.
[10] Keinath A P, Somai B M, Dean R A. Method of diagnosing gummy stem blight in plants using a polymerase chain reaction assay: US20010758073 [P].2001-01-10[2025-02-25].
[11] Somai B M, Keinath A P, Dean R A.Development of PCR-ELISA for detection and differentiation of Didymella bryoniae from related Phoma species[J]. Plant Disease, 2002, 86(7): 710-716.
[12] Sharma A, Kang S S, Sandhu P, et al.Quick and reliable diagnosis of phytoplasmas through nested PCR assay[J]. Plant Disease Research, 2010, 25(1): 48-50.
[13] Diédhiou A G, Borges W L, Sadio O, et al.Assessment of DNA extraction methods for detection of arbuscular mycorrhizal fungi in plant roots by nested-PCR[J]. Acta Scientiarum Biological Sciences, 2014, 36(4): 433-441.
[14] Mansotra R, Vakhlu J.Comprehensive account of present techniques for in-field plant disease diagnosis[J]. Archives of Microbiology, 2021, 203(9): 5309-5320.
[15] 常海城, 李虎, 郑立秋, 等. 向日葵核盘菌环介导等温扩增检测技术[J]. 中国油料作物学报, 2024, 46(6): 1412-1420.
Chang H C, Li H, Zheng L Q, et al.Loop-mediated isothermal amplification assay for detection of Sclerotinia sclerotiorum in sunflower[J]. Chinese Journal of Oil Crop Sciences, 2024, 46(6): 1412-1420.
[16] Zatti M S, Arantes T D, Theodoro R C.Isothermal nucleic acid amplification techniques for detection and identification of pathogenic fungi: a review[J]. Mycoses, 2020, 63(10): 1006-1020.
[17] Notomi T, Okayama H, Masubuchi H, et al.Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Research, 2000, 28(12): e63. doi: 10.1093/nar/28.12.e63.
[18] 连文旭, 王萌, 张宇, 等. 橡胶树胶孢炭疽菌复合群LAMP检测方法的建立及应用[J]. 植物病理学报, 2022, 52(5): 1-11.
Lian W X, Wang M, Zhang Y, et al.Development and application of a loop-mediated isothermal amplification assay for detection of Colletotrichum gloeosporioides species complex on rubber trees[J]. Acta Phytopathologica Sinica, 2022, 52(5): 1-11.
[19] Notomi T, Mori Y, Tomita N, et al.Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects[J]. Journal of Microbiological Methods, 2015, 53(1): 1-5.
[20] Fischbach J, Xander N C, Frohme M.Shining a light on LAMP assays-a comparison of LAMP visualization methods including the novel use of berberine[J]. Biotechniques, 2015, 58(4): 189-194.
[21] Mori Y, Notomi T.Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases[J]. Journal of Infection and Chemotherapy, 2009, 15(2): 62-69.
[22] Hong L, Li J, Lü J, et al.Development and evaluation of a loop-mediated isothermal amplification assay for clinical diagnosis of respiratory human adenoviruses emergent in China[J]. Diagnostic Microbiology and Infectious Disease, 2021, 101(1): 115-401.
[23] Khan M, Wang R B, Li B J, et al.Comparative evaluation of the LAMP assay and PCR-based assays for the rapid detection of Altemaria solani[J]. Frontiers in Microbiology, 2018, 9: 2089. doi: 10.3389/fmicb.2018.02089.
[24] Niessen L, Vogel R F.Detection of Fusarium graminearum DNA using a loop-mediated isothermal amplification (LAMP) assay[J]. International Journal of Food Microbiology, 2010, 140(2/3): 183-191.
[25] 王大会, 赵蕊, 刘丹丹, 等. 猕猴桃软腐病菌的环介导等温扩增快速检测技术[J]. 植物保护学报, 2022, 49(6): 1811-1812.
Wang D H, Zhao R, Liu D D, et al.Rapid detection of kiwifruit soft rot pathogen using loop-mediated isothermal amplification technology[J]. Journal of Plant Protection, 2022, 49(6): 1811-1812.
[26] 余毅鑫, 于健, 张新娜, 等. 基于环介导等温扩增技术检测德里腐霉(Pythium deliense)[J]. 植物病理学报, 2025, 55(3): 478-486.
Yu Y X, Yu J, Zhang X N, et al.Loop-mediated isothermal amplification-based detection of Pythium delicense[J]. Acta Phytopathologica Sinica, 2025, 55(3): 478-486.
[27] Tian Q, Lu C C, Wang S S, et al.Rapid diagnosis of soybean anthracnose caused by Colletotrichum truncatum using a loop-mediated isothermal amplification (LAMP) assay[J]. European Journal of Plant Pathology, 2016, 148(4): 785-793.
[28] Kandan A, Akhtar J, Singh B, et al.Application of loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive detection of fungal pathogen, Colletotrichum capsici in Capsicum annuum[J]. Journal of Environmental Biology, 2016, 37(6): 1355-1360.
[29] Katoh H, Fukuda T, Nishigawa H, et al.Rapid detection of Colletotrichum gloeosporioides in infected strawberry plants using loop-mediated isothermal amplification[J]. Journal of General Plant Pathology, 2016, 82(4): 190-198.
[30] Zou H Y, Li T W, Zhang J, et al.Rapid detection of Colletotrichum siamense from infected tea plants using filter-disc DNA extraction and loop-mediated isothermal amplification[J]. Plant Disease, 2024, 108(1): 35-40.
[31] 程开鑫, 杨凯欣, 邓雅元, 等. 山茶炭疽菌对茶树的致病性及其对杀菌剂的敏感性研究[J]. 茶叶科学, 2023, 43(1): 55-66.
Chen K X, Yang K X, Deng Y Y, et al.Pathogenicity and fungicide sensitivity of Colletotrichum camelliae from tea plant (Camellia sinensis)[J]. Journal of Tea Science, 2023, 43(1): 55-66.
[32] Wang Y C, Hao X Y, Wang L, et al.Diverse Colletotrichum species cause anthracnose of tea plants (Camellia sinensis (L.) O. Kuntze) in China[J]. Scientific Reports, 2016, 6(1): 35287. doi: 10.1038/srep35287.
[33] Wang Y C, Tu Y Y, Chen X L, et al.Didymellaceae species associated with tea plant (Camellia sinensis) in China[J]. MycoKeys, 2024, 105: 217-251. doi: 10.3897/mycokeys.105.119536.
[34] Lü W Y, Yu Y T, Zhong X F, et al.Identification and characterization of Cladosporium species associated with tea plants (Camellia sinensis) in China[J]. Plant Pathology, 2023, 72(5): 868-880.
[35] Tu Y Y, Wang Y C, Jiang H, et al.A loop-mediated isothermal amplification assay for the rapid detection of Didymella segeticola causing tea leaf spot[J]. Journal of Fungi, 2024, 10(7): 467. doi: 10.3390/jof10070467.
[36] 王玉春, 刘守安, 卢秦华, 等. 中国茶树炭疽菌属病害研究进展及展望[J]. 植物保护学报, 2019, 46(5): 954-963.
Wang Y C, Liu S A, Lu Q H, et al.Research progress and prospects of Colletotrichum species causing tea plant diseases in China[J]. Journal of Plant Protection, 2019, 46(5): 954-963.
[37] 赵媛媛, 沈丹宇, 于佳, 等. 基于环介导等温扩增技术检测由强雄腐霉引起的玉米茎腐病[J]. 植物保护学报, 2019, 46(3): 626-633.
Zhao Y Y, Shen D Y, Yu J, et al.Rapid detection of corn stalk rot caused by Pythium arrhenomanes using loop-mediated isothermal amplification[J]. Journal of Plant Protection, 2019, 46(3): 626-633.
[38] Liu Y, Ji Y, Han Y C, et al.Loop-mediated isothermal amplification and PCR combined assay to detect and distinguish latent Colletotrichum spp. infection on strawberry[J]. Journal of Plant Pathology, 2021, 103: 887-899. doi: 10.1007/s42161-021-00873-7.
[39] 赖宝春, 姚锦爱. 蜜柚间座壳黑点病菌(Diaporthecitri)LAMP可视化检测技术的建立[J]. 福建农业学报, 2022, 37(11): 1470-1475.
Lai B C, Yao J A.Establishment of a LAMP assay for rapid detecting Diaporthe citri on pomelo[J]. Fujian Journal of Agricultural Sciences, 2022, 37(11): 1470-1475.
[40] Gardes M, Bruns T D.ITS primers with enhanced specificity for basidiomycetes: application to the identification of mycorrhizae and rusts[J]. Molecular Ecology, 1993, 2(2): 113-118.
[41] 曹继芬, 户艳霞, 王新中, 等. 基于微管蛋白基因的烟草根黑腐病菌分子检测[J]. 中国烟草科学, 2016, 37(6): 60-65.
Cao J F, Hu Y X, Wang X Z, et al.Molecular detection of tobacco black root rot pathogen based on β-tubulin gene[J]. Chinese Tobacco Science, 2016, 37(6): 60-65.
[42] Chen Q, Hou L W, Duan W J, et al.Didymellaceae revisited[J]. Studies in Mycology, 2017, 87: 105-159. doi: 10.1016/j.simyco.2017.06.002.
[43] Battaglia M, Olvera-Carrillo Y, Garciarrubio A, et al.The enigmatic LEA proteins and other hydrophilins[J]. Plant Physiology, 2008, 148(1): 6-24.
[44] Mertens J, Aliyu H, Cowan D A.LEA proteins and the evolution of the WHy domain[J]. Applied and Environmental Microbiology, 2018, 84(15): e00539-18. doi: 10.1128/AEM.00539-18.
[45] Sales K, Brandt W, Rumbak E, et al.The LEA-like protein HSP 12 in Saccharomyces cerevisiae has a plasma membrane location and protects membranes against desiccation and ethanol-induced stress[J]. Biochimica et Biophysica Acta, 2000, 1463(2): 267-278.
[46] Hall B M, Owens K M, Singh K K, et al.Distinct functions of evolutionary conserved MSF1 and late embryogenesis abundant (LEA)-like domains in mitochondria[J]. Journal of Biological Chemistry, 2011, 286(45): 39141-39152.
[47] Lan C Z, Yao J N, Yang X J, et al.Specific and sensitive detection of the guava fruit anthracnose pathogen (Colletotrichum gloeosporioides) by loop-mediated isothermal amplification (LAMP) assay[J]. Canadian Journal of Microbiology, 2020, 66(1): 17-24.
[48] Wang S C, Zhang X Y, Zhang Z C, et al.Fusarium-produced vitamin B₆ promotes the evasion of soybean resistance by Phytophthora sojae[J]. Journal of Integrative Plant Biology, 2023, 65(9): 2204-2217.
[49] 陈玉涵, 马心瑶, 田夏夏, 等. 陕西榆林山药根腐病病原菌的分离与鉴定[J]. 西北农业学报, 2022, 31(11): 1521-1533.
Chen Y H, Ma X Y, Tian X X, et al.Isolation and identification of yam root rot in Yulin of Shaanxi[J]. Acta Agriculturae Boreali-occidentalis Sinica, 2022, 31(11): 1521-1533.
[50] 林善海, 黄思良, 覃丽萍, 等. 广西香蕉真菌性叶斑病病原菌种群结构分析[J]. 植物保护, 2011, 37(5): 181-183.
Lin S H, Huang S L, Qin L P, et al.Investigations on the fungal populations associated with banana leaf spot diseases in Guangxi[J]. Plant Protection, 2011, 37(5): 181-183.
[51] 赵伟, 汪涛, 戚仁德. 利用环介导等温扩增(LAMP)技术快速检测辣椒疫霉菌[J]. 植物病理学报, 2015, 45(1): 93-96.
Zhao W, Wang T, Qi R D.Rapid detection of Phytophthora capsici by a loop-mediated isothermal amplification (LAMP) assay[J]. Acta Phytopathologica Sinica, 2015, 45(1): 93-96.

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