热带作物学报2025，Vol.46Issue(7)：1733-1744,12.DOI:10.3969/j.issn.1000-2561.2025.07.020
海南万宁咖啡炭疽病菌Colletotrichum siamense的分离鉴定及LAMP快速检测体系的建立
Isolation,Identification of Colletotrichum siamense Causing Coffee Anthracnose in Wanning,Hainan,and Development of a Rapid LAMP Detection System
摘要
Abstract
Xinglong coffee,a pillar of the local characteristic agriculture in Wanning,faces severe threats to its sustain-able production from anthracnose.This disease can cause leaf scorching,branch ulcers,and berry brown rot,making it one of the major diseases in the industry.In this study,samples of coffee anthracnose were collected from the main cof-fee-growing areas in Wanning and the pathogenic fungi were isolated.The pathogenic fungi were identified through the combined analysis of multiple genes,including ITS,TUB2,CHS-1,ACT and GAPDH.Furthermore,a rapid detection system based on the loop-mediated isothermal amplification(LAMP)technique was established.50 typical disease samples were collected,and through tissue separation and purification,a total of 24 Colletotrichum isolates were ob-tained.The pathogen population structure in the five planting areas of Wanning was clarified to comprise four species of Colletotrichum spp.:C.tropicale,C.karstii,C.fructicola,and the dominant species was C.siamense(accounting for 45.8％).Based on the specific region of TUB2,a specific LAMP primer set(Tub-L1)was designed and screened.The optimized reaction parameters were established as follows:isothermal amplification at 63 ℃ for 50 minutes,outer-inner primer concentration ratio of 8∶1,Mg2+8 mmol/L,dNTPs 0.8 mmol/L,and without the need for betaine.Validation experiments demonstrated that the detection sensitivity of this system reached 100 pg/μL(10 times higher than conventional PCR).Furthermore,the system showed no positive reactions to seven closely related species within Colletotrichum spp.(including C.tropicale,C.fructicola,C.karstii)or seven non-target plant pathogens[including Hemileia vastatrix(coffee leaf rust),Diaporthephaseolorum,and others].Using the optimized LAMP detection system,the diseased leaf samples collected from the field were analyzed.C.siamense was successfully detected in all samples,and the results were completely consistent with those obtained by conventional PCR methods.In summary,the LAMP-based rapid detection system for Colletotrichum sp.established and optimized in this study demonstrates sig-nificant advantages,including simplified operation,high reaction efficiency,strong specificity,enhanced sensitivity,and visualizable results,making it highly suitable for field-based rapid detection of C.siamense.This system would provide reliable technical support for the precise identification and efficient detection of coffee anthracnose fungus,and have important practical significance and application value for the early warning,disease monitoring,and integrated control of coffee anthracnose.关键词
咖啡炭疽病/病原鉴定/环介导等温扩增(LAMP)/检测Key words
coffee anthracnose/pathogen identification/loop-mediated isothermal amplification(LAMP)/detection分类
农业科技引用本文复制引用
温思为,高圣风,田甜,刘世超,苟亚峰,张若男,薛超,孙世伟..海南万宁咖啡炭疽病菌Colletotrichum siamense的分离鉴定及LAMP快速检测体系的建立[J].热带作物学报,2025,46(7):1733-1744,12.基金项目
海南省自然科学基金项目(No.321QN329) （No.321QN329）
国家现代农业产业技术体系项目(CARS-11). （CARS-11）
