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Xinglong coffee, a pillar of the local characteristic agriculture in Wanning, faces severe threats to its sustainable 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 coffee-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 obtained. 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), Diaporthe phaseolorum, 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 significant 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.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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