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Loop-mediated isothermal amplification and PCR combined assay to detect and distinguish latent Colletotrichum spp. infection on strawberry

文献类型： 外文期刊

作者： Liu, Ya 1 ; Ji, Ying 2 ; Han, Yongchao 3 ; Song, Lili 4 ; Zhang, Liqing 2 ; Ning, Zhiyuan 5 ; Yan, Weizhong 6 ; Gao, Qing 1 ;

作者机构： 1.Shanghai Ocean Univ, Coll Food Sci, Shanghai 201306, Peoples R China

2.Shanghai Acad Agr Sci SAAS, Forestry & Fruit Tree Res Inst, Shanghai Key Lab Protected Hort Technol, Shanghai 201403, Peoples R China

3.Hubei Acad Agr Sci, Inst Ind Crops, Wuhan 430064, Peoples R China

4.Agr Serv Ctr Nanping, Zhuanglang 744600, Pingliang, Peoples R China

5.Anhui Acad Agr Sci, Hort Res Inst, Hefei 230031, Peoples R China

6.Shanghai Agr Technol Extens Serv Ctr, Shanghai 201103, Peoples R China

关键词： Strawberry; Colletotrichum spp; DNA diagnosis; PCR; LAMP

期刊名称：JOURNAL OF PLANT PATHOLOGY （ 影响因子：1.729； 五年影响因子：1.681 ）

ISSN： 1125-4653

年卷期： 2021 年 103 卷 3 期

页码：

收录情况： SCI

摘要： Strawberry is economically important. Anthracnose caused by Colletotrichum spp. has been a serious threat to this crop globally. To detect and distinguish latent Colletotrichum spp. infection on asymptomatic plants at an affordable cost is crucial for this disease control and the sustainability of strawberry industry. Loop-mediated isothermal amplification (LAMP) assay is superior for the higher sensitiveness, efficiency, and lower cost than other methods for pathogen diagnosis. In this study, six previously reported barcodes were evaluated against DNA templates from strawberry fungal pathogens including six Colletotrichum species and five beyond this genus, and further verified in 40 Colletotrichum isolates. Based on the discernibility revealed, a LAMP assay was developed for the diagnosis of Colletotrichum spp. by designing six primers recognizing the conserved regions in beta-tubulin 2 gene from 13 Colletotrichum species of C. gloeosporioides and C. acutatum complexes. The specificity and accuracy of LAMP assay was tested against six Colletotrichum species of two complexes, with a detection sensitivity of 100 pg/mu L genomic DNA. Current LAMP assay beginning with a 10 min plant lysis enabled a direct and quick diagnosis of Colletotrichum spp. in strawberry within one hour. Followed by PCR using primers specific to ApMat, Marker 2, and Marker 1, this assay could specifically differentiate Colletotrichum species, at least for the dominant species C. fructicola and C. siamense in China, realizing a diagnosis of latent infection at a species level. Current LAMP-PCR combined protocol allows for a sensitive and efficient detection and differentiation of latent Colletotrichum infection on strawberry, which will be useful for disease management and monitoring pathogen population.

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