Method to detect and quantify colonization of anthracnose causal agent <i>Colletotrichum gloeosporioides</i> species complex in strawberry by real‐time PCR

Abstract

Abstract Anthracnose, a destructive disease in strawberry worldwide, is caused by the pathogenic fungus Colletotrichum ssp.. To date, numerous studies focusing on its identification, pathogenicity and biological characteristics have been published. However, research on quantifying the colonization of this pathogen is still lacking, which is crucial to the study of anthracnose resistance. In this study, we developed a sensitive primer pair targeting the cutinase gene that is capable of distinguishing Colletotrichum gloeosporioides ( C . gloeosporioides ) species complex, the main species causing strawberry anthracnose in China, including C . fructicola , C . gloeosporioides , C . aenigma and C . siamense , from other Colletotrichum species and typical fungal pathogens. On this basis, we established a quantitative real‐time PCR (qRT‐PCR) assay to accurately quantify pathogen amounts from both pure cultures and infected strawberries, with detection limits of 10 5 –10 1 copies. Using this method, we characterized quantitative traits of C . fructicola colonization in strawberry leaves and discovered differences in pathogen populations at different positions. Moreover, monitoring of pathogen growth during infection showed a dynamic change in the amount of C . fructicola . Finally, a comparison of the absolute and relative quantification results further proved this method's validity. Therefore, we propose that this specific PCR method enables rapid detection and quantification of C . gloeosporioides species complex colonization in strawberry and will be a useful tool for anthracnose management and strawberry resistance studies.