Species reassignment and rapid molecular detection of Colletotrichum associated with persimmon anthracnose in Brazil

Anthracnose is the main fungal disease affecting persimmon (Diospyros kaki Thunb.) and causes significant economic losses. In Brazil, the disease is caused by Colletotrichum species belonging to the Colletotrichum gloeosporioides (CGSC) and C. acutatum (CASC) species complexes. Accurate species identification is essential for effective disease management strategies. However, phylogeny-based approaches are often costly and labor-intensive. This study aimed to clarify the identification of C. fructicola strains associated with persimmon anthracnose in Brazil using multigene phylogenetic analysis and to develop species-specific PCR primers for C. horii, C. chrysophilum, C. nymphaeae, and C. melonis, the causal agents of persimmon anthracnose. The primers were validated using isolates collected from four persimmon-producing regions in Brazil, and their applicability was also assessed using Colletotrichum isolates obtained from apple (n = 8) and pear (n = 1). Multigene phylogenetic analysis based on ITS, GAPDH, and TUB2 sequences demonstrated that seven isolates previously identified as C. fructicola clustered within the C. chrysophilum clade, leading to their reassignment. Based on partial TUB2 gene sequences, species-specific primers were developed and validated using a collection of 183 isolates from persimmon orchards across four Brazilian states. Colletotrichum horii was the predominant species, representing 76% of the isolates and accounting for more than 90% of the population in Paraná, Santa Catarina, and Rio Grande do Sul. In contrast, C. nymphaeae was more frequent in São Paulo (45%), followed by C. horii (41%) and C. chrysophilum (14%). The primers also correctly identified Colletotrichum isolates obtained from other hosts, although cross-amplification was observed between C. melonis and C. limetticola. The new primers developed in this study provide a rapid and reliable tool for detecting the main Colletotrichum species associated with persimmon anthracnose and may also support species identification in other hosts.