Multi-locus study of Colletotrichum humuli from an ancient herbarium specimen and description of C. humulicola sp. nov. from Humulus scandens

Colletotrichum humuli was originally described from diseased leaves of Humulus lupulus collected in 1890. Due to the absence of molecular data, an isotype specimen was examined to clarify its systematic position. The nuclear ribosomal ITS2 region of this specimen was successfully amplified and sequenced using the primer ITS4 along with a newly designed primer, ITSCF1. Additional gene sequences (GAPDH, CHS-1, HIS3, and ACT) were also retrieved, marking the first successful recovery of multiple loci from an ancient Colletotrichum herbarium specimen. Multi-locus phylogenetic analyses demonstrated that C. humuli is genetically distinct from all other known species. Furthermore, comparison with isolates from H. scandens in South Korea revealed a novel species, described here as C. humulicola; together, these two species form a new species complex within the genus Colletotrichum. This study provides a straightforward protocol for obtaining DNA sequences from historical herbarium material.