Nine Johnson grass accessions' excised leaf blades and rhizomes, as well as the leaf blades of two sorghum accessions, BTx623 and SC748-5, were injected with four strains of Colletotrichum sublineola. Seven Johnson grass accession-CS sublineola isolate combinations showed successful acervuli forms. This is the first study to prove Johnson grass rhizomes can be colonized by C. sublineola, notwithstanding the rarity of infection leading to pathogen proliferation.