A Novel Epiphytic Clonostachys chloroleuca Strain that Effectively Controls Cucumber Anthracnose

Cucumber anthracnose is a destructive disease caused by Colletotrichum orbiculare that affects global cucumber production. Although plant-associated fungi are promising sources of biological control agents, their efficacy against cucumber anthracnose has not been established. In the present study, 108 spore-forming endophytic and epiphytic fungal strains were isolated from the leaves of seven plant species and screened for biocontrol activity against C. orbiculare using detached cucumber cotyledon assays and pot experiments. Three highly suppressive strains (GUSF-44, GUSF-66, and GUSF-153) identified in detached cotyledon assays were further evaluated in pot experiments, in which GUSF-66 exhibited the most consistent and pronounced disease suppression, reducing the lesion area by approximately 75%. A multilocus phylogenetic anal-ysis based on the internal transcribed spacer region, RNA polymerase II second largest subunit gene, and translation elongation factor 1-alpha gene sequences identified GUSF-66 as Clonostachys chloroleuca. Microscopic observations showed that this strain successfully colonized the cucumber cotyledon epiphytically. In vitro assays revealed that GUSF-66 produced lytic enzymes, including chitinase, β-1,3-glucanase, and protease, and inhibited the conidial germination and hyphal growth of C. orbiculare. Although some C. chloroleuca strains have been implicated in soybean root rot, GUSF-66 caused no visible symptoms in soybean plants. To the best of our knowledge, this is the first study to demonstrate the biocontrol potential of C. chloroleuca against cucumber anthracnose.