To screen biocontrol strains with high herbicidal potential， the pathogenic fungus JZ-28 was isolated from naturally infected leaves of Viola philippica in Qinghai Province. Through morphological characterization and multi-gene （ITS， EF-1α， LSU） phylogenetic analysis， the isolate was identified as Clonostachys rosea. Pathogenicity assays demonstrated potent herbicidal activity of strain JZ-28， inducing chlorosis in detached leaves of four dominant broadleaf weed species from the Qinghai-Xizang Plateau（Malva verticillata， Elsholtzia densa， Senecio vulgaris， and Chenopodium album）within 7 days post-inoculation （dpi）. Disease incidence rates in whole-plant assays reached 83%， 95%， 100%， and 70%， respectively. Safety evaluation on six major crops and three cool-season vegetables in Qinghai revealed that JZ-28 was safe for four crops： Zea mays， Vicia faba， Pisum sativum， Brassica napus， and three vegetables： Cucumis sativus， Solanum lycopersicum， and Solanum melongena. Lightly Susceptible was observed in highland barley Hordeum vulgare and Triticum aestivum. These findings indicated JZ-28’s potential as a broad-spectrum bioherbicide against broadleaf weeds in multiple cropping systems. Culture optimization identified sucrose and soybean meal as optimal carbon and nitrogen sources for enhancing mycelial growth and sporulation. This study provided the first evidence of Clonostachys rosea’s bioherbicide potential in alpine ecosystems， addressing a critical research gap in microbial herbicide development and offering sustainable weed management solutions for fragile environments.

Weed control  /  Clonostachys rosea  /  Microbial herbicides