Novel Mycoparasitic Mechanisms and Colonization Patterns on Poplar Revealed by GFP-Tagging of the Biocontrol Fungus Clonostachys reniana.

Abstract

Clonostachys rosea has long been the primary model for studying mycoparasitism within its genus; however, the potential of other species remains largely unexplored. In this study, we established a PEG-CaCl2-mediated protoplast transformation system for Clonostachys reniana. Our results demonstrate for the first time that this species is amenable to genetic manipulation and produces transformants with genetic stability, providing a reliable platform for functional genomic research in this fungus. Using Green Fluorescent Protein (GFP) tagging, we achieved stable transformants that retained wild-type physiological traits. Crucially, our data indicated that C. reniana utilizes a distinct mycoparasitic mechanism, which differs from the well-established sequential process of adhesion, coiling, and lysis seen in C. rosea. Confocal and scanning electron microscopy (SEM) revealed that C. reniana, following initial coiling and invasive structure formation, penetrates the host hyphae of Botryosphaeria dothidea. It then grows longitudinally within the hyphal lumen, absorbing nutrients and eliminating the pathogen from the inside. Furthermore, C. reniana hyphae can colonize the intercellular spaces of the stem periderm in 84K poplar (Populus alba × P. glandulosa), while displaying a distinct tissue-specific behavior in the roots by forming a mantle on the root surface resembling that of ectomycorrhizae. These findings establish C. reniana as a highly promising secondary model species within the Clonostachys genus. By uncovering a novel "internal-consumption" mycoparasitic mode, this study expands our understanding of the ecological diversity of biocontrol fungi and provides a powerful genetic toolset for future functional genomic research.