Simplified and efficient Agrobacterium-mediated genetic transformation of Botrytis cinerea using mycelia.

Botrytis cinerea is a necrotrophic fungal pathogen responsible for grey mold disease, causing significant crop losses globally. Effective genetic manipulation of this pathogen is crucial for understanding its biology and developing better disease management strategies. However, current transformation methods typically use conidia spores or protoplasts, processes that are labor-intensive, inefficient, and often yield inconsistent results. We developed a novel Agrobacterium-mediated transformation protocol that utilizes B. cinerea mycelia, eliminating the need for sporulation or protoplast generation and simplifying genetic manipulation. Using a newly constructed binary expression vector encoding green fluorescent protein and hygromycin resistance, we transformed four different B. cinerea strains. All transformations resulted in stable integration and robust green fluorescent protein expression, confirmed by quantitative polymerase chain reaction and confocal microscopy. Although transformants exhibited altered colony morphology compared to wild-type strains, they remained viable and stably expressed the integrated transgene, supporting the method's utility for genetic studies in B. cinerea. This streamlined method provides a reliable, efficient, and scalable approach for genetic studies in B. cinerea, significantly enhancing fungal functional genomics research and plant pathology investigations.