Visualization of the Infection and Colonization Process of Dendrobium officinale Using a Green Fluorescent Protein-Tagged Isolate of Fusarium oxysporum.

Dendrobium officinale soft rot is a widespread and destructive disease caused by Fusarium oxysporum that can seriously affect yield and quality. To better understand the fungal infection and colonization, we successfully created an F. oxysporum labeled with green fluorescent protein using the Agrobacterium tumefaciens-mediated transformation method. Transformants had varying fluorescence intensities, but their pathogenicity did not differ from that of the wild type. Fluorescence microscopy revealed that F. oxysporum primarily entered the aboveground portion of D. officinale through the leaf margin, stomata, or by direct penetration of the leaf surface. It then colonized the mesophyll and spread along its vascular bundles. D. officinale exhibited typical symptoms of decay and wilting at 14 days postinoculation, accompanied by a pronounced fluorescence signal in the affected area. The initial colonization of F. oxysporum in the subterranean region primarily involved attachment to the root hair and epidermis, which progressed to the medullary vascular bundle. At 14 days postinoculation, the root vascular bundles of D. officinale exhibited significant colonization by F. oxysporum. Macroconidia were also observed in black rot D. officinale tissue. In particular, the entire root was surrounded by a significant number of chlamydospore-producing F. oxysporum mycelia at 28 days postinoculation. This approach allowed for the visualization of the complete infection process of F. oxysporum and provided a theoretical foundation for the development of field control strategies.

Natural product osthole can significantly disrupt cell wall integrity and dynamic balance of Fusarium oxysporum.

Dendrobium officinale Kimura et Migo is a traditional Chinese herbal medicinal plant. However, the frequent occurrence of soft rot disease (SRD) is one of the most harmful diseases in D. officinale production in recent years, which can seriously affect its yield and quality. In this study, the major pathogenic fungus (SR-1) was isolated from D. officinale with typical symptoms of SRD, and was identified as Fusarium oxysporum through morphological and molecular identification. The biological activities of five natural products were determined against F. oxysporum using a mycelial growth inhibition assay. The results showed that osthole had the highest antifungal activity against F. oxysporum, with an EC50 value of 6.40 mg/L. Scanning electron microscopy (SEM) showed that osthole caused F. oxysporum mycelia to shrink and deform. Transmission electron microscopy (TEM) showed that the organelles were blurred and the cell wall was thickened in the presence of osthole. The sensitivity of F. oxysporum to calcofluor white (CFW) staining was significantly enhanced by osthole. Relative conductivity measurements and propidium iodide (PI) observation revealed that osthole had no significant effect on the cell membrane. Further experiments showed that the activity of chitinase and ß-1,3-glucanase were decreased, and expression levels of chitinase and ß-1,3-glucanase related genes were significantly down-regulated after treatment with osthole. In conclusion, osthole disrupted the cell wall integrity and dynamic balance of F. oxysporum, thereby inhibiting normal mycelial growth.