Effects of difenoconazole on the mycelial morphology and ultrastructureof Valsa mali

Abstract:【Objective】Apple tree Valsa canker is primarily caused by the ascomycetous Valsa species.Valsa mali is a worldwide and destructive disease in apple production areas, which can cause the bark rotof apple trees and then lead to serious economic losses. At present, the prevention to this disease mainlydepends on the chemical agents, difenoconazole is a type of triazole fungicide, which could effectively prevent and cure Valsa canker, by inhibiting spore germination and mycelial growth. In this study, our aim isto determine the operation of difenoconazole on Valsa mali, and then to provide an important theoreticalbasis for the prevention and control of Valsa canker.【Methods】A 5 mm diameter disc from the V. mail culture was inoculated in the center of a PDA plate, and cultured at 25 ℃. One day after being cultured, thepetri dishes were used for fungicide treament. Concentration of the fungicide was diluted from a stock solution 10 g·L^-1 to 40 mg·L^-1 with germfree double-distilled water. Sepsis filter paper strips (0.5 cm×5 cm)were immersed in the fungicide and then placed in a square (5 cm×5 cm) around the fungal colony. Forcontrol, filter paper strips were immersed in 0.4% acetone. After treatment, the petri dishes were continu⁃ally cultured at 25 ℃ 12, 24, 48 h after fungicide treatment, and then small pieces of PDA from the colonyedge were taken for mycelial morphology and ultrastructure studies.【Results】Observations from scanningwith an electron microscope discovered that the growth of apical hyphae was inhibited and the top expand⁃ed into a sphere with a rough surface at 12 h after fungicide treatment compared with the control. Then 24h after fungicide treatment, fungal colonies grew slowly and appeared to show a serious abnormality: hy⁃phae were irregularly swelled and excessively branched, the hyphal cell walls gradually ruptured, andeven showed the phenomenon of protoplasm extravasation. After 48 h with fungicide treament, coloniesstopped their growth, and the hyphae collapsed, and the majority of the hyphal cell walls seriously rup⁃tured. The part of the hyphae was swollen, and the hyphae were badly shriveled and even necrotic. Obser⁃vations using a transmission electron microscope discovered that cell structure changed significantly at 12 h after treatment. The important change was the hyphal cell walls, which were irregularly thickened accompanied by an internal extending cytoplasm. On the other hand, the number of the mitochondria in thecytoplasm increased and its morphology was abnormally thickened. Moreover, it was also found that thenucleus of the cells changed to a certain extent: the number of nuclei increased and the nucleolus gradual⁃ly spreaded and the area of nucleolar also decreased at the same time. When the hyphae were treated for 48 h with fungicide, the structure of the hyphea cell became worse: the whole cell walls became thickenedand some of their exosporium disintegrated, and the membrane of the mitochondria showed irregularlythickened and malformed, and there was extracellular deep staining which maybe caused by cytoplasmleakage material. At this moment, the cell had some septum and part of the septum became irregularlythickened and malformed, and they could not even develop into a complete diaphragm. In addition, thevacuolization of the mycelial cell was very serious, some intracellular organelles disorganized even theydisappeared completely, and the cytoplasms became necrosis. It is worthy of being mentioned that therewas a new discovery: a daughter hypha could form in the original degenerated hypha. The cell walls became irregularly thickened, and the cytoplasms of the daughter hyphae with disorganized organelles werenecrosis. However, the original degenerated hypha cell walls disappeared.【Conclusion】As a type of fungi⁃ide difenoconazole can affect the structure of the cell wall and the normal growth of the whole cell by affecting the morphology of the cell wall of Valsa mali, resulting in the hyphal malformation and cell necrosis. Specifically: the hyphae irregularly swelled and excessively branched and contorted, protoplasm extravasation occurred, cell walls irregularly thickened and disintegrated until completely obiterated, themembranes of the mitochondria irregularly thickened and malformed, the number of nuclei increased andthe nucleolus gradually spread, the area of nucleolar decreased, a daughter hypha formed within the origi⁃nal degenerated hypha and the cell walls disappeared.