Preliminary Study On The Inhibitory Effects Of Antofine On Citrus Green Mold And Its Antifungal Mechanism

Green mold is one of the main diseases,causing huge economic losses to China's citrus industry every year.Due to the excessive use of chemical fungicides,the Penicillium digitatum resistant strains is produced,and the prevention and control effects are weakened.The natural plant-derived substances can overcome the above defects and have the potential to prevent and control the post-harvest diseases of citrus fruit.Antofine is a plant-derived alkaloid,which is widely found in plants of the family of Asclepiadaceae,such as Baiwei,which can inhibits the growth of a variety of pathogenic fungi and bacteria.This study aim to analyze the inhibitory effect of antofine on Penicillium digitatum sensitive strain Pds01 and resistant strain Pdw03,compared of the difference of the antifungal mechanism of antofine against two strains.And provide theoretical basis for postharvest disease control of citrus,the main results are presented as follows:(1)Effect of antofine on citrus green moldAntofine had good inhibitory effects on Penicillium digitatum Pds01 and Pdw03 strains.The minimum inhibitory concentration(MIC)of Penicillium digitatum Pds01 and Pdw03 strains was 1.5625 ?g/mL,The minimum fungicidal concentration(MFC)was 12.5 ?g/mL.In vivo experiments showed that 1 × MFC and 10 × MFC antofine soaking treatment could effectively reduce the incidence of green mold caused by Penicillium digitatum Pds01 and Pdw03.When the control group of Penicillium digitatum Pds01 strain was rotted,the incidence rate of 1 × MFC and 10 × MFC antofine treatment were 91%±4% and 27%±1%,respectively;when the control group of Penicillium digitatum Pdw03 strain was rotted,The incidence rate of 1 × MFC and 10 × MFC antofine treatment group was 93%±0% and 11%±4%.Scanning electron microscopy(SEM)results showed that 1/2 MIC and MIC antofine treatment for 120 min,the surface of the mycelium appeared rough,wrinkled,and even damaged.(2)Study on the antifungal mechanism of antofine against Penicillium digitatum Pds01 and Pdw03 strains.Experiments of CFW cell wall fluorescence staining showed that antofine did not damage the cell wall of Penicillium digitatum Pds01 strain.Results of PI staining showed that antofine treatment disrupted the cell membrane of Penicillium digitatum Pds01 strain.After 30 minutes of treatment with antofine,the content of membrane structural substance lipid and ergosterol decreased,indicating that the cell membrane began to damage at 30 min,but the content of intracellular malondialdehyde(MDA)and oxygen(ROS)did not changed,indicating that the membrane lipid peroxidation did not occurred in Penicillium digitatum Pds01.Mitochondria membrane potential(MMP)decreased,suggesting the mitochondrial began to damage.When the mycelium treated with 1/2 MIC and MIC antofine for 30 min,the intracellular ATP content of Penicillium digitatum Pds01 strain decreased,and the intracellular ATP content of the treatment group was 2.39 ± 0.13 ?g/g,which was significantly lower than that of the control group 5.22 ± 0.45 ?g/g.This indicates that an imbalance in energy metabolism was occured.The rate of decrease of dissolved oxygen decreased after the treatment with antofine,indicating that the respiratory metabolism has been inhibited.The superposition rate experiment indicated that it may has a influence on the tricarboxylic acid cycle.Further study revealed that cell membrane damage occurred after 30 min and mitochondrial membrane damage occurred at 20 min,indicating that cell membrane damage was later than mitochondrial membrane damageExperiments of CFW cell wall fluorescence staining showed that antofine did not damage the cell wall of Penicillium digitatum Pdw03 strain.Results of PI staining showed that antofine treatment disrupted the cell membrane of Penicillium digitatum Pdw03 strain.When the mycelium treated with antofine for 30 min,the lipid content of the membrane structure of mycelium decreased and the ergosterol content remained stable,indicating that antofine had no effect on the ergosterol synthesis pathway.However,the content of intramolecular malondialdehyde(MDA)and reactive oxygen species(ROS)in mycelium increased sharply,indicating that antofine induced membrane lipid peroxidation in Penicillium digitatum Pdw03 strain.The mitochondrial membrane potential(MMP)of Penicillium digitatum Pdw03 strain decreased after 30 min treatment with antofine,indicating that mitochondrial function was impaired.Moreover,the intracellular ATP content and the total energy load decreased,indicating an imbalance of energy metabolism occurred in Penicillium digitatum Pdw03 strain;cell membrane damage occurred at 20 min,while mitochondrial membrane damage occurred at 30 min,the damage of the cell membrane was earlier than that of the mitochondrial membrane.In summary,this study determined that antofine has a good inhibitory effect on sensitive and resistant strains of Penicillium digitatum,and the antifungal mechanism of sensitive and resistant strains is different,The mitochondrial membrane damage occurred before the cell membrane damaged of Penicillium digitatum Pds01 strain,and the Pdw03 strain was reversed.This study can provide a theoretical basis for the prevention and control of citrus green mold.