A Study On Antifungal Activity Of Osthol

Osthol[7-methoxy-8-3-methyl-(2-buetenyl)]coumarin],extracted from a Chinese herb Cnidium monnieri(L.) Cuss,is reported having anti-arrhythmia effects,immuno enhancement effects,anti-inflammatory effects,anti-virus effects and antagonizing effects against hepatic damage in pharmacology.Its antifungal activity was proven on Alternaria alternate,A.ergillus sp.,Cryptococuus neoformans,etc.In this study, osthol displayed a wide antifungal activity and its potential as biopesticides.Therefore, Sphaerotheca fuliginea and Fusarium graminearum were used in this dissertation to investigate the bioactivity of osthol as a fungicide.And the antifungal mechanism of action of osthol was further studied.In addition,osthol derivatives were synthesized and studied by bioassays.Results were showed as the following:1.Antifungal Spectrum and Pesticide ActivityIn vitro studies,osthol showed a wide antifungal activity,with the EC₅₀ value ranging from 21μg·mL^-1 to 60μg·mL^-1.EC₅₀ values of osthol against S.fuliginea were respectively 42.28 and 30.19μg·mL^-1 using seedlings bioassays and detached leaves bioassays.100μg·mL^-1osthol(without additives) was proven having good performance preventing and controlling pumpkin powdery mildew with the EC₅₀ values 77.41%and 68.72%.1%osthol emulsion in water developed in writer's lab showed higher protection efficiency(83.45-94.59%) against cucumber powdery mildew than 15%triadimefon wettable powder when used in the dosage of 33.75g.ai/hm²,which was similar to the protection efficiency of 10%difenoconazole water dispersible granule.2.Antifungal Mechanism of Action2.1 Effects on pathogensMorphology observation revealed that osthol could affect all life stages of S. fuliginea.In water agar plates and detached leaves,germination rates of S.fuliginea could be decreased 90%after treated with 100μg·mL^-1 osthol for 48h.And the same osthol treatment also led to observably decreasing of conidia amounts.By local cells DAB(3,4-diaminobenzidine) staining of host-pathogen interaction system,the penetration rate of germinating spores can be analyzed and the results indicated that osthol could prevent 75%spores invadation avoiding the further adhesive growth of S. fuliginea on host cuticles.By microscope observation,we found osthol could also inhibit the growth of S.fuliginea hyphae.At 48h after inoculation,the average colony length of S.fuliginea was about 400μm in the control,while it was only 122μm in 100μg·mL^-1 osthol treatment.Moreover,osthol impaired the adhesion of S.fuliginea to pumpkin cuticles by inhibiting the secretion of non-specific esterases from S. fuliginea cells,which hampered the pathogen's taking in.Scanning and transmission electron microscope photos showed that the morphology of osthol-treated S.fuliginea cells became shrinking and wizened with melting cell walls and lysine cell organelles accompanying by the releasing of homogeneous cell contents.By FACS fluorescence microscope observation,we discovered that short-term treatment with osthol could not change the cytoplasmic membrane penetrability of S.fuliginea cells,which showed that cell membrane could not be the direct action target of osthol.Compared with S.fuliginea,F.graminearum,an ascomycotina fungus,had similar sensitivity to osthol and showed low germination rate after treated with osthol. With its vitro culture characteristic,F.graminearum was then used to study osthol's biochemical mechanism.Results showed that the hyphae of F.graminearum broke into fragments in aliquous culture after treated with 100μg·mL^-1osthol for 24 hour.In the study of cell wall synthesis-and lysis-related substances and enzymes,N-GlcNAc contents and chitinase activity in osthol-treated F.graminearum hyphae increased whereasβ-1.6-glucanase activity remained unchanged.¹⁴C glucose was added into the culture to detect the hyphal sugar absorption changes.When F.graminearum was treated with 100μg·mL^-1 osthol for 3h,glucose content decreased to the lowest level, only 51%of the control,while at the same time chitinase activity was 3 times more than the control,which suggested glucose starvation might induce the expression of chitinase.And it was proposed that osthol treatment increased chitinase activity, which led to the degradation of chitins required for biosynthesis of fungus cell wall and led to obstacle of hyphal cell wall synthesis.When F.graminearum was treated with 100μg·mL^-1osthol for 12h,calcium in F.graminearum decreased by 40%,which suggested that osthol inhibited uptake of calcium required for Ca²⁺ dependent vacuole transportation and growing hyphae tip.2.2 Effects on pumpkin seedlingsBesides its direct antifungal activity to S.fuliginea,osthol could induce the activity increasing of chitinase peroxidase and phenylalanineammonialyase in pumpkin leaves,which conduced to the defence reactions of host plants.Results of reverse transcription-PCR indicated that osthol could increase the expression of pumpkin chitinase gene in transcript level.In conclusion,osthol had a multiple mode of action against pumpkin powdery mildew fungi not only for its direct antifungal activity to pathogen and its penetration but also for its resistance induction to pumpkin seedlings.3.Bioactivity of Synthesised DerivativesFive osthol derivatives with pyran ring(JS-A,B,C,D,E) were synthesised in lab and tested to show antifungal activity against seven phytopathgens on vegetables, fruite trees and crops.Among these phytopathgens,Phytophthora capsici showed the most sensitivity to compound JS-B with its EC₅₀ value 41.78μg·mL^-1,which was similar to that of osthol.JS-B could inhibit hyphal growth,restrain the formation of sporangium and decrease the release of zoosporangiospores of P.capsici.Microscope observation showed that JS-B could change morphology of P.capsici hyphae,while the osthol-treated P.capsici hyphae had no changes.