Study On The Fungicidal Activity Of Mikania Micrantha H. B.K

Research and development of natural products is one of primary and effective ways to develop novel pesticides. As dangerous invasive species from abroad, control and utilization of plants killer, Mikania micrantha H.B.K is urgently needed. Previous study has shown that the extract of Mikania micrantha H.B.K have a strong inhibition activity against many plant pathogens. In order to utilize Mikania micrantha H.B.K resource efficiently, make the best use of the situation while avoiding disadvantages, and provide theoretical basis for novel plant-derived fungicide developing, the chemical constituents and fungicidal activity of Mikania micrantha H.B.K were investigated on the basis of some previous study. The main result obtained from this study as follows:1.17 compounds were isolated from Mikania micrantha H.B.K based on bioactivity-guided isolation, and the chemical structures of 15 compounds were identified. Those chemicals include Deoxymikanolide, Dihydromikanolide, Mikanolide, Isabelin, Scandenolide, Mikanokryptin,1β,6α-dihydroxy eudesmane-4(15)-alkene, eudesmane-4 (15), 7 (11)-12,8β-lactone, Mikanin, Stigmasterol,β-Sitosterol, eudesmane-4(15),7(11), 7(11)-triene-12,8β-lactone, luteolin,β-Daucosterol, Eupatolitin. The result shows that 15 identified compounds can be divided into 3 categories:sesquiterpene lactones; flavonoid compounds and steroidal saponins.2. The fungicidal activities of Stigmasterol, Deoxymikanolide, Dihydromikanolide, and Isabelin were evaluated against plant pathothic fungi. The results showed that Stigmasterol can inhibit the mycelium growth of 12 pathogens, which EC50 value is within 365.48-1438.59 mg/L. Stigmasterol showed highest antifungal activities against Botrytis cinerea Pers with an EC50 value of 365.48 mg/L. The results also revealed that there are significant differences between the inhibition effects of Stigmasterol on different pathogens spore germination, Stigmasterol showed highest antifungal activities against Fulvia fulva and Botrytis cirerea Pers. et Tris with an EC50 value of 151.47 and 174.65mg/L, respectively. Furthermore, Stigmasterol displayed certain control effect (68.60% at 1500 mg/L) against B. cirerea in vivo. And Stigmasterol also displayed prominent systemic protective effect (66.80% at 1500 mg/L and 43.10% at 1000 mg/L) and control effect (50.60% at 1500 mg/L and 25.70% at 1000 mg/L) against wheat powdery mildew Blumeria graminis in vivo. Deoxymikanolide displayed a higher inhibition on mycelium growth of 10 pathogens, witch EC50 value was within 5.61-46.48 mg/L, Deoxymikanolide showed highest antifungal activities against Rhizoctonia cerealis Vander Hoeven with an EC50 value of 5.61 mg/L. The bioassay showed that there is a significant difference between the inhibition effect of Deoxymikanolide on different pathogens spore germination, Deoxymikanolide showed highest antifungal activities against Botrytis cirerea Pers. et Tris with an EC50 value of 10.49mg/L. The fungicidal activity of Dihydromikanolide was lower than Deoxymikanolide, Dihydromikanolide only showed a similar antifungal activities with Deoxymikanolide against Phytophthora capsici with an EC50 value of 12.84mg/L. There is a little difference between the inhibition effect of Dihydromikanolide on different pathogens spore germination, the EC50 value of Dihydromikanolide effects on spore germination of 5 pathogens was 20～75 mg/L. The EC50 value of Isabenlin against B. cirerea, R. cerealis and Phytophthora capsici was 221.25,432.21 and 714.69 mg/L, respectively. Based on the bioassay results combined with isolation and identification results of compounds, sesquiterpene lactones and steroidal saponins were considered to be primary fungicidical components in M. micrantha, and steroidal saponins has a certain prospect of developing and utilization.3. In the basis of previous extraction technology of essential oils from M. micrantha with supercritical CO2, the chemical constituents of M. micrantha extracts acquired with 3 methods: hydrostillation(HD), SCE, and extracted with petrol ether(PEE) were analyzed by GC/MS, and the toxicity of those 3 extracts against Fusarium graminearum and Rhizoctonia cerealis was tested. The result showed that the optimized extraction condition was 35 MPa and 55℃with 40 mL/g supercritical carbon dioxide after 20-mintue static extraction, and the extraction rate of essential oil by SCE was higher than other 2 methods. The main components of essential oil from M. micrantha are polyolefins, terpenoids, alcohols, fatty acids, esters and steroidal compounds, and there are significant differences of chemical constituents between essential oil extracted with different methods. The essential oil of M. micrantha showed selective antifungal activities against different plant pathogens, the bioassay showed that essential oil has the highest antifungal activities against Fusarium graminearum and Rhizoctonia cerealis and Botrytis cinerea Pers and weaker inhibitory effect against Fusarium oxysporum f.vasinfectum (Atk.) Snyder et Hansen. There are significant differences of antifungal activities between essential oil acquired by different methods. The toxicity of M. micrantha essential oil extracted by SCE was higher than the essential oil extracted by HD and PEE against Fusarium graminearum and Rhizoctonia cerealis.4. Effect of Stigmasterol against Blumeria graminis were observed by electron microscopy technology. The results showed that stigmasterol can lead to abnormal hyphal growth of B. graminis such as twisted and malformed mycelia, surface roughening, growing point collapse and faster aging. Stigmasterol can also reduce conidia quantity and lead to large area of surface pit in spore. Furthermore, stigmasterol treated fungi cause intracellular protoplasm electronic density to increase, organelles degenerated, mycelia putrescence, and cause mycelia and haustorial body heavy vacuolization and higher density substances sedimentary in mycelia cell. It could also lead to electronic density substances uneven in mycelia cell, enlarge vesicle and reduce quantity of vesicle. The outer plasma membrane haustorium of B. graminiwere was found to be irregulary thicken by stigmasterol. Those results showed that stigmasterol mainly effect biosynthetic process of plant pathogen, meanwhile, it can also effect biological oxidation of plant pathogen to a certain extent. Further research was needed to confirm those hypotheses.5. The powder of M. micrantha has been formulated to 70% wettable powder through the surface active agent and carrier screening. The field trial showed that 70% M. micrantha WP can provide 70% control effect against B. cirerea at 7500 mg/L.The dissertation preliminarily definited the chemical character of antifungal compounds from M.micrantha, and several novel compounds with fungicidal activity were found. The chemical constituent's character and antifungal activity of essential oil from M. micrantha have also been studied. The fungicidal spectrum and activities of Stigmasterol and Deoxymikanolide were investigated, and the application prospect as a novel control agent was evaluated. The formulation of wettable powder of M. micrantha was formulated and developed successfully. Those research results provide theoretical basis for utilization Mikania micrantha H.B.K resource efficiently and novel plant-derived fungicide developing.