Study On Extraction Optimization And Mechanism Of Lysurus Mokusin Metabolites

In the area of the prevention and control of plant diseases and insect pests,people still focus on the use of chemical pesticides.With the increase in the time of single pesticide varieties,forest pests and pathogens have produced certain drug resistance,which has led to an increase in the use of pesticides and fungicides in recent years.Extensive use of chemical pesticides has caused very serious environmental pollution and economic losses to human society.Under the advocacy of the state,the proportion of bio-pesticide use has increased year by year,and it is particularly urgent to seek green bio-pesticides that are high-efficiency,safe and pollution-free.The fungus contains a large amount of insect resistance,antibacterial and antiviral active substances.In the previous experiments,this paper proves that the Lysurus mokusin has antibacterial activity,which shows that L.mokusin has potential for application and development in the field of biological control,and has important development prospects as a biological source pesticide resource.In this study,L mokusin were collected in the field,and the insecticidal and bacteriostatic active substances were extracted by different extraction methods,and the extraction conditions were optimized.The crude extracts of L.mokusin were separated and purified,and we made bioassays of the biological pests of forest pests and pathogens.The effects of crude extraction on the enzyme activities of pests and pathogens and their mechanisms were studied.The following research results were obtained:(1)The active substances from the fruiting bodies of L.mokusin was extracted with ethanol,and the five factors affecting the ethanol extract were screened using Plackett-Burman and Box-Behnken experimental design methods.Then the key factors were optimized.The optimal conditions for the determination of ethanol extracts were:ethanol concentration 60%,ratio of material to liquid 1:80(g/mL)and ultrasonic time 80 min.Under these conditions,the extraction rate is 31.21%,which is similar to the theoretical value of extraction rate of 31.36%.(2)Scanning electron microscopy was used to observe the different effects on the surface of L.mokusin extract powder,in ultrasonic assisted extraction and extraction.The results showed that the ultrasonic extraction-assisted extraction rate of L.mokusin was 31.23%,and the method using warm water extraction was 24.10%.And the surface tissue extracted by warm water extraction was wavy and smooth,while that of the ultrasonically processed sample was loose and rough.(3)Using GC-MC to analyze the chemical constituents in the ethanol extract of L.mokusin,it was found that Pentanoic acid,Hexadecanoic acid and ethyl ester and other seven compounds were present in the sample.(4)The position of the antifungal active substance of the L.mokusin was measured,and then the extraction conditions were optimized.The results showed that the active substance was present in the fermentation broth.The best extraction solvent for the active substance in the fermentation broth was n-butanol,the optimum ratio of material to liquid was 1:3,and the optimal extraction times were 3 times.(5)Separation and purification techniques such as silica gel column chromatography were used,and thin layer chromatography was used for tracking and detection.The n-butanol extract of L mokusin was extracted and purified,then a fungicidal extract L134 was obtained.(6)The antifungal mechanism of L.mokusin L134 against Pestalotiopsis sp.was preliminary clarified by studying how L134 affected spore germination,mycelial growth,membrane permeability,cell wall integrity and key enzymes.The results showed that L134 well-inhibited spores germination and mycelial growth with an EC50 of 0.305 mg/mL.And it affected the activity of mycelium SOD,POD,PPO,CAT and CL,resulting in decreased pathogenicity,obstacles in oxygen free radical scavenging system,increased MDA content and membrane peroxidation.Also,L134 had an adverse effect on the external morphology of cell wall and mycelial growth.(7)The results of the stability test of L134 showed that L134 was stable under different pH treatments and was relatively stable below 65℃.When treated with different metal ions,there was no significant difference.In the test of the oxidant and reductant treatment,the oxidant affected the stability of the antifungal active substance,which was stable under ultraviolet irradiation.(8)Finally,in order to verify the test results,the biological activities of different extracts on gypsy moth and various pathogens were tested.The results showed that the ethanol extract had a good insecticidal activity against the gypsy moth larvae,whose mortality rate reached 59.77%.While n-butanol extract all had inhibitory activities to Sphaeropsis visci,Pestalotiopsis sp.,Cytospora chrysosperma,Fusarium oxysporum,Botrytis cinerea,etc.