Extraction And Enrichment Of Coumarin From Fig Leaves And Its Mechanism Of Resistance To Cucumber Powdery Mildew

In order to comprehensively utilize fig resources,a novel microwave-assisted EDTA extraction method（MAE-EDTA）was established and optimized by single factor experiments and Box-Behnken design（BBD）response surface methodology.A new process for the enrichment of fig leaves coumarin was developed.Through the control effect of coumarin,spore respiration intensity,cucumber resistance-related enzyme activity and gene expression,the effects of enriched fig leaves coumarin on powdery mildew,the main pathogen of cucumber powdery mildew（Podosphaera xanthii）was investigated and its induction of cucumber powdery mildew resistance.1.A new method for extracting three coumarins from fig leaves was proposed and optimized.The results showed that under the optimal technological conditions of EDTA concentration of 0.07 mol/L,ethanol volume fraction of 56%,microwave time of 16 min,extraction temperature of 70℃and microwave power of 500 W,MAE-EDTA extracted Psoralen,umbelliferone and bergapten were 27.24 mg/g,1.36 mg/g and 4.95 mg/g respectively,and the extraction rate of total coumarin reached 33.55 mg/g,which is 2.03 times as that of Thermal reflow method,which is 1.70 times as that of the microwave-assisted extraction method.2.The D101 resin-supported 1-ethyl-3-methyl acetate adsorbent[C₂mim]OAc-D101 was prepared and screened,characterized and analyzed,and used as a new natural active substance purification material.Coumarin was enriched.The purity of coumarin after the final enrichment was 63.2%,which was 7.8 times higher than that of 8.1%before enrichment,and13.7%higher than that of D101 resin without ionic liquid.The average recovery of coumarin was 81%.The contents of psoralen,umbelliferone and bergapten lactone were 51.31%,2.57%and 9.33%,respectively.3.The control effect of fig leaves coumarin on cucumber powdery mildew was investigated by leaf disc bioassay method,and the preventive protection effect of coumarin at a concentration of 0.04 mg/m L on the front of leaves was the best.The final concentration of 0.1mg/m L coumarin inhibited the respiratory intensity of powdery mildew spores by 6.80%,and the superimposed inhibition rate with sodium phosphate was the smallest.Its effect on cucumber powdery mildew spore respiratory metabolism was mainly on the pentose phosphate pathway effective.Microscopic observation showed that spore germination and mycelial growth slowed down after coumarin protection treatment;conidia on leaves were significantly reduced and dispersed after coumarin treatment of infected leaves.The results of SEM and TEM showed that fig leaves coumarin extract can destroy or even decompose the spores and germ tubes of powdery mildew,inhibit or delay the germination of powdery mildew spores,and inhibit the formation of powdery mildew haustorium to varying degrees.It can significantly inhibit the growth process of cucumber powdery mildew.4.Chlorophyll in cucumber leaves and resistance-related enzymes peroxidase（POD）,polyphenol oxidase（PPO）,phenylalanine ammonia lyase（PAL）,chitinase,the activity of 3-glucanase（β-1,3-glucanase）,fig leaves coumarin can inhibit the activity of POD and PPO in cucumber leaves,prevent the loss of chlorophyll,enhance the activity of PAL,chitinase andβ-1,3-glucanase.Then three typical enzyme genes PAL,POD and Chitinase were selected,and the relative expression levels were analyzed by real-time fluorescence quantitative PCR technology.The results showed that coumarin at a concentration of 0.04 mg/m L could affect the expression of related enzyme genes.The peak was reached 3 to 4 days after drugging.The study results play a certain role in better utilization of fig leaves resources,and provide data support for the development of new botanical pesticides to control crop diseases.