Genome Sequencing Of Aureobasidium Pullulans PA-2 And Isolation And Identification Of Herbicidal Active Substances

A fungal strain of PA-2 was isolated from naturally diseased poplar leaves in this laboratory and identified as Aureobacidium pullulans by 18 S r DNA sequence analysis.Previous studies have shown that the fermentation solution of this strain has significant weed control activity and is safe for main plant crops in Qinghai Province,with the potential to be developed into a microbial source herbicide.Therefore,the laboratory carried out whole genome analysis and secondary metabolite prediction of Mycobacterium avium PA-2 using modern isolation and purification.Methods to explore active substances in secondary metabolites.The study resulted in the following conclusions.1.The whole genome of strain PA-2 was sequenced,and the low-quality data were filtered,and the filtered high-quality data were used for subsequent functional annotation and product prediction.The results showed that the total number of Reads for strain PA-2 was 25,615,586,the total number of bases was 3,673,018,658,and the accuracy of base recognition was above 99.9% for 95.28% of the total bases and 49.83% for GC.Functional annotation of GO,KOG,and KEGG identified 10839 protein coding sequences whose gene products are focused on biological processes such as metabolite transport and translocation.The gene prediction identified 229 non-coding RNAs,the largest number of which were t RNAs,most actively involved in intracellular regulation in the bacterium.In the product prediction,the synthesis of secondary metabolites of strain PA-2 was associated with the type-polyketone synthase(T1PKS)-like pathway,and among the four T1 PKS gene clusters,the possible secondary metabolites were melanin and 1,3,6,8-tetrahydroxynaphthalene,asfuranone,azadone A,falciparum,streptozotocin,pyranoflavin E,falciparic acid,and pentanolactone.2.The strain PA-2 was incubated in liquid medium for shaker fermentation,and the fermentation solution was extracted by volume with different polar organic solvents such as petroleum ether,chloroform,ethyl acetate,n-butanol,etc.,and the optimal extraction solvent was determined by the seed germination method.The results showed that at a concentration of 100 ?g/m L,the coarse extract of the chloroform phase had the best effect on wild oat seeds with 69.07% germ inhibition and 76.58% root inhibition,followed by the n-butanol phase.Considering economic and policy factors,n-butanol was chosen as the next extraction agent.3.A product fraction(Compound 1)was obtained by silica gel column chromatography,thin layer chromatography and high performance liquid chromatography,and the structural formula of Compound 1 was inferred by NMR and mass spectrometry(MS).The results showed that the TLC plate exhibited herbicidal activity with an Rf value of 0.68 and maximum absorption peak at ?=220 nm under dichloromethane:methanol(15:1),and MS and NMR identified compound 1 as oloquine with a molecular weight of 250.048.The IC50 of compound 1 for wild oat germ/root inhibition activity was 35.2/14.4 ?g/m L.In this experiment,the whole genome of Mycobacterium botrytis strain PA-2 was sequenced,and the herbicidal active substances produced by the strain were isolated and identified.The development into microbial pesticides or lead compounds is of great importance.