Isolation And Identification Of Endophytes In Aconitum And Its Secondary Metabolites

Aconitum carmichaeli is Ranunculaceae,a perennial herb that can use as a medicine and has insecticidal and bactericidal activity.Aconitine is the main active ingredient and often obtain by plant extraction,but its source is limited and its yield is limited.Studies have shown that endophyte present in healthy plant tissues will produce the same or similar chemical composition as the host plants.In order to explore the microbial source of aconitine,this research take endophyte that isolate and purify from Aconitum plants,and conduct more systematic studies on it.The main contents of the research are as follows:Tissue separation method use to isolate endophyte from healthy roots,stems,leaves,seeds,and seeds of Aconitum carmichaelii and Aconitum kusnezoffii respectively.12 strains of endophytic fungi and 11 strains of endophytic bacteria with different morphological characteristics are isolated and purified by experiments and named according to their source.By HPLC analysis,it determine that the peak time of aconitine is 20.022 min.SNCWT-003 strain,SNWT-010 strain,SNWT-011 strain,SNWT-013 strain and SNWT-014 strain have absorption peaks around 20.022 min,indicating the above strains,aconitine may produce secondary metabolites.Finally,it is determined that the aconitine produce by the SNCWT-003 strain is 5.64×10-3 mg·m L-1,and the aconitine produce by the SNWT-014 strain is 4.4×10-4 mg·m L-1 in the fermentation broth by HPLC-MS.The genus of aconitine-producing endophytic fungi determine by morphological observation and Internal Transcribed Spacer(ITS)sequence analysis.SNGWT-002 strain is identified as Aspergillus flavus,SNCWT-003 strain is Aspergillus parasiticus,and SNWT-014 strain is Penicillium rubrum.In order to expand the practical use of aconite endophytes,the antifungal activity of aconite endophytes on various plant pathogenic fungi explore.The study find that the inhibition rate of endophytic fungus SNCWT-003 on Helminthosporium turcicum,Thanatephorus cucumeris and Sclerotinia sclerotiorum reaching 95.88%.SNWT-013 strain has the best antifungal activity effect on Cordana musae,with an antifungal activity rate of94.12%.The test proves that the fermentation products of SNGWT-002 strain,SNCWT-003 strain,and SNWT-014 strain isolate from Aconitum carmichaeli contain the same secondary metabolite-aconitine as the host plant,which can use as aconitine microbial sources.These researches provide new ideas for the acquisition of aconitine and the possibility of furtherlyexpand the applications of aconitine.