Research On Isolation,Identification,and Biological Characteristics Of Endophytes From Changium Smyrnioides Wolff

Changium smyrnioides Wolff is rare species of a single genus of Umbelliferae in China,and it belongs to the national tertiary key protected plant.The purpose of this paper is to explore the microbial population gene bank of rare Chinese herbal medicine,Changium smyrnioides Wolff,to explore the biological function of the microbial population of Changium smyrnioides Wolff,to screen out endophytes that can antagonize pathogenic strains,and degradation strains which can effectively degrade the widely used ulfonylurea herbicides—Tribenuron-methyl.Furthermore to research the endophytes of Changium smyrnioides Wolff have biotransformation effects on its three furocoumarin components.Research results indicate:(1)In this experiment by three-step disinfection method and plate culture method,29 endophytic bacteria and 18 endophytic fungi were isolated from the roots,stems and leaves of fresh Changium smyrnioides Wolff plants.There were some differences in the number of endophytes of Changium smyrnioides Wolff in different tissues.The number of endophytic strains was the highest in the roots and the least in the stems.Through culture characteristics,morphological characteristics,biochemical characteristics,16 S r DNA gene sequence analysis,genetic map analysis and automatic microbial mass spectrometry detection,most of the endophytic bacteria were found to be Bacillus.(2)Using mixed strains method and after the experimental preliminary select,5strains of endophytic bacteria of Changium smyrnioides Wolff showed better bacteriostasis on Staphylococcus aureus.By rescreening,among them the diameter of inhibition zone of strain NMG5 against Staphylococcus aureus reached 28.5 mm.The diameter of inhibition zone of strain NMJ10 was 20.30 mm.It shows that they had a relatively strong bacteriostatic effect and strain NMJ10 had broad spectrum bacteriostatic effect.According to the biological identification,strain NMJ10 was Bacillus strains(Bacillus sp.NMJ10),and strain NMG5 was Pseudomonasaeruginosa(Pseudomonas aeruginosa NMG5).(3)In order to solve the residual problem of Tribenuron-methyl,the strains capable of effectively degrade Tribenuron-methyl was screened out in this experiment and we looked forward to manufacturing a compound bacterial agent higher degradation rate.Using enrichment acclimation and culture separation method,we had screened out multiple strains capable of degrading Tribenuron-methyl.By rescreening,the strains PMG3 and NMY4 were obtained that they can efficiently degrade ulfonylurea herbicides Tribenuron-methyl.After strain degradation,the degradation efficiency was highest on the first day,and the degradation rate reached89% on the third day.After three days of degradation,the degradation rate of the degradation of Tribenuron-methyl by the strain PMG3 can reach 89.01%-90.39%,and the degradation rate of strain NMY4 was 88.97%-89.13%.According to the biological identification,strain PMG3 was Bacillus strains(Bacillus sp.PMG3).(4)In order to explore the biotransformation of three furocoumarin components by endophytes of Changium smyrnioides Wolff,the content of three furanocoumarin components of imperatorin,isoimperatorin and psoralen was determined by HPLC method,after fermentation of Changium smyrnioides Wolff raw powder culture medium of endophytes from Changium smyrnioides Wolff.As the strain transformation,after Changium smyrnioides Wolff original liquid was fermented by the endophytes of Changium smyrnioides Wolff,the content of furanocoumarin components of imperatorin,isoimperatorin and psoralen was increased,and the metabolites were mainly concentrated in the supernatant of the fermentation liquid.During the fermentation process of Changium smyrnioides Wolff culture solution,after fermentation by strain NMG10,the growth ratio of imperatorin,isoimperatorin and psoralen was 97.72%,2.38% and 4.62%,respectively.Relatively the total amount of furanocoumarin was increased by 39.60%.Through a series of experiments,it can be seen that the transformation of three furocoumarins by strain NMG10 was the best.During the whole fermentation process,the strains had a significant effect on the content of imperatorin,which proved be more conducive to the transformationsynthesis of imperatorin component.According to the biological identification,strain NMG10 was Bacillus pseudomycoides.