Isolation And Identification Of Fritillaria Ussuriensis Maxim Endophytes And Screening Of Antagonistic Activity Strains

Endophyte was defined as the microorganism that colonized at asymptomatic plant tissues at some stage of the life cycle of host plant. Since Strobel isolated one strain of endophyte which can compound Paclitaxel from Taxus brevifolia, researcher focus on the research of medicine plant endophyte. Pingbeimu is the drybulbs of Fritillaria ussuriensis, Liliaceae family, which is one of the traditional Chinese medicine. In the theory of TCM, Pingbeimu has the efficacy of cleaning the heat and dispelling phlegm, nourishing the lung and relieving the cough, it was used to treat the cough and phlegm, its activity componets are steroidal alkaloids. Wild Fritillaria ussuriensis widely distribute in China Northeast, and the plant output sharply decreased in recent years for the medicine plant resource was excessively exploited, ecology system was destroyed. Therefore, there is important therical and practical meaning to carry out the rearch on endophyte resource of Fritillaria ussuriensis, and furtherly conduct the effective component production utilizing the endophytes. Five samples of Fritillaria ussuriensis were taken from JiLin province in this study, and isolated endophyte from them, then identified them and analyzed the diversity with biological methods and morphological observation, also screened them for antagonistic activity.This paper research content mainly has the following several aspects:(1) The different combation of HgCl2 and NaClO were used in surface sterilized with different treated time, the optimal sterilizing strategy of Fritillaria ussuriensis tissue was choosed: bulb sueface is 0.2% HgCl2 for 12 min, leaf and stem is 2% NaClO for 5min.(2) We isolated endophytes belonged 17 genuses 32 species from sterilized Fritillaria ussuriensis tissue, and among them, fungi endophyte belong to 9 genuses 17 species, bacteria endophyte belong to 8 genuses 15 species, no actinomycetes. Fusarium and Lysinibacillus are dominant microorganism, account to 21.85% and 15.63% of the total number.(3) Data were analyzed with SPSS Statistics 19 to conduct the cluster analysis with ward method, the diversity of endophyte was obtained. The results show that the endophytes in the bulb are belong to 15 genuses and 25 species, and its richess index, eveness index and dominant index are all the higher than in the stem and leaf, they are 2.2121, 0.8624 and 0.6391, respectly. There are endophytes in the stem are belong to 7 genuses and 12 species, and there are endophytes in the leaf are belong to 10 genuses and 16 species, but all of them are lower than in the bulb.(4) Data were analyzed with SPSS Statistics 19 to conduct the cluster analysis with ward method, the diversity of endophyte, richess index, eveness index and dominant index were obtained. The results show that the endophytes in the JingYuⅡare the highest, the three indexes are 2.2450, 0.8752 and 0.8876, respectly. There are endophytes belong to 5 genuses and 7 species in JingYu I, 7 genuses and 9 species in Tonghua I, 3 genuses and 5 species in Tonghua II, 11 genuses and 17 species in Changchun.(5) It was screened the endophyte with antagonistic activity with flat panel standoff method to Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, and 7 endophyte strains were obtained. Then screened fermentation again with paper method, 5 endophyte fungal strains were obtained, which have better antagonistic activity.