Eucommia Skin Associated With The Active Ingredient In The Study Of Fungi Students

Purpose:By collecting series Eucommia barks from some typical habitats, The changes of the community structure of endophytic fungi in Eucommia barks between different seasons or different habitats would been found out, which are related to the climate factors. The HPLC/Q-TOF-MS fingerprint of Eucommia barks from different habitats was been constructed in order to compare the differences of effective components in different seasons or habitats and analyze the dynamic accumulation trends of active constituents. Then, through analyzing the metabolites in fermentation of some endophytic fungi, we try to search for the same compounds as the Eucommia ulmoides extract. All above draw a clue to get access to the relationship between the endophytic fungi and the content of main compounds in Eucommia barks.Methods:(1) Series of endophytic fungi wre been isolated from the barks of Eucommia ulmoides in different three regions (Lueyang country, Zunyi country, Cili country) using PDA plates. Based on morphological characteristics and analysis of ITS sequences, we identified and do some statistical analysis of the endophytic fungi which had been separated. Choosing typical strains belong to Phomopsis and its perfect stage——Diaporthe to phylogenic analysis and calculate the genetic distances.(2)The same method was been used to separate, identify and count for endophytic fungi from the barks of Eucommia ulmoides in different seasons. A correlation analysis was also made on the community structure of endophytic fungi and climatic factors.(3) The fingerprint was established by HPLC/Q-TOF-MS which had given the differences between these regions.(4) The contents of effective components were calculated based on HPLC/Q-TOF-MS. A linear and regression analysis (the relative frequencies of different genera as dependent variables, the contents of effective components as independent variables was done in the statistic procedure for evaluating the relationship between endophytic fungi and its host plants.(5) The typical strains had been fermented, and the fermented product had been detected by HPLC/Q-TOF-MS. We compared the compounds in the fermented product with the Eucommia ulmoides extract to find the similarities.Results:(1) A total of152strains of endophytic fungi were isolated from the barks of Eucommia ulmoides in different three regions, these strains were identified into8genera. Thereinto Phomopsis, Diaporthe and Alternaria were common genera to Eucommia barks from different sites. But the dominant genus was different. According to the similarity coefficient, the composition of the endophytic fungi was distinctly different between the barks from three sites. The diversity and species richness in Lueyang country and Cili country were found higher than in Zunyi country. The NJ phylogenetic trees and the genetic distances showed that, there was very high genetic diversity in the Phomopsis and Diaporthe.(2) The diversity and species richness show the barks of Eucommia ulmoides in November had rich genra of endophytic fungi, and the barks of Eucommia ulmoides in March or July had simple community structure of endophytic fungi. The main climatic factors which had influence on the community structure of endophytic fungi were the minimum relative humidity, precipitation and mean relative humidity. But the influence was different between the genra.(3) HPLC/Q-TOF-MS Fingerprint of22common peaks was established preliminarily,14main characteristic components had been identified. The cluster analysis show a large gap between the barks of Eucommia ulmoides from Yunnan, Sichuan and others.(4) The main effective components had a great difference between the barks of Eucommia ulmoides from different regions and seasons. The quality of Eucommia barks from Lueyang and Cili in March and May was excellent. The result of linear and regression analysis indicates Phomopsis and Diaporthe had close relationship with the components.(5) By comparing the TIC or UV spectra of the fermented product with the spectra of the Eucommia ulmoides extract,4common peaks had been found, but after analysis of the MS data, the compounds were different from each other. Conclusion:The community structure and diversity of endophytic fungi were significant different in Eucommia barks from the three habitats and different seasons. The main climatic factors which has influence on the community structure of endophytic fungi in Eucommia barks from different seasons are the minimum relative humidity, precipitation and mean relative humidity. The Eucommia barks from different regions has different chemical substances and the contents, which is relative to the endophytic fungi, especially the two genra-Phomopsis and Diaporthe. The same compounds with the Eucommia ulmoides extract were not detected in the fermented product. We have speculated that chemical substances of the Eucommia ulmoides extract are indirectly affected by endophytic fungi. So much more researches should been done to explore the interactions between the endophytic fungi and its host plants.