Isolation And Screening Of Antagonistic Fungal Endophyte From Eucommia

Eucommia ulmoides Oliver is a deciduous tree Eucommia Eucommiaceae, China’s unique economic trees, two national protected plants. It’s secondary metabolite such as chlorogenic acid,Aucubin, geniposide so has antibacterial, anti-tumor and other pharmacological activity. plants Endophyte commonly found in a variety of plants, many of endophytes can produce the same or similar metabolite of host plants, and many are new substances not found. Use of endophytes screening bioactive components or lead compound has become an important way to find natural medicines, and many compounds have significantly inhibited to animal and plant pathogens, Therefore, the field of medicine and pesticides, plant endophytes is a hot research currently.At present, eucommia endophytic fungi research mainly concentrated in shanxi, sichuan, Shanghai and other places, more important is the lack of research on separation of endophytes active ingredients. The same plant grown in different environments, diversity and biological activity of its endophytes are different. Kaifeng in Henan Province, close to the Yellow River, located in central and northern China demarcation point, its unique geographical location and climatic conditions for Screening endophyte provides a unique natural resource. The experimental material collected from Henan University Botanic Garden, hoping to separating to have good bacteriostatic effect of endophytic fungi, in order to lay the foundation of further extract good biological activity and novel chemical structures of endophytic fungi metabolites.This study used tissue separation to separat endophytic fungi from eucommia’s leaf and fruit growing in medicinal botanical garden of henan university, screening by observing the colony’s character, size, color.Nutrient solution with high performance liquid chromatography(HPLC) testing nutrient solution whether contains chlorogenic acid, aucubin, etc.the main active ingredients of eucommia ulmoides. Confrontation training evaluation of each strain’s antibacterial activity. Have antibacterial activity of the strains to confrontation method, then by growth rate method to evaluate the antibacterial activity. For good antibacterial effect endophytic fungi, determination of ITS fragments, identification of the type with blast comparison, the results are as follows:1. Isolation and identification of bacteria: By 1/4 scribing method to isolate and purifie Endophytes. By microscopic detection of Endophytes’ hyphae and spores were detected classification, the results show: A total of separated 91 plants different form strains. 2. The determination of the chemical composition of Endophytes’ secondary metabolites: seven Endophytes can produce Aucubin; two Endophytes can produce chlorogenic acid; no Endophyte can produce flavonoids. 3. Eucommia ulmoides endophytes bacteriostatic experiment: Using confrontation and mycelial growth rate method to measure bacteriostatic activity of eucommia ulmoides’ endophytes. The fungus for test were Rhizoctonia cerealis, Drechslera Sofokiniana, Fusarium graminearum, Bacillus cereus, Staphylococcus aureus, Monilia albican, Salmonella choleraesuis, Escherichia coli. By bacteriostatic experiment results can be seen that endophytes have inhibitory effect on pathogens for test isolated from the plant more than pathogens for test isolated from the animal, and endophytes has better inhibition source of plant than source of animal. 4. Molecular identification: Extract eucommia endophytes’ DNA with a kit, PCR amplification, sequencing by biological company, the sequence of blast in Gene Bank, molecular identification of the endophytes. Results show that: 1、2、7、9、11、16、18、23、26、27、29、30、42、46 strains are Alternaria;41 strain is Phoma;4 strain is Niqrospora;31、49 strains are Cladosporium.