Isolation And Screening Of Active Endophytic Bacteria From Eucommia

Endophyte are microorganisms that live asymptomatically inside plant tissues for all or part of their life cycle.Some of them can produce plant-derived and novel bioactive compounds,any of which could be developed into novel antimicrobial,antioxidant and anti-tumor agents like the host.Screening the endophytic bacteria which has all kinds of activities to take the place of the plant is the hotspot in current research.Eucommia ulmoides is a traditional medicinal plant of China,which is a living-fossil plant,and the only species both in its genus and its family.It has all sorts of function like antimicrobial,antioxidant anti-tumor and ect.This natural medicinal medicinal resource is now short supply because of the over-collection of the wild plant,which is now protected in China.So it is very important to find an alternative way to satisfy the pharmaceutical demand.There are a number of studies about E.ulmoides endophytic fungi and we have found the antimicrobial activity of E.ulmoides endophytic fungi.Meanwhile,There are few researches on endophytic bacteria.So,now we will screen endophytic bacteria which have antimicrobial and antioxidant activity.In this paper,37 different endophytic bacteria were successfully isolated from the bark of healthy and non-pest insects by 1/4 scribing method.Among them,7 could not be passaged and 30 could pass on.Subsequently,the DNA of each strain was extracted by colony cleavage method.The bacterial 16S rDNA universal primers were used as PCR primers,and a large amount of bacterial DNA was obtained by PCR reaction.Then,the DNA was sequenced and compared with the sequence published by NCBI,Nine Endophytic bacteria can not detect their DNA sequence,while the remaining 21 strains belong to Bacillus sp.,Staphylococcus sp.,Alcaligenes sp.,Acinetobacter sp..The isolation rate was 55%of Bacillus sp.,followed by Acinetobacter sp.and Staphylococcus sp..The isolation rate was 20%,respectively.Finally,the isolation rate isolated from Alcaligenes sp.was 10%.The antimicrobial activity of endophytic bacteria was studied by mycelial growth rate method.The culture for test were Fusarium graminearum Schw,Bacillus cereus Frankland,Xanthomonas campestris pv.citri,Bacillus subtili,Staphylococcus aureus,Monilia albican.Eight of 21 endophytic bacteria strains had bacteriostasis activities.There were 3 strains with 4 kinds of pathogenic bacteria which have bacteriostatic effects.The three strains were EU1,EU3 and EU12.We compared EU1 and EU3 by fermentation with n-butyl alcohol and ethyl acetate extraction.The fermented detectd the bacteriostatic activity,still found metabolites of plant source of endophytic bacteria has bacteriostatic action.We detected antimicrobial activity by Kirby-Bauer way of fermentation products.Fermentation prducts had better effect on phytopathogen and the general trends was that ethyl acetate extract have better antimicrobial activity.EU1 is Bacillus amyloiquefaciens and EU3 is Acinetobacter sp.There were 11 bacteria with activity of T-AOC.Then the activities of the ability of removing O^2-,DPPH and·OH had been tested.We found that EU3（Acinetobacter sp）,EU16（Bacillus sp.）and EU20（Bacillus pumilus）had well activities.Further antioxidant experiment showed that the fermented products of EU3 had obvious effects on the three free radicals of O^2-,DPPH and·OH,EC₅₀ of EU3 are 4.25 mg/mL,4.52 mg/mL,5.37 mg/mL.EC₅₀ of EU16 are 7.04 mg/mL,8.42 mg/mL,8.94 mg/mL.EC₅₀ of EU20 are7.06 mg/mL,7.10 mg/mL,10.48 mg/mL.EU3 is Acinetobacter sp.,EU16 is Bacillus sp.and EU20 is Bacillus pumilus.The results showed that the two endophytic bacteria EU1 and EU3 isolated from E.ulmoides had good biological activity,and the endophytic bacteria of EUI had good antibacterial activity,while EU3 had good antioxidat activity.The discovery of the above two strains not only for the preparation of related drugs to provide a new way of thinking,but also for the follow-up study laid a solid foundation.