Jun Jujube, The Inhibitory Action Of Endophytic Bacteria Isolation, Identification And Screening

ObjectiveIn this study, Jiaocheng Jun jujube was selected as the material to isolate endophytic bacteria. The community diversity of endophytic bacteria was investigated by using culture-independent and culture-dependent approaches. Then find a more suitable medium for isolating endophytic bacteria and filtering out endophytic bacteria those could be able to antagonism pathogenic bacteria of plant and human body.Methods1.7common mediums were used to isolate endophytic bacteria. Such as Tryptic Soy Agar Medium(TSA), Ringer’s Medium(R2A), King’s Medium B(KMB), Broth Peptone Agar Medium(BPA), Luria-Bertani Medium(LB), Yeast extract Peptone Medium(YPM), Free-nitrogen Medium.2. Endophytic bacteria were isolated by using culture-dependent method. And morphological performance, physiological and chemical characteristics of antibiotic bacteria were observed. Including catalase test, oxidase test, starch hydrolysis, nitrate reduction, indole test, V-P test, salt tolerance, maximum tolerable temperature. Combine the result of16S rDNA sequencing and physiological and biochemical characteristics make preliminary identification.3. Screening of endophytic antibiotic bacteria: determined antibiotic activity of endophytic bacteria that are able to antagonism pathogenic bacteria of plant and human body.4. The community diversity of endophytic bacteria was investigated by using culture-independent approach:①surface sterilization;②genomic DNA extraction of jujube;③PCR amplification and purification;④clone library construction;⑤enzyme segmentation type;⑥sequencing of represent strip;⑦diversity analysis.Results1. The result of medium choice:The tissue of jujube diluted to104-106times is more suitable for the isolation of endophytic bacteria. This experiment used7kinds of isolated medium, KMB medium and Free-nitrogen medium have the best effect.2.16strains of endophytic bacteria and2strains of endophytic fungi were obtained, including Bacillus sp., Lysinibacillus sp., Staphylococcus sp., Paenibacillus polymyxa, Rhodococcus sp., Kocuria sp., Arthrobacter sp., Micrococcus sp., Acinetobacter sp..3. Filter out endophytic bacteria that are able to antagonism pathogenic bacteria of plant and human body, including Acinetobacter sp., Arthrobacter sp., Lysinibacillus sp., Paenibacillus polymyxa. 4. To construct a clone library,15OTUs, including151positive clones were assigned to amplify16S rDNA segments. The results showed that E.coli was the most dominant group (3OTUs,27.8%of the total clones), followed by Bacillus cereus (2OTUs,18.5%of the total clones) and Paenibacillus sp.(3OTUs,15.2%of the total clones).Conclusion1. There was a large number of endophytic bacteria in jujube.18strains of endophytic bacteria and endophytic fungi were obtained.2. KMB medium and Free-nitrogen medium had the bast effect on isolating endophytic bacteria.3. Filter out some endophytic bacteria that are able to antagonism pathogenic bacteria of plants and human body.4. Culture-independent and culture-dependent method are quite different on population distribution.