| In this experiment,a strain of bacteria which has a strong inhibitory effect on Aspergillus niger has been isolated from a contaminated Aspergillus niger plate.Plate fermentation method and Oxford cup method were used to detect the inhibitory effect of the broth of this strain on various plant pathogens such as plant root rot diseases,like Rhizoctonia solani Khiin and Xanthomonas oryzae.This means,the bacteria have important application value in the prevention and control of agricultural plant diseases and fruit preservation.In this article,Taxonomy,bioinformatics,separation,purification and preliminary identification were used to describe the bacteria.The main research contents are as followings:1.From the genotypic characteristics theories,we can determine that the isolated strain YL1 was Bacillus velezensis.Sequence alignment of 16 S rDNA showed that its' similarity with the Bacillus velezensis CR-502 was 99.85%.Alignment by genomic ANI,the homology of this strain and Bacillus velezensis CR-502 was 98.29%.So we can confirmed that this strain is the same species with Bacillus velezensis CR-502.Due to the different strains,this strain was named as Bacillus velezensis YL1.2.After get the whole sequence of Bacillus velezensis YL1 and predict its possible antibiotics.The complete genome sequence of this bacteria was obtained by splicing the second-generation sequencing genome sequence,and then analysis the genome using the antismash website,compared with the known antibiotic synthesis genes to predict the genes and types of possible antibiotics.The genome of the strain was found to contain the complete synthesis genes of Bacillibactin,Bacilysin,polyketides Bacillaene and Macrolacin.And also some genes of this strain has high similarity with lipopeptides Fengycin and Surfactin synthetic genes,some unknown synthetic genes of terpenoids and polyketides were also found.3.This article also explored the properties of Xanthomonas oryzae in fermentation broth.It was found that the activity of Xanthomonas oryzae produced by the fermentation broth has no significantly reduce after treatment under 100°C for 2h;when the pH of thefermentation broth was adjusted to the range 2-10 and keep for 4h,the activity has no greatly change;The substances in the fermentation broth which can against Xanthomonas oryzae are insensitive to trypsin and pepsin.In addition,the components are soluble in various organic reagents such as methanol,ethyl acetate and acetonitrile,and insoluble in petroleum ether.4.Separation and purify period obtained a variety of antibiotics produced by the bacteria.This experiment first concentrated the fermentation broth and then extracted it with methanol.The peak time of the active substance was determined by HPLC method.It was found that the bacteria produced not only one kind of antibiotic,after futher extracted with acetonitrile,antibacterial substances could been obtained.And then further purified by HPLC to obtain a higher purity of active substances that can against Xanthomonas oryzae.Due to the strong polarity of antifungal substances,it is difficult to extract via other organic reagents,using LH-20 gel column pairs to initially purify the methanol leaching material and then use HPLC method for further purify to obtain a higher purity active material.5.The isolated antibiotic molecular weight was determined and also analysis found that the corresponding antifungal antibiotic was Surfactin.The molecular weight of antifungal substance measured by LC-MS was 1044.56 and 1058.57,and the molecular weight of the antibacterial substance was 447.24.Through the prediction of genomic metabolites and compare with natural product dictionary database,we can surmise that the molecular weights of 1044.56 and 1058.57 were the lipopeptides Surfactin B and Surfactin C,and the molecular weight of 447.24 did not find similar molecular weights materials in the genomic predicted materials.Also there was no substance of similar nature found in the antibiotic database,we need further experiments to determine it. |
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