| Laiyang pears are a specialty of Laiyang City,Yantai,Shandong Province.The occurrence of pear diseases limits the healthy development of the Laiyang pear industry.Black spot is a fungal disease caused by Alternaria alternata.It mainly infects the leaves,fruits and new shoots of pears.It often occurs in the storage period,resulting in fruit rot,which seriously affects the yield and quality.Effective control of black spot is particularly important in the production and storage of Laiyang pears.At present,the control of pear black spot mainly adopts chemical control methods.The use of chemical preparations will lead to a series of problems such as environmental pollution,pesticide residues,and the production of drug-resistant fungle.Therefore,it is very important to discover new antifungal preparations that are environmentally friendly,efficient and do not pose a threat to human health.In this study,from the perspective of biological control,a strain of Bacillus amyloliquefaciens that can stably antagonize black spot was screened out,and the antifungal substances produced by the active bacteria were extracted,purified and preliminarily identified,and its biocontrol effect was studied.Providing certain support for the field of plant disease control and food safety.The main experimental results of this paper are shown bellow:In the first part,the bacteria in the soil,sea mud,sea sand,seawater and marine organisms around the coast of Yantai were isolated and cultured by the plate culture method,and 79 strains of bacteria were finally obtained.Through the plate confrontation method,the pathogenic black spot fungus Alternaria alternata 196 was used as the indicator bacteria to select the bioactive bacteria from the obtained 79 bacteria and the existing bacterial library in the laboratory.Finally,one strain can stable antifungle was selected,named bioactive bacteria E14-2.DNA was extracted by boiling method,and the sequence was successfully amplified using bacterial universal primers 27 F and 1492 R.After 16 S r DNA sequencing,BLAST comparison was performed on the NCBI website,and appropriate references strain were selected to construct a phylogenetic tree.The results showed that the bioactive bacteria E14-2 was Bacillus amyloliquefaciens,named Bacillus amyloliquefaciens E14-2.In the second part,the experiment extracted,purified and preliminarily identified the antifungal substances produced by bioactive bacteria E14-2.Taking the width of the inhibition zone determined by the plate confrontation method as a reference,the activity of the antifungal substances produced by the active bacteria within 72 hours of culture in BHI broth medium and NO.3 liquid medium was determined,and the culture conditions of the active bacteria were finally determined as using BHI broth medium was incubated at 37 °C for 48 h with constant temperature shaking at 210 r.The antifungal substances were extracted by acid precipitation and organic solvent extraction.It was found that the acid precipitation method could effectively extract the active antifungal substances.Both ethyl acetate and chloroform extraction could not extract the antifungal substances from the fermentation broth.Therefore,acid precipitation was selected.method for crude extraction of antifungal substances.Antifungal substances were further purified and extracted by HLB solid phase extraction column,analyzed and identified by high-performance liquid chromatography tandem high-resolution mass spectrometry,and the mass spectrometry data were optimized by MZmine software and uploaded to the GNPS website for comparison and analysis.As a result,it was found that the antifungal substance produced by the bioactive bacteria E14-2 was a derivative of Iturin A.In the third part,Yantai Laiyang pear was selected for the experiment,the pathogenic black spot fungus Alternaria alternata 196 was inoculated on the surface of Laiyang pear fruit,and the antifungal substance produced by the bioactive bacteria E14-2 was inoculated with the pathogenic black spot fungus as the control group.The pears were cultured at a constant temperature and humidity of 25 °C,and the size of the lesions was recorded and sampled at 3 d,5 d,and 10 d.The samples were extracted for toxins,and the samples and standards were detected by liquid chromatography tandem high-resolution mass spectrometry.Morphological results showed that the antifungal substances could significantly inhibit the growth of lesions and prevent fruit rot.Toxicity analysis showed that the production of five toxins AOH,AME,ALT,TEN,and Te A were suppressed by orders of magnitude due to the protection of antifungle substances.58 species were found in Laiyang pear samples not inoculated with antifungal substances,and 24 species were found in samples inoculated with antifungal substances.After inoculation with antifungal substances,the glucose-binding phase II metabolites of black spot fungus 196 were significantly reduced,but no glucose-binding cryptic toxin could be found.The experiment proved that the antifungal substances produced by the bioactive bacteria E14-2 had obvious control effect on Laiyang pear infected by black spot fungus. |
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