Activity Detection And Fermentation Conditions Optimization Of Lipopeptide Antibiotics Synthesized By Biocontrol Bacillus Subtilis Z-14

Take-all of wheat,a devastating soil borne fungal disease that is caused by the infection of wheat root by Gaeumannomyces graminis var.tritici?Ggt?.Nowadays,resistant cultivars of wheat to this disease are not available and methods to control this pathogen by chemicals are insufficient.Crop rotation can control this disease,but its application in large-scale of wheat planting area is limited.In this case,biological control is expected to become the effective measure to control take-all of wheat.Based on the endophytic Bacillus subtilis Z-14,which has significant control effect on take-all of wheat,this study isolated and identified the lipopeptide antibiotics produced by Z-14 strain,the antagonistic mechanism,antifungal effect and fungi diversity of fengycin and iturin A were studied,and the fermentation conditions of the synthetic lipopeptide antibiotics were optimized.Lipopeptide antibiotics synthesized from B.subtilis Z-14 were isolated,identified using HPLC and MALDI-TOF-MS.The mobile phase components were?A?0.1%trifluoroacetic acid in water and?B?acetonitrile?60:40,v/v?,with a flow rate of 0.8 ml/min at 25°C.UV detector detection wavelength was set to 230 nm.The results showed C14^C17 iturin A and fengycins were successfully separated.And a single production of fengycin and C14 iturin A,C15 iturin A was prepared.In order to study the antifungal mechanism of fengycin and iturin A against Ggt,the effects of fengycin and iturin A on the morphology of Ggt mycelium were studied by microscopy and scanning electron microscopy.The effects of fengycin and iturin A on the structure of Ggt cells were observed by transmission electron microscopy.And the plate biocontrol test was carried out to test the biocontrol effect of take-all of wheat.The experimental results showed that fengycin mainly destroy the internal structure of Ggt cells,so that Ggt stop growing.Mainly in the cytoplasm and various organelles to digest,the formation of a large number of vacuoles,hyphae dry twist distortion.When Ggt was treated by iturin A,the cell wall disappeared,the cell membrane degenerated,the intracellular material shrinked,and the hyphae breaked into fragments,there by stopping development.In the plate biocontrol test,100%control effect was achieved when Ggt was treated with fengycin solution at a concentration of 1.0 mg/mL,while 100%control was achieved when treated with a concentration of 5.0 mg/mL of iturin A solution,so the control effect of fengycin was better than that of iturin A.High-throughput sequencing was used to examine the effects of fengycin and iturin A on fungal diversity in soil.The dilution curves and Shannon curves of all samples indicate that the data measured in this test was sufficient to cover most of the microbial diversity in the sample,and the results of all data analyses were reliable.Through Alpha diversity analysis,all indicators?Chao1,Ace,Shannon,Simpson?reflected that the sequencing results can represent the real situation of microorganisms in the sample.Specifically,at the level of the phylum,the effects of iturin A and fengycin on soil fungal diversity were not significantly different.Iturin A and fengycin could alleviate the reduction of Ascomycota,increase the relative abundance of Chytridiomycota,Olpidiomycota and Olpidiomycota,and reduce the relative abundance of Basidiomycota and Glomeromycota.At the genus level,both iturin A and fengycin could increase the relative abundance of Chaetomium and Alternaria,and reduce the relative abundance of Aspergillus and Gibberella.Iturin A could reduce the number of Mortierella and Myrothecium,and fengycin increases the number of Mortierella and Myrothecium.Iturin A increased the relative abundance of Fusarium in soil,and fengycin reduces the relative abundance of Fusarium.The Beta diversity assessment of microbial community in soil samples,iturin A and fengycin can change the soil fungi diversity to some extent,but the effect of iturin A on soil fungal diversity had a great influence on the treatment time,while fengycin had a variety of soil fungi.The effect of fengycin was basically balanced after 7 days of treatment.Regarding the impact on bacterial diversity,at the level of the phylum,fengycin and iturin A had no significant effect on soil bacterial diversity.At the genus level,iturin A had no significant effect on soil bacterial diversity,and fengycin reduced the abundance of Sphingomonas to increase the relative abundance of Steroidobacter.Beta diversity assessment showed that fengycin significantly affect soil bacterial diversity at 7 d,and iturin A had no significant effect on soil bacterial diversity.In order to improve lipopeptide production capacity of B.subtilis Z-14,the culture conditions of B.subtilis Z-14 were optimized by response surface method.The carbon source,nitrogen source and inorganic salt which had great influence on the antifungal activity of B.subtilis Z-14 fermentation supernatant by single factor were corn flour,soybean meal and FeSO4·7H2O,which had great influence.The culture condition factors were speed,inoculum amount and pH.The optimal medium and culture conditions obtained by response surface optimization were 3.85%corn flour,1.57%soybean meal,0.03%FeSO₄·7H₂O,0.02%NaH₂PO₄·2H₂O,0.04%Na₂HPO₄·2H₂O,and initial pH 7.0,inoculation 8.5%,speed 190 r/min,cultured at 37°C for 33 h.Fermentation culture was carried out using the optimized medium and culture conditions.Finally,the antifungal band width of the fermentation supernatant on take-all of wheat pathogen increased from 0.8 cm to 1.45 cm.The content of fengycin,C14 iturin A and C15 iturin A increased by 4.19,5.30and 2.53 times,respectively.In the pot culture control experiment,the fermentation supernatant with the best application was 30 mL,which could prevent and control the take-all of wheat by 88.11%.