Identification Of An Antimicrobial Protein AMEP412 And Its Application In The Control Of Potato Scab

Potato scab is a bacterial disease caused by Streptomyces spp.,which severely damages the roots of potato.In recent years,with the expansion of potato planting area,this disease becomes more and more serious.Chemical control of potato scab has caused a serial of problems like environmental pollution.Therefore,biological control with safety and efficiency becomes the focus of the current research.In this study,a biocontrol strain was isolated and screened against S.scabiei from the disease soil of potato scab and the strain was identified according to its morphological characteristics,physiological and biochemical properties and molecular identification.The antimicrobial protein was separated and purified by ion exchange chromatography,etc.The target band on SDS-PAGE was cut and sent to mass spectrometry sequencing.Then,the properties of the antimicrobial protein were analyzed including the minimum inhibition concentration.On this basis,the yield of the antimicrobial protein was used as an index to optimize the culture conditions.Finally,the biocontrol effect of the fermentation broth on potato scab was further tested by pot and field assays.These results were shown in detail as follows:In this experiment,50 bacteria strains were isolated from the diseased soil of potato scab by plate dilution method.Strain BU412 showed a significantly inhibitory effect on potato scab,which was identified as B.subtilis according to its morphological characteristics,physiological and biochemical properties and molecular identification results.To isolate the antimicrobial protein,the supernatant was applied for Source^TM Q ion exchange and Superdex^TM 75 size exclusion chromatography.The target protein sample with antimicrobial activity showed a main megascopic band on SDS-PAGE,which was cut off and analyzed by mass spectrometry sequencing.The MS sequencing result revealed an uncharacterized protein containing 76 AA?WP₀17418614.1?,which was then named as AMEP412.The comparison and analysis of the sequence of AMEP412 in the antimicrobial peptides database resulted no similar sequence with the protein,which confirmed that AMEP412 was a novel protein with antimicrobial function.The minimum inhibition concentration of AMEP412 result showed that the MIC was 0.05mg/m L against S.scabiei,the stability test results showed that AMEP412 had good stability against thermal and in wide p H range.Its antimicrobial activity was not sensitive to metal ionsand proteinase K.However,its activity was completely abrogated with Tween 80 treatment;Several antimicrobial peptide segments were designed and synthesized,which determined the three functional areas of AMEP412?GS21,GY20 and GY23?;AMEP412 was labelled with FITC,the results showed that it could interact with aerial mycelia and spores;the antimicrobial spectrum analysis showed that AMEP412 had good antagonistic effect on several plant and animal pathogens such as Fusarium graminearum Schw and Staphylococcus aureus.The yield of AMEP412 was optimized by Plackett-Burman and Central Composite Design experiments according to the single factor assay.The main factors affecting the antimicrobial protein yield were screened and the response equation was constructed.The optimal culture conditions were determined: yeast extract?35.97 g/L?,dextrose?10 g/L?,culture time 66.98 h,culture temperature 27.14 °C,initial p H 7.0,rotational speed 160 rpm,inoculation amount 0.6%.The biocontrol effect of strain BU412 fermentation broth on potato scab was further verified by pot and field assays.In the pot assay,three treatment groups were set up,including CK,treatment group 1?1×10⁷ cfu/g soil?and treatment group 2?2×10⁷ cfu/g soil?.The result showed that incidence and disease index in group 1 decreased significantly after treatment with the fermentation broth.There was almost no obvious disease spot on the surface of potato in group 2.The disease index was down to 25.63%,and the relative control effect reached 63.13%.In the field assay,two treatment groups were set up,including CK and treatment group?2×10⁷ cfu/g soil?.Incidence at 42.85% and the relative control effect reached 51.67% after treatment with the fermentation broth,which further confirmed that AMEP412 had a remarkable biocontrol effect against potato scab.In this paper,a biocontrol strain BU412 was isolated and identified against S.scabiei and a novel antimicrobial protein AMEP412 was identified.The antimicrobial function of the protein was characterized,then its related properties were analyzed,and the optimal fermentation conditions for high yield of the protein were determined.Finally,this study proved that strain BU412 fermentation broth had a good control effect on potato scab by pot and field assays.The study can not only enrich the antimicrobial proteins of Bacillus species,but also provide the material and basis for the biocontrol research of potato common scab.