| Gray mold disease of plant caused by Botrytis cinerea is a worldwide disease that causes significant economic losses to agricultural production.In terms of its prevention and control,with the emergence of the drawbacks of chemical pesticides,biological control methods that are highly efficient,safe,reliable and environmentally friendly have received increasing attention.Therefore,the development and utilization of biocontrol bacteria and their antifungal substances have become a research hotspot in recent years.In previous work,our research group isolated a strain of Bacillus velezensis named S3-1 from a soil sample collected around roots of cucumber plants.The strain S3-1 had a widely resistance against pathogen fungal such as Botrytis cinerea,Fusarium graminearum and Rhizoctonia solani.In this study,the antifungal proteins were isolated from the fermentation supernatant of strain S3-1.And for further application of this strain,the stability,control effect and antifungal mechanisms of its antifungal proteins were also investigated.Furthermore,the antifungal proteins were separated and identified by using electrophoresis and mass spectrometry analysis.In order to improve the ability of strain S3-1 to producing antifungal proteins,the best fermentation condition was determined through optimizing test.The results were as follows:1.The fermentation supernatant of strain S3-1 had an antifungal activity on B.cinerea.It was precipitated with solid ammonium sulfate and the results showed that the optimal saturation of ammonium sulfate was 40 % for precipitating antifungal proteins.2.The stability test results showed that the antifungal proteins had a good thermal stability;they were stable in a wide range of p H value but sensitive to strong acid or strong alkaline environment;they were not sensitive to ultraviolet light,some organic reagents and inhibitors.3.Control efficacy test results showed that the antifungal proteins had an obvious control effect on gray mold of cucumber and cherry tomato.4.To ascertain the antifungal mechanisms of the antifungal proteins to B.cinerea,morphological alterations of the hyphae were examined by using both optical microscope and scanning electron microscope.The results showed that the hyphae became malformation,uneven and broken surfaces,irregular widths and clear swellings after treated with antifungal proteins.Also,the antifungal proteins could change the cell membrane permeability and inhibit the germination of spores of B.cinerea.5.The antifungal proteins were separated into three fractions namely AP-1,AP-2and AP-3 by using native-PAGE.Antifungal activity test results showed that AP-1 had no antifungal activity,but the AP-2 and AP-3 had strong antifungal activities against B.cinerea.6.Both AP-2 and AP-3 exhibited the obvious and single band on SDS-PAGE and their molecular weights were in the range 25.0-35.0 k Da and 18.4-25.0 k Da,respectively.The analysis by MALDI-TOF-TOF-MS/MS suggested that the AP-3 was highly similar to the Endo-1,4-beta-xylanase with a molecular weight 23248.1 Da,its protein sequence coverage reached 53 %.However,no high matching rate proteins were blasted for AP-2,it was presumed to be a new antifungal protein.7.In order to improve the ability of strain S3-1 to producing antifungal proteins,the fermentation medium and conditions of the strain were optimized by using the response surface analysis method.The results showed that the optimal fermentation medium was composed of glucose 14.09 g/L,yeast extract 7.80 g/L and Na Cl 7.31g/L.And the optimal fermentation condition was fermentation period of 65 h,rotation speed of 200 r/min,cultural temperature of 26 °C,initial p H value of 6.5,inoculation quantity of 1 %,medium volume of 115 m L in 500 m L flask,bacterial inoculation age of 12 h.Under such conditions,the inhibition rate of the equal volume antifungal protein solution increased by 46.9 % and the amount of extracted protein increased by 45.1 %. |
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