Preliminary Study On The Optimization Of Acid Producing System Of Pichia Anomala 0732-1 And Its Biocontrol Mechanism To Bacterial Fruit Bloth Of Hami Melon

Objective:In order to effectively control bacterial fruit spot of hami melon by using Pichia anomala 0732-1,this paper studied the acid production condition,colonization condition and inhibition mechanism of crude extract of fermentation broth on pichia anomala for controlling bacterial fruit blotch of hami melon.The study of colonization characteristics and antagonistic mechanism provided theoretical references for the development and application of P.anomala0732-1.Methods:Response surface method was used to optimize the bacteriostasis and fermentation conditions of P.anomala 0732-1.The colonization ability of P.anomala 0732-1 on the surface of hami melon leaves and its influencing factors were studied by plate colony counting method.The effects of the fermentation crude extract of P.anomala 0732-1 on the cell wall,cell membrane and related guard enzymes of Acidovorax avenae subsp.citrulli(Aac)were studied by spectrophotometry.Result:The amount of dissolved oxygen had a great effect on the fermentation of P.anomala0732-1.The pH of fermentation broth decreased slowly and the lowest was 3.7 because of the low dissolved oxygen content in stationary culture,which was not as good as the acid production in shaking culture.The fermentation conditions of P.anomala 0732-1 were optimized by response surface method.The diameter of the inhibition circle was 17.9 mm.With the increase of the treatment time of different medicaments and fermentation broth,the germination rate of hami melon seeds decreased.Based on the comprehensive germination rate and the size of the bacteriostasis circle,the fermentation liquid was suitable for the hami melon seed treatment.The conditions of high humidity(90%),high concertration(10~8 cfu/mL P.anomala 0732-1)and 28℃were favorable for the colonization and acid production of P.anomala 0732-1,respectively.The colonization and acid-producing ability of P.anomala 0732-1 on leaf surface was influenced by the inoculation sequence of Aac.Inoculating P.anomala 0732-1 for 24 hours and then inoculating pathogen had little effect on the colonization and acid production of P.anomala 0732-1.The fermentation crude extract treatment of P.anomala 0732-1 had a certain effect on the cell wall of Aac.However,the fermentation crude extract treatment increased the metabolic enzymes ALT and AST,but the membrane of Aac did not completely lose its selective permeability.Fermentation crude extract treatment decreased the activities of peroxidase(POD),superoxide dismutase(SOD)and catalase(CAT)in Aac.Conclusion:Under static culture,due to the low dissolved oxygen content,the pH of fermentation broth was higher than that under shaking culture condition.The dissolved oxygen quantity decreases with the acid production of P.anomala 0732-1 in the early stage.The response surface method was used to obtain the optimum fermentation conditions for P.anomala 0732-1 acid.High concentration of P.anomala 0732-1 suspension(10~8 cfu/m L),high temperature(28℃)and high humidity(90%)were beneficial to the colonization and acid production of P.anomala0732-1.The colonization and acid-producing ability of P.anomala 0732-1 on leaf surface was influenced by the inoculation sequence of Aac.The activity of peroxidase(POD),superoxide dismutase(SOD)and catalase(CAT)in Aac decreased after treatment by the fermentation crude extract of P.anomala 0732-1.The cell membrane and cell wall of the Aac are damaged,resulting in the continuous leakage of proteins and small molecular ions in the cytoplasm,which leads to the increase of protein content and the increase of electrical conductivity in the liquid of bacteria.Finally,the ability of growth and reproduction of Aac was greatly reduced or even lost,and the bacteriostasis effect was achieved.