Preparation And Study On Immobilized Biocontrol Pellets Against Pseudostellariae Heterophylla Leaf Spot

Pseudostellariae heterophylla is an herbaceous herb that can be used for both medicine and food.In recent years,with the development and utilization of medicinal active ingredients in Pseudostellariae heterophylla,the health care products with Pseudostellariae heterophylla are increasingly the increase in its high application value and commercial value has made it widely concerned,and the outbreak of fungal diseases has seriously affected the yield and quality of Pseudostellariae heterophylla.Immobilized microbial technology is an important biological control measure that can easily control the concentration of bacteria and protect the strains from reducing the loss of strains.In accordance with the discovery of the Bacillus subtilis JK05 which has been discovered by our laboratory against the Phyllosticta commonsii of Pseudostellariae heterophylla leaf spot.The antagonistic active substance produced by JK05 and the preparation of JK05 immobilized biocontrol pellets by using composite materials were studied.And I alsostudied the effect on the quality of Pseudostellariae heterophylla by using immobilized biocontrol pellets,the research results are significant important for controling the fungal diseases such as Pseudostellariae heterophylla leaf spot.The main research results of the thesis are as follows:（1）Extraction and identification the antagonistic active substance of Bacillus subtilis JK05.It demonstrated that the antagonistic active substance of Bacillus subtilis JK05 is an antagonism protein,and the antagonism protein precipitated at an ammonium sulfate concentration of 30-70%.The protein antagonistic activity is extremely significant by precipitating 40-60%.While the protein precipitated at an ammonium sulfate concentration of 30-40%and 60-70%,it`s antagonistic activity is very low.As the heat treatment temperature increases,the antagonistic activity of protein decrease,but it did not change significantly in the range of 30-80°C（P>0.05）.When the temperature was higher than 80°C,it decreased significantly（P<0.05）,but still on a higher level.It demonstrates that the antagonistic activity of antagonism protien has good thermal stability.The antagonistic activity of the antagonism protein is significantly reduced while too acidic（pH 4）or too alkaline（pH 10）.The effect of acid and alkali treatment was not significant while the pH is in the range of 5-9（P>0.05）.Only one protein component were obtained from the crude antagonism protein by DEAE Sepharose Fast Flow Ion exchange chromatography and Sepadex G-100 gel chromatography which showed a single band on the SDS-PAGE map and significant antagonistic activity on Phyllosticta commonsii.The antagonism protein BSAP of Bacillus subtilis JK05 was initially identified as Dihydrolipoamide dehydrogenase by LC-MS/MS.FT-IR showed that the antagonism protein BSAP had a characteristic absorption peak amide I at 1631.028 cm^-1 due to the C=O in-plane stretching vibration,it belongs to theβ-sheet structure;And a characteristic absorption peak amide III at 1296.915 cm^-1 due to the N-H in-plane bending vibration,it belongs to theα-helical structure.（2）Preparating biocontrol pellets which embed Bacillus subtilis JK05 by immobilized microbial technology.Based on the mechanical strength,mass transfer performance and antagonistic activity of immobilized biocontrol pellets,the best composite carrier material for immobilized biocontrol pellets was 9%polyvinyl alcohol,3%sodium alginate and 2%activated carbon.The optimal concentration of Bacillus subtilis JK05 in the pellets was 2?10⁸ cfu/mL.The immobilized biocontrol pellets is porous and there is full of JK05 in the porous showed by scanning electron microscopy（SEM）.The structure of JK05 is complete and full in shape.while the temperature is in the range of 15²4°C,the antagonistic activity of immobilized biocontrol pellets significantly increaced with increasing temperature（P<0.05）,and decreased when the temperature reached 27°C,but compared with 24°C.The difference was not significant（P>0.05）;When the pH is in the range of 4-8,immobilized biocontrol pellets had great antagonistic activity.The relative biological content of air dried immobilized biocontrol pellets keep above 80%within 60 days.After 120 days later,the relative biological activity was keep above 60%.The result showed the immobilized biocontrol pellets has good storage resistance.（3）Analysing the effect of applying immobilized biocontrol pellets on the quality of Pseudostellariae heterophylla during cultivation by using the change of main chemical constituents of Pseudostellariae heterophylla as a norm.The polysaccharide content of Pseudostellariae heterophylla was significantly increased（P<0.05）.The maximum value of polysaccharide content was 12.49%when the quantity of application is 0.1 g per plant.There was no significant change in water content,saponin content and ash content（P>0.05）.Application of immobilized biocontrol pellets reduced the content of Fe and Mn in Pseudostellariae heterophylla,Mg,Rb,Co had no significant change,and the content of K,Ca,Zn and Cu increased.The kind of volatile constituents were decreased,and there was no significant difference between all amino acid but Methionine.