Study On Biological Control Against Gentian Leaf Blight By Mixed Culture And Anti-infective Constituents In The Strain

Endophytic fungi LD421 and LD455 have been separated and selected form gentiana manshurica during the previous experiments,which have good antagonism on gentiana leaf blight.Based on this,the mixed culture conditions of LD421 and LD455 have been studied to improve the antibacterial activity on the gentiana Ieaf blight.Meanwhile,the biocontrol effect of the broth is further studied.The main antibacterial activity ingredients in LD421 are further tracked,to provide theoretical basis for the effect of biocontrol fungi on the prevention of Gentian.Taking gentiana leaf blight as the indicator bacteria,the mixed culture conditions of endophytic fungi LD421 and LD455 are studied by the growth rate method,which includes culture methods,culture rate and culture time.The results show that the mixed seed liquid of LD421 and LD455 with the ratio of 3:1,which is then added to a 500mL conical flask containing 300mL PD culture medium,could provide the best antibacterial activity after 13-day culture,with the antibacterial rate of 92%.Cultivated gentiana is taken as the experimental material in the greenhouse biocontrol.Inactivation metabolic liquid for the endophytic fungi is added to the experimental group,and 70%thiophanate 1000-time liquid is added to the positive comparison group.Gentiana leaf blight is added to the experimental group and the comparison group after 4 weeks.The results show that the mixed culture could realize the antibacterial rate of 88.59%,which is close to that of the 70%thiophanate 1000-time liquid,with longer prevention period.The antibacterial rates of LD421 and LD455 broth are 85.06%and 75.93%respectively.Besides,the prevention period of LD455 broth is relatively lower.Biennial fungi is adopted in field biocontrol.Inactivation metabolic liquid for the endophytic fungi is added to the experimental group,70%thiophanate 1000-time liquid is added to the positive comparison group,and only water is added to the blank group regularly.The results show that the mixed culture broth can realize the antibacterial rate of 90.29%for gentiana leaf blight,which is close to that of the 70%thiophanate group from early July to middle September,with the disease index lower than that of the blank group.The antibacterial rate of LD421 broth can achieve 90.00%,which is close to that of 70%thiophanate group from early July to early August;the rate of LD455 broth is relatively lower,79.15%.The antibacterial activity ingredients in LD421 broth are tracked.Five macroporous resins D101、D301、AB-8、HPD100 and X-5 are selected and the experimental results show that the alkalescent anion polymeric adsorbent D301 could provide the best separation effect according to the antibacterial rate of gentiana leaf blight.Water elution and 20%ethanol elution are carried out for physical and chemical identification and TLC analysis to determine the activity ingredients.Further experimental results by urea-SDS-PAGE show that glycopeptides substances in the LD421 broth are the main antibacterial ingredients.