Isolation And Screening Of Active Substances From Streptomyces Roseoverticillatus Sr-63 Strain And Study On The Mechanism Of Inhibiting Xanthomonas Oryzae Pv.Oryzae

Bacterial leaf blight（BLB）disease caused by Xanthomonas oryzae pv.oryzae（Xoo）is considered to be one of the most serious bacterial diseases in rice,leading to severe yield losses.Microbial biological control agents（MBCAs）are applied to crops for biological control of plant pathogens where they act via a range of modes of action.Production of antimicrobial secondary metabolites with inhibiting effects against pathogens is one of the modes of action.Actinomycetes synthesize approximately two-thirds of all naturally derived antibiotics currently used in medicine,veterinary medicine and agriculture.Majority of these molecules originate from Streptomyces genus.Consequently,it’s necessary to select effective antibacterial producing strains,and identify and screen the key compounds produced by Streptomyces against Xoo.It provides strain resources for biological control of bacterial leaf blight.This research was based on the Streptomyces roseoverticillatus 63 strain preserved in laboratory.The secondary metabolites produced by Sr-63 were separated and purified by silica gel and sephadex LH-20 column chromatography,and six metabolites were obtained.The structures of metabolites were analyzed by ¹H-NMR,¹³C-NMR and HR-ESI-MS.The key effective antibacterial compound against Xoo was screened by Oxford Cup method,as well as to gain insight into the underlying mechanisms responsible for the antagonistic effect against Xoo.The major findings are mentioned as follows:1.Six metabolites were obtained from the ethyl acetate extract of Sr-63 strain fermentation broth,and the six compounds were identified as carbazomycin A,carbazomycin B,carbazomycin G,actiphenol,pimprinethineand3-Methoxy-2-methyl-carbazole-1,4-quinone according to the literature.2.The carbamycin B was screened to had a strong activity against Xoo using Oxford Cup method,the diameter of the inhibition zone of 100μg/m L carbamycin B against Xoo reached 25 mm.The results indicated that the minimum inhibitory concentration（MIC）value of carbazomycin B against Xoo was 8μg/m L,while for bismerthiazol the MIC value was found to be 16μg/m L by unsing 96-well plate microdilution method.The results showed carbamycin B inhibited the growth of Xoo.3.The antibacterial mechanism of carbamycin B on Xoo was preliminarily explored,and the effects of carbamycin B on cell morphology,biofilm formation,cell membrane permeability,components of the cell membrane,extracellular polymeric substance（EPS）,cell surface hydrophobicity,extracellular enzymes,protein synthesis,nucleic acid synthesis,and cell energy metabolism were determined.Scanning electron microscopy showed that the carbazomycin B caused morphological changes in Xoo.The carbazomycin B caused Xoo cell contents（electrolytes,proteins,β-galactosidase,etc.）varying degrees of leakage because the cell membrane structure or permeability changed.The fourier transform infrared spectroscopy study clearly showed the indicating chemical and physiological changes of the components of the cell membrane in Xoo,inhibited the activity of malate dehydrogenase and the synthesis of protein in Xoo cells,but there was no obvious inhibitory effect on the synthesis of DNA.4.The feasibility of Sr-63 and the carbamomycin B resistant to bacterial leaf blight was studied.The bio-control experiment showed that the fermentation broth of Sr-63 had a remarkably good prevention of rice bacterial blight disease,and the lesion inhibition rate of Zhonghua 11 rice reached more than 95.38%,which was better than the control effect of 500μg/m L of bismerthiazol（91.88%）.The lesion lengths observed in rice plants inoculated with untreated Xoo cells was 70.3 mm in average,while the lesion length of leaves inoculated with treated Xoo cells（4×MIC carbazomycin B）was 19.0 mm in average,the inhibition rate decreased by 72.98%.