Diversity Of Endophytic Bacteria In Rape And Its Antagonism To Sclerotinia Sclerotiorum

This study collected the roots,stems and leaves of Brassica napus Xiang-You 15 from the experimental field,which belonging to the College of Bioscience and Biotechnology at Hunan Agricultural University.Using gradient dilution method,the endophytic bacteria were isolated and purified.Based on the phylogenetic analysis of 16 s rRNA gene sequences the diversity of endophytic bacteria was indicated.Agar diffusion method was used to screen anti-pathogen strains and the plate confrontation method was for isolating the strains that inhibiting Sclerotinia sclerotiorum.According to above results,the mechanisms of antagonism were further studied.This work provided the experimental foundation and the theoretical guidance for further researching endophytic bacteria resource and biological protection within Brassica napus.1.The culture mediums(LBA,NA,NIU,T)were selected as the isolation medium and the 68 strains of endophytic bacteria were obtained from the rhizome leaves of the rape sample.Based on morphological comparison,the 18 strains with obvious difference were carried out for next steps.2.In this work,a series of bacterial strains were selected grounded on 16 S rRNA gene sequencing and phylogenetic analysis.Statistical results show that the 18 strains of endophytic bacteria belong to the bacteria in the domain of three branch: Bacillaceae Pseudomonadaceae and Enterobacteriaceae,four genera: Bacillus,Pseudomonas,Rahnella and Escherichia.Among them,Bacillus and Pseudomonas are dominant groups.3.Regarding Bacillus subtilis,Staphylococcus aureus,Sarcina lutea,Escherichia coli,Bacillus proteus and Aspergillus niger as indicator bacteria,68 strains of endophytic bacteria were screened by agar diffusion method.The results indicated that 7 strains(Y171004 Y172014 Y173005,Y174007,Y174017 Y177001 Y177002)among of them possessed antimicrobial activity,and their physiological and biochemical characteristics were also studied.Y171004,Y172014,Y174007,Y177001,Y177002 were Bacillus sp,Y173005 was Pseudomonas sp,and Y174017 was Escherichia sp.The successful separation of the above endophytes,to a certain extent,fills the blank of the endogenous bacteria in rape and provides a reference for the study of the beneficial strains and the connection between the rape and the endogenous bacteria.4.Two strains with strong antagonistic abilities were selected and the antagonistic mechanism of the two strains was studied.Y171004 and Y177002 had an obviously antagonistic effect on the Sclerotinia scleroterum.Under the light microscope observation,the mycelium was wrinkled,thickened or discolored.The crude extract of fermented liquid protein,secreted by strain Y171004,had antibacterial activity.The activity lost after 95 water bath for 30 minutes,exhibiting its poor tolerance to high ?temperature.In terms of the non-antibacterial activity of crude Y177002 protein,the antibacterial substance of Y177002 fermentation is not protein.According to SDS-PAGE,the molecular weight of the crude Y171004 protein was about 45~65KDa.