| Clubroot caused by Plasmodiophora brassica is a typical soil-borne disease.It is one of the serious diseases in Brassica napus and causes severe economic losses every year.Biological control potentials of two biocontrol strains F85 and T113 were studied in this paper.Through analysis of 16 S r DNA,gyr A and 22 housekeeping gene sequences,F85 and T113 were identified.We improved the biocontrol effect of biocontrol strains through optimization of fermentation conditions.The methods of UPLC,LC-MS and genomics were used to analyze antibiotic substances in biocontrol bacteria.Effects of biocontrol bacteria on plants were further explored by RNA-seq.The main research results are shown as follows:1.Phylogenetic trees were separately constructed by 16 S r DNA,gyr A and 22 housekeeping genes,and F85 and T113 were identified as Bacillus velezensis and B.amyloliquefaciens,respectively.The fermentation conditions of the two strains were optimized.The optimal medium for F85 was: 1% sucrose,0.3% paste yeast extract,0.3% yeast extract and 0.5% sodium chloride.The optimal fermentation conditions were: 37?,p H 8,5% of inoculation amount,120 ml/250 ml of bottling volume,150-200 r/min of rotation speed,and 48 h of incubation time.The optimal fermentation condition for T113 is 2% glucose,0.5% sucrose,0.5% yeast extract,1% sodium chloride,and 0.1% dipotassium phosphate.The optimal fermentation conditions are: 28-30°C,p H 7-8,5%-7% of inoculation amount,80-100 ml/250 ml of bottling volume,150-200 r/min of rotation speed and 60 h of incubation time.In pot and field control experiments,the pot control effeciency of the biocontrol F85 was 80.36%,and the field control effeciency was 52.94%.The pot control effeciency of T113 was 72.67%,and the field control effeciency was 47.06%.2.The crude extract of fermentation liquid of F85 was extracted by acid precipitation method,and the crude extract was detected by ultra high-phase liquid chromatography UPLC and LC-MS.Eleven lipopeptide antibiotics were produced,which mainly contained homologues of iturin and bacillomycin L and surfactin.To explore the biological control mechanism of biocontrol bacteria deeply,the genomes of F85 and T113 were sequenced and many antibiotic synthesis-related genes were predicted in the genomes,including lipopeptides,peptides,polyketones,terpenes,bacteriocins,antibacterial protein,ansamycins,carbapenem,streptomycin,monobactam,phenylpropanoid,penicillin,cephalosporin,novobiocin and more.Some antibiotics were selectively validated by PCR or RT-PCR.3.RNA-seq was used to analyze the transcriptome level of Arabidopsis plants treated with F85 fermentation liquid.In leaves,10287 differentially expressed genes were found,of which 5022 genes were down-regulated and 5265 genes were upregulated.In roots,13,716 differentially expressed genes were found,of which 6995 genes were down-regulated and 6721 genes were up-regulated.In GO enrichment analysis,the differential genes in leaves were mainly enriched in response to metal ions,bacteria,Thylakoids,photosynthetic membranes,m RNA activity and more.The differential genes in roots were mainly enriched in premises metabolism and energy,photosynthesis,secondary metabolism and more.In KEGG enrichment analysis,differential genes in leaves are mainly enriched in carbon metabolism,amino acids Synthesis,TCA cycle,and Glycolysis/Gluconeogenesis.Differential genes in roots were mainly enriched in ribosomes,carbon metabolism,amino acid synthesis,and proteasomes.KEGG pathway is significantly enriched in the 2-oxocarboxylic acid metabolism pathway,amino acid metabolism pathway,plant hormone signal transduction pathway and flavonoid synthesis pathway,which were related to plant disease resistance.Many disease resistance-related genes including pathogenesisrelated proteins,detoxification-related proteins,reactive oxygen species and nitric oxide and calcium signaling pathways were detected to be up-regulated in Arabidopsis leaves and roots. |
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