Efficacy Of Biocontrol Bacterium FD6 Against Black Rot Of Cabbage And Function Analysis Of Two-component Regulatory System CbrA/CbrB

Black rot of cabbage is an important disease of cruciferous plants caused by Xanthomonas campestris pv.campestris(Xcc).In order to determine whether Pseudomonas protegens FD6 could inhibit the expansion of black rot disease,we test the biocological control effect of FD6 after the cabbages were treated at different time.The results showed that the biocontrol bacterium FD6 had a better control effect on the pathogen,and the control effect was better when treated in advance.Previous studies have shown that pyoluteorin(PLT)and 2,4-diacetyphloroglucinol(2,4-DAPG or PHL)are the main antimicrobial substances produced by FD6.Avariety of two-component regulatory systems have effect on the production of these two compounds.A two-component regulatory system typically consists of receptor proteins and regulatory proteins.They are encoded by two different genes,usually adjacent to each other,and form an operon.CbrA/CbrB is widely found in Pseudomonas genus and consists of membrane-bound sensor histidine kinase(CbrA)and cytoplasmic response regulatory protein(CbrB).CbrA/CbrB is mainly involved in carbon and nitrogen metabolism and plays an important role in the process of bacterial adaptation to the environment.This study aims to clarify whether CbrA/CbrB in biocontrol bacterium is involved in the regulation of biocontrol related traits expression.The results are as following:The deletion of 593 bp and 640 bp fragments of cbrA and cbrB genes were obtained by SOE-PCR method.Phenotypic analysis showed that compared with the wild type FD6,the CBRA-deficient mutant had significantly higher competition production and swarming ability,significantly reduced biofilm formation,and did not produce extracellular proteases.The HPLC data showed that the production of PLT and 2,4-DAPG decreased significantly after mutation of CbrA.The PLT and 2,4-DAPG production decreased by 4 times and 14 times,respectively.This result indicated that CbrA positively regulated the synthesis of PLT and 2,4-DAPG.qRT-PCR data also showed that CbrA had a positive effect on the transcription levels of 2,4-DAPG and PLT antibiotic synthesis-related genes,including pltA,pltL,pltM,pltR,phlD and phlH genes.The cbrB deletion led to significantly increase of bacterial growth rate,siderophore production and biofilm formation.However,the extracellular protease production and swarming movement were not significantly affected.HPLC results showed that the production of PLT and 2,4-DAPG increased by 1.25 times and 3.11 times,respectively,after cbrB was mutated.These results indicated that CbrB negatively regulated the synthesis of PLT and 2,4-DAPG antibiotics.Our results revealed that the CbrA/CbrB two-component regulation system was involved in regulating the expression of various biocontrol related factors in P.protegens FD6.