Molecular Basis On Action Of Pseudomonas Fluorescens SN15-2 Against Ralstonia Solanacearum

Ralstonia solanacearum is a devastating soil-borne pathogen,and the traditional methods to control R.solanacearum wilt disease have limited success.In recent years,biological control has become a hot spot for the prevention and control of plant diseases because it is environmentally friendly and safe to humans and animals.We collected soil samples from natural decay soil of tomato bacterial wilt in Shanghai area.SN15-2 which showed a strong antagonistic activity to R.solanacearum was isolated and screened by antagonistic experiments,and then identified as Pseudomonas fluorescens.We evaluated the ability of.fluorescens SN15-2 to produce antibiotics and colonize rhizosphere,and explored biocontrol efficacy of P.fluorescens SN15-2 against R.solanacearum through greenhouse experiment.Furthermore,transmission electron microscope technology and RNA-Seq technology were used to explore the mechanism of P.fluorescens SN15-2 against R.solanacearum.Firstly,the isolated biocontrol strain SN15-2 was identified as.fluorescens by morphological,physiological,biochemical and molecular biological methods.By PCR amplification and gene sequencing,we discovered that.fluorescens SN15-2 can produce 2 4-,two acetyl benzene three phenol(2,4-DAPG),pyrrolnitrin(PRN),pyoluteorin(PLT),and HCN.R fluorescens SN15-2 was marked with rifampin,and then the colonization experiment of.fluorescens SN15-2 in the rhizosphere of tomato was carried out.The result showed P.fluorescens SN15-2 has a good ability of colonization in tomato rhizosphere.On the sixtieth day,the concentration of P.fluorescens SN15-2 in tomato rhizosphere still had 3.67x105CFU/g(dry soil).Biocontrol efficacy of P.fluoreescens SN15-2 against R.solanacearum was investigated through greenhouse experiment,and result showed the biocontrol efficacy of P.fluorescens SN15-2 against R.solanacearum was 46.58%which was significantly higher than that of streptomycin sulphate(32.67%).Secondly,the half-maximal inhibitory concentration of P.fluorescens SN15-2 metabolites against R.solanacearum was calculated to be 23.4%by linear fitting method.The effect of the half-maximal inhibitory concentration P.fluorescens SN15-2 metabolites on R.solanacearum was studied by transmission electron microscope negative staining and ultrathin section methods,and results showed P.fluorescens SN15-2 destroyed cell walls and membranes of R.solanacearum,and further caused cytoplasmic overflow of R.solanacearum.Thirdly,molecular mechanism of P.fluorescens SN15-2 against R.solanacearum was studied by RNA-Seq technology,and RNA sequencing identified 264 genes that were significantly differential expression in response to.fluorescens metabolites.Further analysis of differential expression genes showed.fluorescens SN15-2 metabolites destroyed the cell membranes of R.solanacearum by inhibiting biosynthesis of integral membrane protein;P.fluorescens SN15-2 metabolites destroyed the cell walls of R.solanacearum by upregulating the expression of murein hydrolase and downregulating the expression of diaminopimelic acid;P.fluorescens SN15-2 metabolites depressed growth and reproduction of R.solanacearum by inhibiting biosynthesis of nucleic acids,but stimulate catabolism of nucleic acids;and P.fluorescens SN 15-2 metabolites inhibited growth and metabolism of R.solanacearum by causing iron absorption pressure on R.solanacearum by siderophore.On the other hand,small heat shock protein,organic hydroperoxide resistance protein,DNA protection during starvation or oxidative stress transcription regulator,and putative choline dehydrogenase and related flavor proteins oxidoreductase were up-regulated,presumably to mitigate damages caused by P.fluorescens SN15-2 to its cell membranes,proteins and DNA.Multidrug resistance transmembrane protein was also up-regulated,presumably to promote efflux of harmful substances.However,due to extreme toxicity of P.fluorescens SN15-2 metabolites,universal stress proteins were inhibited instead of being stimulated,and this result implied some resistance mechanisms of R.solanacearum became invalid under the stress of P.fluorescens metabolites.In general,we isolated and screened a P.fluorescens from natural decay soil of tomato bacterial wilt in Shanghai area,and enriched the microbial resources for the prevention and control of bacterial wilt.Furthermore,molecular mechanism of P.fluorescens SN15-2 against R.solanacearum was studied.This research provided a theoretical and practical basis for biological control of bacterial wilt.