Screening,evaluation And Genome Analysis Of Plant Probiotic Fluorescent Pseudomonas Strains With Type ? Secretion System

Chemical control of postharvest diseases of fruits and vegetables has potential risk affecting food safety.To develop biological control agents using beneficial microbial resources is a new alternative for green food production.Fluorescent Pseudomonas is a group of probiotic bacteria closely associated with plants,which is capable of producing a variety of secondary metabolites to antagonize pathogens,promot plant growth and control postharvest diseases.It was reported recently that some fluorescent Pseudomonas strains contained homologs of plant pathogenic type ? secretion system(T3SS).However,little is known about the distribution of such bacteria and the composition of the T3 SS gene clusters.In this study,we collected plant rhizosphere soil samples throughout the country to isolate and screen fluorescent Pseudomonas strains harboring T3 SS homologs.We further evaluated the potential of plant probiotic capacity and determined the T3 SS gene clusters and genetic characters through whole genome sequencing and analysis.The results are summarized as follows:(1)One thousand six hundred and eleven fluorescent Pseudomonas strains were isolated from 61 plant rhizosphere soil samples collected from 19 provinces and cities in China.Antagonistic assays indicated that 494 strains showed antagonism against Ralstonia solanacearum,8 strains against Pseudomonas syringae,776 strains against Magnaporthe grisea,and 803 strains against Phytophthora sojae.,a screening A primer set hrcC3,designed from the conserved hrcC gene of T3 SS,was used to screen the strain collections.In total,67 strains had a positive detection of hrcC gene,which possesses4.2% of all the fluorescent strains.A further sequence analysis demonstrated 15 strains out of the positive selections had unique hrcC gene sequence.(2)The final 15 strains with T3 SS genes were determined the antagonistic capacity against Penicillium digitatum and Penicillium italicum,two major postharvest pathogens.Among them,strain FP2301 showed 100% inhibition rate against the two pathogens and reduced the disease incidence by50.0% and 45.3%,respectively.The plant probiotic tests indicated that 12 strains produced proteolytic enzymes,15 strains produced siderophores,1-aminocyclopropane-1-carboxylic acid deaminase,indoleacetic acid,and biofilm while all of the 15 strains failed to produce amylase and cellulase.Strains FP1749 and FP1962 could trigger hypersensitive reaction when inoculated on Nicotiana benthamiana.(3)The draft genome of the 15 strains were sequenced and analyzed.The average genome size was 6.40 Mbp,G+C content was 60.3%,and the coding sequences were 5763.Combining Multilocus Sequence Analysis(MLSA)and Average Nucleotide Identity(ANI),the 15 fluorescent Pseudomonas strains were clustered in 4 subgroups:P.fluorescens FP834?FP1501?FP2054?FP690?FP2087??FP1885;P.mandelii FP1743?FP1749??FP1962;P.corrugata FP822?FP1162?FP2301?FP425?FP2262;P.koreensis FP1761.The pan-genome of the strains had 13,265 coding genes,of which 3062 constituted the core genome accounting for 53.1%of the average genome size.The T3SS gene clusters of the strains could be clustered in 5 groups based on a phylogenetic analysis using the full hrcC gene sequence.A BlastP scan showed each strain had a vary number of effector protein homologs.Given the versatile plant probiotic traits of the 15 strains,the potential to produce secondary metabolites was analyzed using antiSMASH.Abundant biosynthetic gene clusters were predicted from different strians to encode polyketides,cyclic lipopeptides,bacteriocins,and siderophores.Remarkably,a gene cluster synthesizing 2,4-diacetyl phloroglucinol(DAPG)was found in strain FP2301,which provides an immediate evidence for anti-pathogen capacity.