Pseudomonas Fluorescens are gram negative bacteria that inhabit soil,water and water surface.Recently they were also detected to live in the surface coat of pine wood nematode (PWN),Bursaphelenchus xylophilus,a pathogen causes pine wilt disease.Studies reveal that without the surface coat microbial,PWN alone would not cause pine wilt disease.Thus microbial in the surface coat of PWN may play a significant role in causing the pine wilts disease.Learning more about the microbial in the surface coat of PWN is an active demand for a better understanding on how to protect pines from the wilt disease.In this study,the genome of P.fluorescens GcM5-1A was sequenced using a Roche 454 GLX sequencer.Althogether,over 1.5 million high quality reads are obtained,which comprise of 600Mb oligonucleotides.These sequences are about ca.100x sequencing depth of the P.fluorescens GcM5-1A genome.Subsequently,the obtained sequences are assembled by the in-house Newbler software.Totally,the assembled sequences cover a physical length about 6.01Mb.Contig N50 is 76kb,and 79 contigs are greater than 500bp.Using the GLIMMER annotation engine,5516 ORFs are predicted.Among these ORFs,2988 of them share significant homology with the other 4 sequenced P.fluorescens isolates,and 1137 ORFs are found to be unique in the GcM5-1A genome.Previous studies revealed that the 4 sequenced P.fluorescens isolates were categorized into 2 lineages,with Pf0-1 and Pf5 formed one lineage,whereas SBW25 and WH6 formed the other lineage.We perform the phylogenetic analysis of the 5 sequenced isolates,and find that GcM5-1A is more similar to SBW25 and WH6.Synteny analyses among these 5 isolates also verify that GcM5-1A is closer to SBW25 and WH6.It is notable that there is a DNA segment,contig24,in the genome of GcM5-1A has nearly no homology with the other P.fluorescens isolates.Strong homology is found among ORFs between contig24 and Pseudomonas phage.We subsequently compare contig24 and Pseudomonas phage PAK_P1,whose gnome size is the most comparable to the size of contig24.We found that they were very similar in terms of genome size,GC content and predicted gene numbers.However,gene sequences diverged significantly between contig24 and Pseudomonas phage.The mystery beyond this finding remains unresolved.Pathogenicity studies on GcM5-1A have revealed that the flagellar protein,FliC,is pathogenic.In this study,we successfully locate FliC in the genome sequence of GcM5-1A.We also detect the complete Type III Secretion System (T3SS) that is responsible for flagellum secretion and act as the virulence factors in pathogenic gram negative bacteria.It is worthy of note that,Beside WH6,GcM5-1A is the second isolates of P.fluorescens that possesses the complete T3SS.With availability of the genome sequence of P.fluorescens GcM5-1A,it will greatly help to study the genes underlying pine wilting,and study the secretion system for pathogenic protein.This study provided fundamental resources for better understanding of the pathogenic microbial of P.fluorescens species,and supplied essential knowledge for help controlling the severe pine wilt disease.