Markerless fliC Knockout in Pseudomonas fluorescens and Preliminary Evaluation on Pinus massoniana Callus

Pine wilt disease is a devastating forest disease caused by Bursaphelenchus xylophilus infection. The associated bacterium Pseudomonas fluorescens and its flagellin protein have been implicated in this process, but the role of the flagellin-encoding gene fliC in pathogenesis remains unclear. Current gene knockout strategies in P. fluorescens are limited by low transformation efficiency and cumbersome procedures, which constrain functional studies of this gene. To address this methodological gap, we established a fliC knockout method based on the pT18sacB-sacB counterselection system. Short homologous arms were constructed by splice overlap extension PCR, and the knockout mutant ΔfliC was obtained through S17-1 λpir-mediated conjugation and sucrose counterselection. The mutant was verified by junction PCR, internal PCR, and sequencing. Using Pinus massoniana callus as the experimental material, we preliminarily compared the wild-type strain and the ΔfliC mutant in individual pathogenicity and in promoting complex infection by B. xylophilus. The ΔfliC mutant showed delayed symptom onset and lower disease severity than the wild type, and its ability to promote complex infection also appeared reduced. These results provide preliminary evidence that fliC contributes to symptom development in this callus assay. The established fliC knockout technique provides a methodological basis for further investigating the role of associated bacteria in pine wilt disease.