Improved and simplified method for aseptic isolation of nematodes and nematode-endosymbiotic bacteria from pine seedlings

Abstract

Pine wilt disease (PWD), caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus, significantly impacts pine species and poses a broader ecological concern. An understanding of these nematode-associated microbes is essential for formulating sustainable PWD management strategies. We introduce a streamlined method for the aseptic extraction of B. xylophilus from pine seedlings, evolving beyond traditional Baermann funnel approaches. The method ensures optimal nematode extraction under sterile parameters, with seedling cutting discs processed using a unique sterile syringe assembly setup. The efficiency and simplicity of this method promise to significantly reduce the time and resources required. It also incorporates endosymbiotic bacterial isolation from isolated nematodes. The robustness of this method is affirmed by the successful isolation and identification of nematodes and bacterial strains as endosymbionts. Collectively, this protocol paves the way for more effective studies of nematodes and associated microbes, promoting the understanding of PWD and offering practical implications for better PWD management.•A simplified, aseptic method for extracting B. xylophilus from pine seedlings, offering a modern alternative to traditional Baermann funnel method.•Utilization of a specialized sterile syringe assembly setup, ensuring controlled and optimal nematode isolation.•Method validation achieved through the successful isolation and identification of bacterial strains as nematode endosymbionts.