Methods to obtain and maintain cultures of pinewood nematodes, <i>Bursaphelenchus xylophilus</i>

The pinewood nematode <i>Bursaphelenchus xylophilus</i>, native from North America, is the causal agent of the pine wilt disease, being considered one of the most important invasive pests in Eurasian pine forests. The establishment of laboratory isolates of <i>B. xylophilus</i> has been crucial to research on this important pathogen of pine trees. However, studies on the effects of <i>in vitro</i> serial passage on the pathogen evolution, and suitability of standard culture media to the wild populations have been surprisingly few. In the present work, we tested the suitability of different culture media for obtaining <i>B. xylophilus</i> lab populations from dauer larvae that have been isolated from pine sawyer beetles (<i>Monochamus</i> spp.), which serve as their vector. We conducted studies in North America and Portugal. Results showed that adding groundwood (xylem from pine trees) to standard agar medium dramatically improves the survival and growth of wild populations of <i>B. xylophilus</i> in the laboratory. Further, malt extract agar proved to be more suitable than potato dextrose agar. Once in culture, <i>B. xylophilus</i> grew well feeding on either of two fungus species: <i>Botrytis cinerea</i> or <i>Ophiostoma minus</i>. Growth rates were highest on <i>B. cinerea</i>, the most widely used fungus to maintain lab cultures of <i>B. xylophilus</i>, but <i>O. minus</i> has the advantage of being a natural associate and putative diet of wild populations. There remains a need for better understanding of how to obtain and maintain laboratory populations of this pathogen such that their natural characteristics are maintained.