Comparison of methods to obtain and maintain cultures of the pinewood nematode, Bursaphelenchus xylophilus

ABSTRACT

The pinewood nematode Bursaphelenchus xylophilus, 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 B. xylophilus has been crucial to research on this important pathogen of pine trees. However, studies on the effects of in vitro 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 B. xylophilus lab populations from dauer larvae that have been isolated from pine sawyer beetles (Monochamus 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 B. xylophilus in the laboratory. Further, malt extract agar proved to be more suitable than potato dextrose agar. Once in culture, B. xylophilus grew well feeding on either of two fungus species: Botrytis cinerea or Ophiostoma minus. Growth rates were highest on B. cinerea, the most widely used fungus to maintain lab cultures of B. xylophilus, but O. minus 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.

KEYWORDS:

• Botrytis cinerea
• culture medium
• fungus
• Ophiostoma minus
• technique

Acknowledgments

We are grateful to Janel diBiccari and Alexandra Ricardo for help on laboratory work, and to Erich Vallery for trapping all pine sawyer beetles in Louisiana. We are also thankful to Robin M. Giblin-Davis and Dale R. Bergdahl for confirming nematode identification, and to two anonymous referees for their valuable comments and suggestions. This research was funded by the Forest Research Centre, a research unit funded by Fundação para a Ciência e a Tecnologia I.P. (FCT), Portugal (UIDB/00239/2020).

Disclosure Statement

No potential conflict of interest was reported by the authors.

Supplementary Data

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Additional information

Funding

This work was supported by the Fundação Luso-Americana para o Desenvolvimento [Sup FCT PTDC/AGR-CFL/098869/2008]; Fundação para a Ciência e a Tecnologia [DL 57/2016/CP1382/CT0009]; Fundação para a Ciência e a Tecnologia [SFRH/BPD/80867/2011]; Fundação para a Ciência e a Tecnologia [PTDC/AGR-CFL/098869/2008]; Fundação para a Ciência e a Tecnologia [SFRH/BPD/46995/2008].