, Volume 241, Issue 6, pp 1325–1336 | Cite as

In vitro co-cultures of Pinus pinaster with Bursaphelenchus xylophilus: a biotechnological approach to study pine wilt disease

  • Jorge M. S. Faria
  • Inês Sena
  • Inês Vieira da Silva
  • Bruno Ribeiro
  • Pedro Barbosa
  • Lia Ascensão
  • Richard N. Bennett
  • Manuel Mota
  • A. Cristina FigueiredoEmail author
Original Article


Main conclusion

Co-cultures of Pinus pinaster with Bursaphelenchus xylophilus were established as a biotechnological tool to evaluate the effect of nematotoxics addition in a host/parasite culture system.

The pinewood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), was detected for the first time in Europe in 1999 spreading throughout the pine forests in Portugal and recently in Spain. Plant in vitro cultures may be a useful experimental system to investigate the plant/nematode relationships in loco, thus avoiding the difficulties of field assays. In this study, Pinus pinaster in vitro cultures were established and compared to in vivo 1 year-old plantlets by analyzing shoot structure and volatiles production. In vitro co-cultures were established with the PWN and the effect of the phytoparasite on in vitro shoot structure, water content and volatiles production was evaluated. In vitro shoots showed similar structure and volatiles production to in vivo maritime pine plantlets. The first macroscopic symptoms of PWD were observed about 4 weeks after in vitro co-culture establishment. Nematode population in the culture medium increased and PWNs were detected in gaps of the callus tissue and in cavities developed from the degradation of cambial cells. In terms of volatiles main components, plantlets, P. pinaster cultures, and P. pinaster with B. xylophilus co-cultures were all β- and α-pinene rich. Co-cultures may be an easy-to-handle biotechnological approach to study this pathology, envisioning the understanding of and finding ways to restrain this highly devastating nematode.


Maritime pine Monoxenic culture Pinewood nematode Relative water content Shoots structure Volatiles 





Days after inoculation


European and Mediterranean Plant Protection Organization


Gas chromatography


Gas chromatography coupled to mass spectrometry


Indole-3-butyric acid


Light microscopy


Periodic acid–Schiff’s reagent


Pine wilt disease


Pinewood nematode (Bursaphelenchus xylophilus)


Retention index


Relative water content


Scanning electron microscopy


Schenk and Hildebrandt medium


Schenk and Hildebrandt elongation medium


Schenk and Hildebrandt multiplication medium





The authors would like to thank Dr. Célia Miguel (Forest Biotechnology, Instituto de Tecnologia Química e Biológica) for her technical advice, Eng. João Sanches (Mata Experimental do Escaroupim, Instituto da Conservação da Natureza e das Florestas) for supplying maritime pine seeds. M. Mota was partially supported by the EC 7th Framework project REPHRAME KBBE.2010.1.4-09. P. Barbosa and M. Mota also supported by Fundação para a Ciência e a Tecnologia (FCT) under PEst-C/AGR/UI0115/2011 and PEst-OE/AGR/UI0115/2014. Jorge Faria is grateful to FCT for the PhD grant SFRH/BD/43738/2008. This study was partially funded by FCT, under Pest-OE/EQB/LA0023/2011 and research contract PTDC/AGR-CFL/117026/2010.

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Jorge M. S. Faria
    • 1
  • Inês Sena
    • 1
  • Inês Vieira da Silva
    • 1
  • Bruno Ribeiro
    • 2
  • Pedro Barbosa
    • 2
  • Lia Ascensão
    • 1
  • Richard N. Bennett
    • 3
  • Manuel Mota
    • 2
  • A. Cristina Figueiredo
    • 1
    Email author
  1. 1.Universidade de Lisboa, Faculdade de Ciências de Lisboa, DBV, CESAM, Centro de Biotecnologia VegetalLisbonPortugal
  2. 2.NemaLab/ICAAM, Instituto de Ciências Agrárias e Ambientais MediterrânicasUniversidade de ÉvoraÉvoraPortugal
  3. 3.Universidade de Trás-os-Montes e Alto Douro, Quinta dos PradosVila RealPortugal

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