Planta

, 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 Figueiredo
Original Article

Abstract

Main conclusion

Co-cultures ofPinus pinasterwithBursaphelenchus xylophiluswere 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.

Keywords

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

Abbreviations

BAP

6-Benzylaminopurine

DAI

Days after inoculation

EPPO

European and Mediterranean Plant Protection Organization

GC

Gas chromatography

GC–MS

Gas chromatography coupled to mass spectrometry

IBA

Indole-3-butyric acid

LM

Light microscopy

PAS

Periodic acid–Schiff’s reagent

PWD

Pine wilt disease

PWN

Pinewood nematode (Bursaphelenchus xylophilus)

RI

Retention index

RWC

Relative water content

SEM

Scanning electron microscopy

SH

Schenk and Hildebrandt medium

SHe

Schenk and Hildebrandt elongation medium

SHm

Schenk and Hildebrandt multiplication medium

t

Trace

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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
  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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