Infection of In Vivo and In Vitro Pines with the Pinewood Nematode Bursaphelenchus xylophilus and Isolation of Induced Volatiles

Summary

The protocol describes the in vivo and in vitro pinewood nematode infection of Pinus pinaster and their volatilome analysis through Gas Chromatography (GC) and GC coupled to Mass Spectrometry (GC-MS).

Abstract

The pinewood nematode (PWN) is a phytoparasite that causes pine wilt disease (PWD) in conifer species. This plant parasitic nematode has heavily contributed to pine deforestation in Asian countries, e.g., Japan, China, and Korea. Over the last two decades, in Europe, Portugal and Spain have been greatly affected. Research on the mechanisms of PWN infection and/or PWD progression in susceptible host species relies on the controlled infection of pine seedlings under greenhouse conditions. This technique is laborious and mobilizes substantial economic and human resources. Additionally, it can be prone to variability that results from the genetic diversity associated with some pine species but also from the interference of external factors. As an alternative, in vitro co-cultures of pine with PWNs offer a more advantageous system for studying biochemical changes since they a) allow controlling single environmental or nutritional variables, b) occupy less space, c) require less time to obtain, and d) are free from contamination or from host genetic variation. The following protocol details the standard in vivo PWN infection of Pinus pinaster, the maritime pine, and the establishment of the novel in vitro co-cultures of pine shoots with the PWN as an improved methodology to study this phytoparasite influence on pine volatiles. PWN-induced volatiles are extracted from in vivo and in vitro infected pines by hydrodistillation and distillation-extraction, and the emitted volatiles are captured by solid phase microextraction (SPME), using fiber or packed column techniques.

Introduction

The pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Bührer 1934) Nickle 1970, is a plant parasitic nematode that mainly parasitizes Pinus species. This phytoparasite is vectored by insects of the genus Monochamus into trees of susceptible pine species during the insect's maturation feeding. The PWN kills the tree by attacking its resin canals and reducing resin flow, and by damaging its vascular tissue, causing interruptions in the water column. Lack of water at the tree canopy induces the first visible symptoms of pine wilt disease (PWD), i.e., the pine needles become chlorotic after the cessation of photosy....

Protocol

1. Growing in vitro pinewood nematode

NOTE: Pinewood nematodes are grown by feeding on the fungal mycelium of a non-sporulating strain of Botrytis cinerea (de Bary) Whetzel¹¹.

1. For routine sub-culture, transfer a culture plug (0.5 cm diameter) from the outermost border of the fungal colony onto a plate of sterile potato dextrose agar (PDA) and keep at 25 ± 1 °C for 7 to 10 days, or until the fungal colony .......

Discussion

The protocol presented here outlines an enhanced methodology to analyze volatile compounds in maritime pine infected by the PWN, where environmental and genetic variability is reduced and does not influence the outcomes. Using pure lines of in vitro maritime pine genotypes, extracted and emitted volatiles can be analyzed as a host response to one of the most damaging biotic threats to pine forests.

Maintenance of reference cultures or the growth of large amounts of PWNs is easily perf.......

Materials

Name	Company	Catalog Number	Comments
38 mesh test sieve	Retsch	60.131.000038
6-Benzylaminopurine (6-BAP)	Duchefa Biochemie	B0904
Charcoal activated	Duchefa Biochemie	C1302
Clevenger apparatus	WINZER Laborglastechnik	25-000-02
Hydrogen peroxide solution	Sigma-Aldrich	H1009-500ML
Indole-3-butyric acid (IBA)	Duchefa Biochemie	I0902
Likens-Nickerson apparatus	VitriLab LDA.	c/IN29/32
Microbox round containers	Sac O2	O118/80+OD118
n-Pentane	Sigma-Aldrich	1.00882
PARAFILM M sealing film	BRAND	HS234526B-1EA
Phyto agar	Duchefa Biochemie	P1003
Potato Dextrose Agar	BD DIFCO	213400
Scalpel blade no. 24	Romed Holland	BLADE24
Schenk & Hildebrandt Basal salt medium	Duchefa Biochemie	S0225
Schenk & Hildebrandt vitamin mixture	Duchefa Biochemie	S0411
SPME fiber assembly Polydimethylsiloxane (PDMS)	Supelco	57300-U
SPME Fiber Holder	Supelco	57330-U
Sucrose	Duchefa Biochemie	S0809
Tenax TA- stainless steel tubes- conditioned + capped	Markes International	C1-AAXX-5003