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Abstract

Biology

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

Published: September 27th, 2024

DOI:

10.3791/67149

1INIAV, I.P., National Institute for Agrarian and Veterinary Research, I.P., 2GREEN-IT Bioresources for Sustainability, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa (ITQB NOVA), 3Centre for Environmental and Marine Studies (CESAM Lisboa), Biotecnologia Vegetal (BV), DBV, Faculdade de Ciências da Universidade de Lisboa, 4Centre for Ecology, Evolution and Environmental Changes (CE3C), Biotecnologia Vegetal (BV), DBV, Faculdade de Ciências da Universidade de Lisboa, 5Hercules Laboratory & Chemistry and Biochemistry Department of Science and Technology School, University of Évora

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.

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