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Optimizing the quantification of the pine wood nematode (Bursaphelenchus xylophilus) in Pinus pinaster through real-time quantitative PCR
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Resumo(s)
Native to Europe, the maritime pine (Pinus pinaster) spreads through mainland Portugal. Multiple economic agents depend on its existence, namely for construction wood, or resin. As the second largest forest species in occupied area, the pine´s importance does not restrict to the economy: Pinhal do Rei is of cultural relevance, and the enourmous biotic chain dependent on the pine forests is extremely important to protect, specially considering the portuguese climatic regime, unsuitable for many species.
Besides forest fires, pests and diseases were also accountable for forest area reductions is the last years. Pine wilt disease (PWD) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, has been particularly damaging. The exotic parasite uses a native vector for dissemination, and is unchallenged while spreading through healthy trees. Measures to reduce dissemination and control the spread of the associated PWD are of utter importance for all Europe, and they must de supported by reliable quantification and detection methods of PWN.
Real-time quantitative PCR parameters were studied with samples from an experiment with three year-old inoculated pine seedlings, collected at five time-points. From 12 genes tested, two were considered adequate by conventional PCR. After PCR-product sequencing and validation, samples were tested in qPCR. A linear regression from knwon-concentration PWN samples created a calibration curve, while primer efficiency was obtained with a dilution series. BxITS2 was proved the most suitable gene, producing more reliable results when amplifying nematode and plant DNA altogether. The positive results of quantification on samples with parasite and plant DNA extracted simultaneously are very promising. Thus, we validated a direct extraction quantification method, but the methodology can still be improved to overcome identified weaknesses
Descrição
Mestrado em Engenharia Agronómica - Proteção de Plantas / Instituto Superior de Agronomia. Universidade de Lisboa
Palavras-chave
nematode Pinus pinaster qPCR regression quantification
Contexto Educativo
Citação
Gouveia, V.P. - Optimizing the quantification of the pine wood nematode (Bursaphelenchus xylophilus) in Pinus pinaster through real-time quantitative PCR. Lisboa: ISA, 2021, 83 p.