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Direct quantitative real‐time PCR assay for detection of the emerging pathogen Neonectria neomacrospora
Author(s) -
Nielsen Knud N.,
Thomsen Iben M.,
Hansen Ole K.
Publication year - 2019
Publication title -
forest pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.535
H-Index - 49
eISSN - 1439-0329
pISSN - 1437-4781
DOI - 10.1111/efp.12509
Subject(s) - biology , pathogen , dna extraction , real time polymerase chain reaction , polymerase chain reaction , internal transcribed spacer , dna , botany , microbiology and biotechnology , gene , genetics , phylogenetic tree
The epidemic outbreak in northern Europe of Neonectria neomacrospora, the causal agent of dieback in Abies spp., led the European and Mediterranean Plant Protection Organization (EPPO) to include the pathogen on its alert list in 2017. Effective monitoring of this pathogen calls for a rapid and sensitive method of identification and quantification. A probe‐based real‐time PCR (qPCR) assay based on the β‐tubulin gene was developed for the detection and quantification of N. neomacrospora in infected wood samples, and directly for ascospores. This study presents the first published species–specific molecular detection assay for N. neomacrospora . The analytical specificity was validated on taxonomically closely related fungal species as well as on 18 fungal species associated with the host ( Abies s p.). The analytical sensitivity was tested on naturally infected wood, on purified pathogen DNA in a matrix of host DNA and on N. neomacrospora ascospores for detection of airborne inoculum. The latter was tested both with a DNA extraction step prior to qPCR and without DNA extraction by direct qPCR on collected ascospores. The assay was specific to N. neomacrospora , with a sensitivity of 130 fg purified DNA, or 10 ascospores by direct qPCR. Omitting DNA extraction and amplifying directly on unpurified ascospores improved assay sensitivity significantly.