Open AccessTesting new PCR primers and a TaqMan™ probe for detection of <i>Phlyctema vagabunda </i>syn. <i>Neofabraea alba </i>
Author(s) -
Michelle J. Vergara,
I.P.S. Pushparajah,
John F. Mackay,
K.R. Everett
Publication year - 2019
Publication title -
proceedings of the new zealand weed and pest control conference/new zealand plant protection/proceedings of the ... national weeds conference/proceedings of the new zealand weed control conference/proceedings of the new zealand plant protection conference
Language(s) - English
Resource type - Journals
eISSN - 0370-2804
pISSN - 0370-0968
DOI - 10.30843/nzpp.2019.72.309
Subject(s) - taqman , biology , polymerase chain reaction , microbiology and biotechnology , real time polymerase chain reaction , ribosomal dna , gene , genetics , phylogenetics
Phlyctema vagabunda syn. Neofabraea alba is a fungal pathogen that causes bull’s eye rot (BER) of apples. Polymerase chain reaction (PCR) primers complementary to the inter-transcribed spacer region of ribosomal DNA (ITS) and the β-tubulin gene region, and a TaqMan™ probe assay were developed to detect this pathogen. These assays were compared in quantitative PCR (qPCR) reactions for amplification of DNA extracted from several fungal species and from apple tissue. Although the ITS and the β-tubulin primers amplified all N. alba isolates, both primers also amplified a few other fungal species. The TaqMan™ probe used with published primers for N. alba only amplified N. alba isolates. The TaqMan™ assay resulted in the lowest crossing threshold (Ct) values for DNA extracted from apple fruit, leaves, and spores collected on cellophane from eight apple orchards. The TaqMan™ results were correlated with percentage BER (%BER) in a 400-apple sample harvested from the same orchards. The TaqMan™ probe assay was the most sensitive and specific qPCR protocol tested, and Ct values showed the best correlation with %BER.