New real‐time PCR assay for detecting the blueberry and cranberry twig blight pathogen

Abstract

Diaporthe vaccinii is a fungal pathogen of concern to Vaccinium species, responsible for twig blight disease. This fungus may survive in woody tissues without displaying symptoms, which makes the diagnostics more challenging. To provide a rapid and efficient diagnostic tool for D. vaccinii, a new and specific TaqMan real‐time PCR assay was developed using translation elongation factor 1 gene region. The assay was specific to D. vaccinii and did not cross‐react with any other tested Vaccinium pathogens. The sensitivity of the assay was found to be 1 pg of the pathogen DNA in water or in host DNA, which is equivalent to less than 20 hyphal cells. The assay was also used to test infected plant tissues, which determined that the assay can detect the pathogen from symptomatic as well as non‐symptomatic material. Furthermore, D. vaccinii was detected after mixing infected asymptomatic tissue with healthy stem tissues at a proportion of 1:10 by weight. To co‐amplify the D. vaccinii and the plant cytochrome oxidase gene as internal control in a single reaction, a duplex assay was successfully developed without any reduction in sensitivity. Inter‐laboratory testing of the assay showed consistent results, demonstrating that the developed assay is robust and transferrable to other laboratories.