Molecular Diagnostic Assay for Detection of the Butternut Canker Pathogen Sirococcus clavigignenti-juglandacearum

Abstract

Butternut canker, caused by the fungal pathogen Sirococcus clavigignenti-juglandacearum, is present throughout the range of butternut (Juglans cinerea) and is the primary cause for its decline. A quick and reliable method for identification of S. clavigignenti-juglandacearum would provide a valuable tool for the detection of the pathogen on propagative material to avoid spread, as well as assist studies targeted at the epidemiology of this pathogen, in particular the dissemination of the pathogen by seeds of the butternut. The objective of this study was to develop a diagnostic assay to detect S. clavigignenti-juglandacearum in butternut plant tissue. The primers were developed using an alignment of internal transcribed spacer (ITS) sequences from isolates of S. clavigignenti-juglandacearum and several closely related species. These primers were tested on J. cinerea, 48 isolates of S. clavigignenti-juglandacearum recovered from diseased trees, and 26 species of other fungi recovered from butternut tissue. The primers amplified a product from the DNA of all isolates of S. clavigignenti-juglandacearum, detected its DNA at a concentration as low as 1 pg/μl, and detected the pathogen at a concentration of 1 × 103 spore/ml. The primers developed in this study will be a valuable tool for the detection of S. clavigignenti-juglandacearum present on butternut seeds, and as a rapid diagnostic tool for early detection of the pathogen on butternut trees.