Real-time PCR detection and discrimination of the Ceratocystis coerulescens complex and of the fungal species from the Ceratocystis polonica complex validated on pure cultures and bark beetle vectors

Abstract

Eight Ceratocystis Ellis & Halst. species belonging to the Ceratocystis coerulescens complex are pathogens causing blue-stain on Pinaceae. Three of these species, namely C. polonica, C. laricicola, and C. fujiensis, are particularly aggressive and can cause tree mortality. Although currently absent from the North American landscape, they are considered a significant potential threat to the Canadian boreal forest. As they are difficult to distinguish from native North American species belonging to the C. coerulescens complex, we developed a real-time PCR detection test for each of the three species to detect the equivalent of one fungal spore directly from insect vectors. DNA from at least one species of the C. coerulescens complex was detected on 86% of the beetles (Ips typographus (Linnaeus, 1758) and Ips cembrae (Heer, 1836)), whereas C. polonica DNA was detected on 60% of the I. typographus and C. laricicola DNA was detected on 84% of the I. cembrae. Between 20 and 344 225 spore equivalents were detected on the beetle specimens, and no inhibition effect of DNA extract from environmental samples was observed. These molecular detection tools will allow for rapid and reliable detection of C. polonica, C. laricicola, or C. fujiensis, allowing for a rapid implementation of eradication measures in case of introduction into Canada.