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

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 ,o rC. fujiensis, allowing for a rapid implemen- tation of eradication measures in case of introduction into Canada. Resume : Huit especes de Ceratocystis Ellis & Halst. appartenant au complexe Ceratocystis coerulescens sont des agents phyto- pathogenes provoquant le bleuissement du bois chez les Pinaceae. Trois d'entre elles, C. polonica, C. laricicola et C. fujiensis, sont particulierement agressives et peuvent provoquer la mortalite chez les arbres. Bien qu'actuellement absentes du paysage nord-americain, elles sont considerees comme une menace potentielle importante pour la foret boreale canadienne. Etant difficiles adistinguer des especes indigenes appartenant au complexe C. coerulescens, nous avons developpe des tests de detection en PCR en temps reel permettant de detecter aussi peu qu'un equivalent de spores fongiques directement sur des insectes vecteurs. L'ADN d'au moins une espece du complexe C. coerulescens a ete detecte sur 86 % des coleopteres (Ips typographus (Linnaeus, 1758) et Ips cembrae (Heer, 1836)), alors que l'ADN de C. polonica a ete detecte sur 60 % des I. typographus et celui de C. laricicola sur 84 % des I. cembrae. Entre 20 et 344 225 equivalents spores ont ete detectes sur les specimens de coleopteres et aucun effet d'inhibition de l'extrait d'ADN provenant d'echantillons environnementaux n'a ete decele. Ces outils de detection moleculaire permettront une detection rapide et fiable de ces agents pathogenes en cas d'introduction et la mise en place rapide de mesures d'eradication. Mots-cles : Ceratocystis, detection moleculaire, agents pathogenes forestiers exotiques, qPCR, Ips.

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