Morphological and genetic analyses of hellebore leaf spot disease isolates from different geographic origins show low variability and reveal molecular evidence for reclassification into Didymellaceae

Abstract

Hellebore leaf spot, caused by Coniothyrium hellebori, is the most common fungal disease of Helleborus species not only in botanical and ornamental gardens but also in nurseries. To correct the current lack of knowledge regarding this widely distributed pathogen, this study investigated 25 C. hellebori isolates collected from different countries in North America and Europe, primarily Germany. The morphology, pathogenicity and molecular genetic relationships on the basis of random amplified polymorphic DNA (RAPD) of these isolates were studied. RAPD primers produced a total of 394 bands, of which 40% were polymorphic. Genetic distances were calculated, and a dendrogram with bootstrap analysis was constructed by the unweighted pair group method with arithmetic mean (UPGMA) cluster method. All isolates were identified as C. hellebori, the causal agent of the disease. Two C. hellebori subclades were found, which could not be correlated with the geographic origin of the isolate, but with the plant host species and morphological characteristics. Sequence comparisons of the large subunit and internal transcribed spacer loci between C. hellebori and sequences from GenBank revealed that C. hellebori has to be grouped into the Didymellaceae family and rather belongs to Phoma or Microsphaeropsis than to Coniothyrium. This work represents the first study of this plant pathogen causing severe damage in Helleborus stocks and provides important information for the development of future Helleborus resistance breeding strategies. © 2011 Blackwell Verlag GmbH.