Differentiation of Stemphylium vesicarium from Stemphylium botryosum as causal agent of the purple spot disease on asparagus in Germany

Abstract

The purple spot disease of asparagus is the most important disease in German asparagus growing regions. Two different Stemphylium species, S. vesicarium and the closely related species S. botryosum, are described as causal pathogens. Because of the strong phenotypical similarities the morphological differentiation is very difficult. Therefore the development of a suitable alternative to distinguish these species is an important need. The aim of this study was to develop a molecular and genetic based differentiation method for S. vesicarium and S. botryosum, and to analyze asparagus samples from Germany with this method to identify the prevalent causal agent of the purple spot disease in Germany. The sequences of three different DNA-markers were compared to get the most appropriate basis. Additionally to the commonly used ITS regions, parts of the protein-coding genes gapdh (glyceraldehyde-3-phosphate-dehydrogenase) and cytochrome b were analysed. The most significant difference between the two species was a 3 kb intron present in the S. botryosum cytochrome b region but not in S. vesicarium. This difference showed to be suitable for the distinction of these two Stemphylium species by a simple PCR-reaction. In addition to the qualitative analysis, the frequencies of these species were detected directly from asparagus field samples with the help of qPCR. In all german samples collected in 2010, 2011, 2013 and 2014 only S. vesicarium could be identified.