Multiple locus genealogies and phenotypic characters reappraise the causal agents of apple ring rot in China

Apple ring rot inflicts severe economic losses in the main apple producing areas of East Asia. The causal agent of the disease has been variously identified as Macrophoma kuwatsukai, Physalospora piricola and Botryosphaeria berengeriana f. sp. piricola, although B. dothidea is currently the most widely accepted pathogen name. The taxonomic uncertainty has delayed research that is needed to manage effectively this destructive disease. In the present study, genealogical concordance phylogenetic species recognition (GCPSR) was applied to pathogenic fungal isolates from apple and pear from several locations in China, along with several reference isolates. Phylogenetic results based on sequences of four nuclear loci (ITS, EF-1 alpha, HIS and HSP) revealed the existence of two species within the examined isolates. One includes an ex-epitype isolate of B. dothidea and the other includes an isolate that was previously designated as B. berengeriana f. sp. piricola. Morphologically, the latter taxon presented an appressed mycelial mat on PDA whereas B. dothidea displayed columns of aerial mycelia reaching the lids, and conidia of the latter species were longer than B. dothidea. Botryosphaeria dothidea had a faster growth rate than the latter taxon under relatively high temperatures. Pathogenicity tests showed that on pear stems the latter taxon caused large-scale cankers along with blisters whereas B. dothidea was non-pathogenic, but on apple shoots the two fungi induced large and small wart-like prominences, respectively. Overall, this cryptic species demonstrated sufficient genetic variations and biological differences from B. dothidea. As a result of taxonomic study, we described here the latter taxon in a new combination, Botryosphaeria kuwatsukai and designate an epitype. Both B. kuwatsukai and B. dothidea are considered to be the main causal agents for apple ring rot in China and Japan.