Molecular identification and characterization of Elsinoë murrayae (Synonym: Sphaceloma murrayae) from weeping willow

Sphaceloma murrayae is a significant fungal pathogen of Salix spp. It causes greyish‐white leaf spots, which were reported worldwide except in China. Its morphological characteristics were described in the early literature; however, there is a lack of molecular information pertaining to this fungus. This study identified and characterized three fungal isolates that obtained from weeping willow leaf spots in China. Based on disease symptoms, morphological characteristics and single nomenclature rules for fungi, these isolates are proposed to be new combinations of Elsinoë murrayae (Synonym: S. murrayae). Phylogenetic analysis that combined internal transcribed spacer (ITS), large subunit (LSU), RBP2 and TEF1‐α DNA sequences indicated that E. murrayae isolates and Elsinoë salicina—another Elsinoë sp. isolated from Salix sp.—were distinguishable species. With trypan blue staining and stereomicroscopic observation, we found that large‐scale cell death occurred at 2 days postinoculation (dpi) and slight disease symptoms started at 3 dpi when the conidia were inoculated on Salix babylonica leaves. Pathogenicity analysis revealed that three isolates can successfully infect mature leaves of S. babylonica, Salix fragilis and Salix suchowensis, but not Salix matsudana. In addition, a necrosis‐ and ethylene‐inducing‐like proteins’ (NLP) gene, named EmNLP1, was cloned. The cytotoxicity of EmNLP1 was confirmed by transient assay in tobacco. During infection, EmNLP1 dramatically peaked at 2 dpi and maintained a high‐level expression in the necrosis lesion growing stage.