Molecular characterization reveals three Neopestalotiopsis species causing strawberry disease outbreaks in Spain

The emergence of new fungal diseases is one of the major challenges affecting strawberry production worldwide. Among them, Pestalotiopsis -like species have been reported in multiple countries as causal agents of fruit rot, leaf spot, root rot, and crown rot. In recent years, these pathogens have caused numerous disease outbreaks in Spanish strawberry fields, threatening industry sustainability and requiring accurate identification for effective disease management. Phylogenetic characterization, based on internal transcribed spacer (ITS), elongation factor (TEF), and β-tubulin (TUB) regions, led to the identification of phylogenetically distinct groups in Spain: Neopestalotiopsis rosae , Neopestalotiopsis sp., and N. iranensis . Pathogenicity of the isolates was confirmed by inoculating two representative isolates of each species onto three strawberry cultivars, ‘Rociera FNM’, ‘Palmeritas’, and ‘Plared 15105’. Differences in aggressiveness were observed on leaf inoculations and Neopestalotiopsis sp. (Pe_50_1) was the most aggressive in leaf inoculation assays. However, there were no significant differences in aggressiveness among the isolates in crown inoculation tests. This study corrects previous misidentification of N. clavispora in Spain, confirming N. rosae as the predominant species affecting the Spanish strawberry industry. These findings represent the first report of Neopestalotiopsis sp. and N. iranensis causing leaf spot and crown rot in Spain, providing essential molecular tools for accurate pathogen diagnosis and targeted disease management strategies.