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In this study, 3,426 grafted grapevines ready to be planted from 15 grapevine nursery fields in Northern Spain were inspected from 2016 to 2018 for black-foot causing pathogens. In all, 1,427 isolates of black-foot pathogens were collected from the asymptomatic inner tissues of surface sterilized secondary roots and characterized based on morphological features and DNA sequence data of the nuclear ribosomal DNA-internal transcribed spacer region, histone H3, translation elongation factor 1-alpha and β-tubulin genes. Eleven species belonging to the genera Dactylonectria, Ilyonectria, Neonectria, and Thelonectria were identified, including Dactylonectria alcacerensis, D. macrodidyma, D. novozelandica, D. pauciseptata, D. torresensis, Ilyonectria liriodendri, I. pseudodestructans, I. robusta, Neonectria quercicola, Neonectria sp. 1, and Thelonectria olida. In addition, two species are newly described, namely D. riojana and I. vivaria. Twenty-four isolates representing 13 black-foot species were inoculated onto grapevine seedlings cultivar ‘Tempranillo’. The pathogenicity tests detected diversity in virulence among fungal species and between isolates within each species. The most virulent species was D. novozelandica isolate BV-0760, followed by D. alcacerensis isolate BV-1240 and I. vivaria sp. nov. isolate BV-2305. This study improves our knowledge on the etiology and virulence of black-foot disease pathogens, and opens up new perspectives in the study of the endophytic phase of these pathogens in grapevines.

Occurrence and Diversity of Black-Foot Disease Fungi in Symptomless Grapevine Nursery Stock in Spain