Molecular, Cultural and Pathogenic Characterization of Neopestalotiopsis rosae from Strawberry in Mexico

Leaf spot and crown rot, caused by Neopestalotiopsis species, are emerging diseases of strawberry worldwide. This study aimed to: i) identify and characterize isolates of Neopestalotiopsis recovered from different strawberry organs; ii) assess their aggressiveness; and iii) evaluate the response of 13 strawberry varieties against the most common species present in Mexico. Four hundred ninety-eight isolates were obtained from leaves, roots, and crowns in central Mexico (Michoacan, Guanajuato, Puebla, and Mexico states) from 2020 to 2021. A subsample of 160 isolates was characterized by a High-Resolution Melting (HRM) assay, and 26 were identified by multilocus phylogenetic analysis (MLPA) based on ITS, tef1, and β-tub partial genes, along with morphological characterization. Incubation period (IP) and the time to reach 100% leaf severity were evaluated in detached leaf assays. The response of 13 varieties was also assessed using detached leaflet assays. HRM analysis grouped the isolates into three categories: N. rosae (n = 156), Neopestalotiopsis sp. (n = 1), and other Neopestalotiopsis species (n = 3). The MLPA revealed that 22 isolates were N. rosae, one was Neopestalotiopsis sp., one was Pestalotiopsis sp., and two were near N. iranensis. Isolates with a 12-h IP (11%) reached 100% severity within 6–7 days after inoculation (DAI), and those with a 24-h IP (57%) within 8–10 days; both groups were considered aggressive. In contrast, isolates requiring more than 48 h IP (32%) took 11–14 days to reach 100% severity and were considered non-aggressive. 'Marisol' was the least susceptible cultivar, whereas 'Florida Fortuna', 'Albion', 'Strawberry Festival', and 'Fronteras' were the most susceptible. These findings clarify the diversity and aggressiveness of Neopestalotiopsis spp. and support management and breeding efforts.