Detection and Species Differentiation of Neopestalotiopsis spp. from Strawberry (Fragaria × ananassa) in Florida Using a High-Resolution Melting Analysis

Abstract

Since 2017, a new Neopestalotiopsis sp. has been reported causing severe Pestalotia leaf spot (PLS) outbreaks in Florida. The threat of this new species has forced several strawberry growers to destroy their fields early in the season. The need for a fast and accurate diagnosis of PLS came from growers’ concerns and the fact that PLS symptoms can be easily confused with leaf spots caused by other pathogens. Moreover, although N. rosae can be isolated from symptomatic tissues, it is not as aggressive as the new Neopestalotiopsis sp. in Florida. Therefore, this study aimed to develop a high-resolution melting (HRM) assay to accurately detect and differentiate Neopestalotiopsis spp. associated with strawberry and validate this assay for its practical application in a plant diagnostic clinic. Two sets of primers were designed based on the partial beta-tubulin gene (β-tub) and by cross-comparison of their HRM results. This molecular assay was able to differentiate Neopestalotiopsis spp. into three groups: the new Neopestalotiopsis sp. associated with PLS outbreaks, N. rosae, and other Neopestalotiopsis spp. represented by several other species within the genus. The HRM assay had a detection limit of 10 pg of genomic DNA or 10 conidia and was found to be efficient and accurate to be implemented in our plant diagnostic clinic, allowing for a quick turnaround of results. [Figure: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Keywords

• diagnostic
• fungi
• HRM
• molecular
• strawberry
• techniques