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

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. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license

Identification of sequence mutations in Phytophthora cactorum genome associated with mefenoxam resistance and development of a molecular assay for the mutant detection in strawberry (F. × ananassa)

Abstract Phytophthora crown rot (PhCR) caused by Phytophthora cactorum is one of the most damaging diseases of strawberry worldwide. Mefenoxam is one of the major fungicides currently used to manage PhCR. However, the emergence and spread of resistant isolates have made controlling the pathogen in the field problematic. In the present study, using whole genome sequencing analysis, mutations associated with mefenoxam-resistant isolates were identified in six different genomic regions of P. cactorum. The 95.54% reads from a sensitive isolate pool and 95.65% from a resistant isolate pool were mapped to the reference genome of P. cactorum P414. Four point mutations were in coding regions while the other two were in noncoding regions. The genes harboring mutations were functionally unknown. All mutations present in resistant isolates were confirmed by sanger sequencing of PCR products. For the rapid diagnostic assay, SNP-based high-resolution melting (HRM) markers were developed to differentiate mefenoxam-resistant P. cactorum from sensitive isolates. The HRM markers R3-1F/R3-1R and R2-1F/R2-1R were suitable to differentiate both sensitive and resistant profiles using clean and crude DNA extraction. None of the mutations associated with mefenoxam resistance found in this study were in the RNA polymerase subunit genes, the hypothesized target of this compound in oomycetes. Our findings may contribute to a better understanding of the mechanisms of resistance of mefenoxam in oomycetes since serves as a foundation to validate the candidate genes as well as contribute to the monitoring of P. cactorum populations for the sustainable use of this product