P-476 - Resolving Taxonomy of Two Aggressive Neopestalotiopsis Lineages on Strawberry

In 2017, a Neopestalotiopsis sp. emerged causing leaf spot and fruit rot outbreaks in Florida, while N. rosae was linked to leaf spot and crown rot outbreaks in Mexico. These two groups were identified by sequencing ITS, β-tubulin, and EF1-α genes, but species delineation was unclear. Our objectives were to phenotype isolates from strawberry as aggressive or non-aggressive, and use whole-genome sequencing (WGS) to distinguish and clarify the taxonomy of Neopestalotiopsis sp. and N. rosae. A phylogeny using concatenated core genes revealed that isolates representing the two original 2017 outbreaks clustered separately and formed two lineages within N. rosae. Likewise, isolates aggressive on strawberry were placed into these two lineages, whereas other lineages contained only isolates non-aggressive on strawberry. Average Nucleotide Identity (ANI) showed >98.7% similarity between the seven observed lineages and the type isolate of N. rosae, while ANI values < 96.8% to type isolates from other species suggested a 97% threshold for species differentiation. Ten widely used housekeeping genes were selected, but none yielded monophyletic groupings for N. rosae lineages. Concatenated phylogenetic analyses using ITS, EF1-α, β-tubulin, ACT, GAPDH, HIS3, CAL, and RPB2 produced a tree with the same topology as WGS. These genes could be used to resolve taxonomic ambiguities within Neopestalotiopsis. Ongoing research aims to determine the biological implications of these findings, including markers for diagnosis, cultivar susceptibility across different lineages, and fitness of isolates.