Convergent Evolution of C239S Mutation in Pythium spp. β-Tubulin Coincides with Inherent Insensitivity to Ethaboxam and Implications for Other Peronosporalean Oomycetes

Ethaboxam is a benzamide antioomycete chemical (oomicide) used in corn and soybean seed treatments. Benzamides are hypothesized to bind to β-tubulin, thus disrupting microtubule assembly. Recently, there have been reports of corn- and soybean-associated oomycetes that are insensitive to ethaboxam despite never having been exposed. Here, we investigate the evolutionary history and molecular mechanism of ethaboxam insensitivity. We tested the sensitivity of 194 isolates representing 83 species across four oomycete genera in the Peronosporalean lineage that were never exposed to ethaboxam. In all, 84% of isolates were sensitive to ethaboxam (effective concentration to reduce optical density at 600 nm by 50% when compared with the nonamended control [EC₅₀] < 5 μg ml⁻¹), whereas 16% were insensitive (EC₅₀ > 11 μg ml⁻¹). Of the insensitive isolates, two different transversion mutations were present in the 239th codon in β-tubulin within three monophyletic groups of Pythium spp. The transversion mutations lead to the same amino acid change from an ancestral cysteine to serine (C239S), which coincides with ethaboxam insensitivity. In a treated soybean seed virulence assay, disease severity was not reduced on ethaboxam-treated seed for an isolate of Pythium aphanidermatum containing a S239 but was reduced for an isolate of P. irregulare containing a C239. We queried publicly available β-tubulin sequences from other oomycetes in the Peronosporalean lineage to search for C239S mutations from other species not represented in our collection. This search resulted in other taxa that were either homozygous or heterozygous for C239S, including all available species within the genus Peronospora. Evidence presented herein supports the hypothesis that the convergent evolution of C239S within Peronosporalean oomycetes occurred without selection from ethaboxam yet confers insensitivity. We propose several evolutionary hypotheses for the repeated evolution of the C239S mutation.