First report of tar spot on corn caused by Phyllachora maydis in Kentucky.

In September 2021, signs of black circular to oval shaped fungal structures (stromata) were observed on corn (Zea mays L.) leaves on a non-commercial inbred line in Todd County, Kentucky. Signs were only observed in a small pocket within the larger field, with disease levels ranging from 1- 5% incidence and 1-25% severity on individual leaves affected in the field. Corn leaves had senesced and only fungal structures were available to aid in diagnosis. Microscopic examination of stromata uncovered ascomata within the clypei/stromata. Further examination of ascomata revealed multiple asci containing eight hyaline, uniseriate, aseptate, oval to ovoid ascospores ranging in size from 8 to 12 µm x 5 to 7 µm. Observed signs were consistent with published reports of tar spot caused by Phyllachora maydis (Parbery 1967; Valle-Torres et al. 2020). For molecular confirmation of the causal agent, corn leaves were surface sterilized in diluted bleach (10%) for 30 seconds and stromata were excised from the leaves using a sterile scalpel. Five to seven stromata were placed into each microcentrifuge tube. Liquid nitrogen was added to the microcentrifuge tubes and the frozen stromata were ground using a sterilized pestle. The ground stromata tissue was used for DNA extraction using a Synergy 2.0 plant DNA extraction kit (OPS Diagnostics, Lebanon, NJ). A portion of the internal transcribed spacer (ITS) region was amplified by PCR utilizing ITS-4 and ITS-5 primers. Amplicons were subjected to Sanger sequencing to obtain a consensus sequence. Using the BLASTn algorithm the consensus sequence shared 100% similarity to three P. maydis Genbank accessions: MG881848.1, MG8814847.1, MG881846.1. A representative sequence was deposited in GenBank (accession no. OQ034699.1). Due to P. maydis being an obligate parasite, Koch's postulates were not attempted.