First Report of Gray Leaf Spot caused by Pyricularia oryzae on Zoysia japonica in the United States of America

Gray leaf spot (GLS) is a damaging disease of perennial ryegrass, tall fescue, and St. Augustinegrass (Harmon, 2023). Zoysiagrass samples exhibiting GLS-like symptoms were collected from ‘Zenith’ zoysiagrass in Raleigh (KL027603) and from an experimental line of zoysiagrass in Rolesville, NC (KL027571) in late July 2022. Symptoms included oval or round lesions, tan color, and a dark brown border (Fig. 1A). Over time, the affected leaves exhibited dieback from the tip, and significant damage was observed. Temperatures in the Raleigh area averaged 26°C in July 2022 and local precipitation was greater than average. Zoysiagrass is an important turfgrass for golf course fairways and home lawns in the transition zone and Southeastern US. Thus, knowledge of a new host for a devastating disease such as GLS is critical. Following incubation of affected zoysiagrass in a humid environment at 23°C for 24 hours, portions of leaves with typical GLS symptoms were viewed microscopically, where numerous three-celled, pear-shaped conidia were observed (Fig. 1B). Symptomatic leaves were placed onto antibiotic-amended potato dextrose agar (PDA) containing 0.05 g L-1 of chloramphenicol, streptomycin, and tetracycline. After subculturing, 95% of all samples plated yielded colonies that matched colony descriptions for P. oryzae (Harmon, 2023, Ma and Uddin, 2009, and Nanayakkara, U. N. et al., 2008). Once pure cultures were obtained, actively growing isolates were transferred to oatmeal agar and grown at 22°C for 5 days. After 5 days, sterilized alfalfa stems were placed in the cultures to induce sporulation. Cultures were incubated at 26°C under consistent fluorescent light for 10 to 12 days. Spores were harvested by flooding the plates with 15 mL of distilled water, and conidia were scraped from the surface of the plates from ten isolates with a sterilized needle (Ma and Uddin, 2009, and Nanayakkara, U. N. et al., 2008). Five spores per isolate were measured using a micrometer embedded in a Zeiss AX10 Imager A1 compound microscope. Conidia were hyaline, pyriform in shape, two-septate, and averaged 8.5 ± 1.2 (wide) x 27.3 ± 6.3 (length) μm, consistent with the descriptions presented in Harmon (2023). The ITS1, 5.8S, and ITS2 region along with the actin gene from two zoysiagrass isolates were amplified by polymerase chain reaction using fungal ITS5/4 primers (KL027603 / KL027571 - 509 bp) and ACT-512F/ACT-783R (KL027603 - 240 bp / KL027571 - 249 bp)(Couch and Kohn 2002, and White et. al 1990). These sequences were uploaded to NCBI GenBank under accession numbers (KL027603 rRNA: PV477962, KL027603 ACT: PV495209; KL027571 rRNA: PV477982, KL027571 ACT: PV495208). Nucleotide blasting resulted in ≥99% sequence identity to Pyricularia oryzae Cavara accession CBS:365.52. (ITS1,5.8S,ITS2: Genbank accession MH857082.1)(ACT: Genbank accession KM485191.1). The rRNA region and actin gene were concatenated, and a clustal omega alignment was performed. A Kamura-Nei neighbor joining tree with 1,000 bootstraps was generated and visualized in Geneious Prime (Kamura et al 2004) (Fig. 2). KL027603 and KL027571 clustered with P. oryzae with 100% bootstrap support, providing more evidence that these isolates are P. oryzae. Koch’s postulates were performed on ‘Zenith’ zoysiagrass plants established from seed. Pots containing sterilized potting mix were seeded with ‘Zenith’ zoysiagrass and allowed to grow and develop for 7 weeks prior to inoculation. All ten isolates of P. oryzae were plated onto PDA and allowed to grow at 26°C for three days. After 3 days of growth, agar plugs from the edge of the colonies were transferred into sterilized rye grain to produce inoculum. Five infested rye grains were placed in the canopy of ‘Zenith’ zoysiagrass and kept moist by putting them in a clear plastic bin in the greenhouse, where temperatures ranged from 23-28°C. Symptoms developed 5 to 7 days after inoculation and were consistent with those described above and those attributed to GLS on other turfgrasses. We successfully isolated P. oryzae from the affected tissue in the pathogenicity tests. To our knowledge, this is the first report of GLS disease on zoysiagrass in the United States.