First Report of a Leaf Rust Disease on Intermediate Wheatgrass ( <i>Thinopyrum intermedium</i> ) Caused by <i>Puccinia recondita</i> sensu lato in the United States of America

Intermediate wheatgrass [IWG, Thinopyrum intermedium (Host) Barkworth &amp; D.R. Dewey] is a perennial grass species undergoing direct domestication for use as a perennial grain crop, marketed under the trade name Kernza® (Bajgain et al. 2020; DeHaan et al., 2018; Gutknecht et al. 2025; Wagoner, 1990; Wagoner &amp; Schaeffer, 1990). In June of 2025, rust infections were observed in the IWG germplasm nursery located at the St. Paul campus of University of Minnesota (Figure S1-A). Rust-colored pustules containing abundant urediniospores were observed mainly on the adaxial leaf surface. Microscopic examination indicated that the urediniospores were obovoid, ellipsoid, or globoid and measured 25 to 32 × 20 to 25 µm, with two to three germ pores on the surface of each spore (Figure S1-B). No telia were formed during the IWG growing season. To confirm the identity of this leaf rust pathogen, we sequenced the nuclear ribosomal rRNA internal transcribed spacer (ITS) region using the primers ITS1 and ITS4 (White et al. 1990), elongation factor 1 alpha (EF1-a) gene sequence using two sets of primers EF1-526F/EF1-1567R and EFbasidF/EFbasidR (van der Merwe et al. 2007), and beta-tubulin 1 (β-tub) gene segment using primers β-tub 1317F, β-tub 1442F and β-tub 2662bR (van der Merwe et al. 2007) for four single-pustule-derived isolates (K1, K2, K3, and K4), which were derived from leaf samples collected from four different IWG accessions in the germplasm nursery. All isolates are identical for the three gene sequences. The sequence data were deposited in NCBI GenBank (Accession Nos. PZ099283 for ITS, PZ135678 for EF1-a, and PZ135677 for β-tub). They shared the highest identities (99.55%, 99.02%, and 98.93%) with ITS (DQ417411), EF1-a (OZ387426) and β-tub (OZ387443) of P. triticina (= P. recondita f. sp. tritici), respectively, compared to the three gene sequences from other Puccinia species in the NCBI database. A urediniospore suspension at 1 × 108 spores/ml in Soltrol oil 170 was prepared from each of the four single-pustule-derived isolates (K1, K2, K3, and K4) and used to spray-inoculate 7 – 9 days old seedling plants of one IWG accession (MN1802-SYN2), one rye (Secale cereale L.) variety ('Prolific'), two barley (Hordeum vulgare L.) lines ('Aime' and 'Robust'), and four wheat (Triticum aestivum L.) genotypes ('Little Club', 'Thatcher', 'Line E', and 'Morocco'). After 10 to 14 days of incubation, rust pustules appeared on IWG MN180-SYN2, rye Prolific, and barley Aime, whereas only flecks or immune responses were observed on barley Robust and the four wheat genotypes, Little Club, Thatcher, Line E, and Morocco (Figure S1-C). The rust spores collected from the infected plants of IWG MN1802-SYN2 showed the same microscopic morphology as those collected from naturally infected IWG in the field nursery. No record of leaf rust closely related to P. triticina on Th. intermedium was found on USDA Fungal Databases (https://fungi.ars.usda.gov/). Also, no herbarium specimens of Thinopyrum intermedium with leaf rust infections were maintained in Canada and the United States based on MyCoPortal (https://www.mycoportal.org). Therefore, this is the first report of leaf rust caused by P. recondita in IWG. Further research is required to determine if it is a new Puccinia species or a subspecies or forma specialis (f. sp.) of P. recondita sensu lato. This leaf rust disease may pose a potential threat to IWG that is being grown as a perennial grain crop in Minnesota and other regions of the US.