First report of Puccinia glechomatis, a rust fungus of creeping charlie, in Minnesota.

In 2020 and 2021, at four locations around the Twin Cities, Minnesota (Sep. 2021: 44.980, -93.319; Aug 2021: 44.989, -93.186; Sep 2020: 45.000, -93.138; Sep. 2020: 44.870, -92.779), a rust fungus was observed infecting creeping charlie (Glechoma hederacea), a weed of Eurasian origin (Hutchings and Price, 1999). Within stands, severity ranged from 1 to 30 % leaf loss. Telia were reddish-brown when young, starting small (0.1 to 0.5 mm), and growing into round but irregular sori measuring 1 to 5 mm, sometimes coalescing to form larger sori (Fig 1A). Sori are primarily abaxial, forming depressions on the adaxial surface, and sometimes occurring along stems and leaf petioles. Partial leaf necrosis occurs with high foliar infection and leaf dieback with high infection of petioles. Thin-walled, colorless leptosporic teliospores, or leptospores, (Fig 1B) were present in samples. Dark, thick-walled teliospores were not noted. Leaves bearing leptospores were soaked in water for four hours and suspended over young plants in a dew chamber at 20 °C for 16 hours. Plants were maintained in a greenhouse at 20-22 °C. After three weeks, nascent telia were observed on inoculated plants. The ITS region and a segment of EF1-α were selected for sequencing for the samples from the first two listed locations. For the ITS, the primers ITS1rustF10d (Barnes and Szabo, 2007) and ITSRu1 (Rioux et al., 2015) were used, and for EF1-α, a reaction was conducted following van der Merwe (2007). Amplicons were barcoded and sequenced on an Oxford Nanopore MinIon following manufacturer instructions (kits EXP-PBC001 and SQK-LSK109 with R9 flow cells). Reads (PRJNA802185) were filtered for quality (> Q16), sorted and separated by length, and aligned. A consensus sequence was generated for each amplicon with >50x coverage. BLAST searches of the EF1-α sequences OM489402 and OM489403 from the first two locations respectively showed 99.3 % (643/647) and 99.7 % (645/647) similarity with Puccinia glechomatis (EF560587). ITS sequences had not been reported for P. glechomatis, and there are no matches with >96 % homology in GenBank for the sequences OM470970 or OM470969, from the same samples. Morphological and life cycle traits are consistent with this identification. Creeping charlie is a common weed of turf, gardens, orchards, forests, and meadows. It is present in 46 of the lower 48 United States but is most common east of the Great Plains, in the Pacific Northwest, and in neighboring regions of southern Canada (Böllman and Scholler, 2004). P. glechomatis was recorded for the first time in 1992 in north-central Pennsylvania (Böllmann and Scholler, 2006). Examination of herbarium specimens and surveys established its distribution across the eastern U.S. and in a small area in the Pacific Northwest by the early 2000s, and in 2001 its presence was recorded in southern Wisconsin (Böllmann and Scholler, 2006). The basidiospores likely do not travel far, but the fungus may move long distances through plant matter and establish in new locations (Böllmann and Scholler, 2006). P. glechomatis is not known to affect native plants and may have a positive ecological effect, reducing the vigor of its undesirable host. To our knowledge, this is the first report of P. glechomatis in Minnesota. It is evidence of the continued westward spread of this rust in North America (Böllmann and Scholler, 2006). Sequenced samples were submitted to the Arthur Fungarium at Purdue University (PUR N24012 and PUR N24013, respectively).