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).

First report of a rust fungus (Puccinia sp.) infecting lemongrass in Minnesota.

In July 2021 and July - Oct. 2022, in a community garden near Mankato, Minnesota, rust disease was observed on lemongrass (Cymbopogon citriatus). In 2022, all 20 plants in the garden plot were infected. Lemongrass is used in some Asian cuisines and for tea or medicine. It is not hardy in Minnesota but is grown in gardens and outdoors in small-scale production. Uredinia were cinnamon-brown on the abaxial surface of leaf blades. Pustules were small (0.2 - 0.5 x .01 - .05 mm) and numerous, causing large necrotic lesions and leaf dieback (Fig. 1A). Severity ranged from 5 - 50% leaf loss. Urediniospores were finely echinulate, slightly ovular (22-25 x 20-23 µm), thick-walled (2.5-4 µm), with 3-4 roughly equatorial, sometimes scattered germ pores (Fig 1B; 1C). Clavate paraphyses were abundant. Other spore types were not observed. The pycnidia of a mycoparasitic fungus were present within the uredinia. The specimen was submitted to the Arthur Fungarium at Purdue University (PUR N24011). Primers ITS1rustF10d (Barnes and Szabo, 2007) and ITSRu1 (Rioux et al., 2015) were used to generate amplicons for the rust fungus, and ITS4 and ITSF+ (White, 1990) for the mycoparasite. Amplicons were sequenced on an Oxford Nanopore MinIon with R9 flow cells following manufacturer instructions. Reads (PRJNA802078) were filtered for quality (> Q13) and length (> 200 bp), mapped to reference sequences, aligned, and separated based on similarity. Consensus sequences were generated for the amplicons of the rust fungus and of two other fungi. BLASTn searches of the ITS sequences, OM442990 and OM442991, identified an Alternaria sp. (99.8% match (597/598) with MT548677) and Sphaerellopsis filum (syn. Darluca filum; 98.3% (529/538 bp) match with EF600974), a common rust mycoparasite. A BLASTn search of the rust fungal ITS sequence (OM442989) yielded 98.9% (549/555) and 98.6% (633/642) match with MT955206 and MT955207, respectively, both Puccinia cesatii on Bothriochloa ischaemum. The third closest match is P. cymbopogonis on C. citriatus (97.1% (595/613) with KY764115). Urediniospore morphology is consistent with that of P. cesatii (Cummins, 1971). Available evidence suggests the fungus is P. cesatii or a closely related species. Puccinia cesatii has been reported infecting Cymbopogon spp. (Stevenson et al., 1926; Dhar and Rekha, 1984), but lemongrass is not generally considered a host-possibly due to confusion of P. cesatii with P. cymbopogonis, a closely related rust pathogen of lemongrass that is morphologically very similar to P. cesatii. P. cymbopogonis has not been reported in the U.S. Rust diseases of lemongrass have been reported in three states: Hawaii (Gardner, 1985), California (Koike and Molinar, 1999), and Florida (Ploetz et al., 2014). In each case, the rust was identified as Puccinia nakanishikii. Urediniospores of P. nakanishikii are larger (26-36 µm long) (Cummins, 1971) and the ITS2 has no significant sequence similarity. P. cesatii is widespread in Eurasia, the southwest U.S., and Mexico (Cummins, 1971). Cummins lists three genera closely related to Cymbopogon as telial hosts of P. cesatii: Bothriochloa, Capillipedium, and Dicanthium. He lists nine rust fungi that infect Cymbopogon but does not list P. cesatii. Of these nine species, only P. cymbopogonis is morphologically similar. Further research is needed to investigate the potential impact of rust fungi on lemongrass production and to elucidate phylogenetic relationships of rust fungi infecting lemongrass.

Puccinia coronata var. coronata, a Crown Rust Pathogen of Two Highly Invasive Species, Is Detected Across the Midwest and Northeastern United States.

Puccinia coronata var. coronata (Pcc) causes crown rust disease of glossy buckthorn (Frangula alnus) and reed canarygrass (Phalaris arundinacea), two highly invasive plant species in North America. Pcc is closely related to major pathogens of cereals, turfgrasses, and forage grasses. It occurs throughout Europe but was first recorded in North America in 2013. Where its hosts co-occur, such as in wetlands in the Twin Cities metro area in Minnesota, we have observed Pcc causing significant infection that results in defoliation and fruit loss in glossy buckthorns and premature leaf senescence in reed canarygrass. In this research, we mapped the distribution of this likely recently introduced rust fungus and provided a description of disease signs and symptoms and pathogen morphology. Samples were acquired by two primary means: by surveys in Minnesota and by correspondence with users of iNaturalist.org, a social network for nature enthusiasts and community scientists. With an Oxford Nanopore MinION, we sequenced two to four loci from 22 samples across 13 states and identified samples by phylogenetic analysis and sequence similarity. Notably, four pure isolates appear to have intragenomic variation of the ITS region. We found that Pcc is present throughout the range of glossy buckthorn in the eastern United States. In Minnesota, Pcc is not common outside the range of glossy buckthorn despite the presence of susceptible grass hosts.