FIRST REPORT OF BENIOWSKIA SPHAEROIDEA LEAF BLIGHT, SWITCHGRASS MOSAIC AND TILLETIA MACLAGANII (HEAD SMUT) ON SWITCHGRASS IN INDIANA Gail E Ruhl (1); Tom Creswell (1); Sara Bratsch (4)Brooke Stefancik (1); Megan Romberg (2); Shawn Kenaley (3); Gary Bergstrom (3); Bong-Suk Kim (1); Chris Speers (1); Keith Johnson (1)

(1) Purdue University, West Lafayette, IN. (2) USDA-APHIS-PPQ,-Beltsville, MD. (3) Cornell University, Ithaca, N.Y. (4) University of Minnesota, St. Paul, MN

Introduction Beniowskia sphaeroidea Switchgrass Mosaic Virus Switchgrass ( L.), a native, perennial C4 grass found across North America has been used as a forage for grazing livestock, food and shelter for wildlife and soil conservation. From 2012-2017, a Purdue research initiative focused on utilizing switchgrass as a herbaceous energy crop (HEC). When unusual signs and symptoms were noted on switchgrass growing in the HEC plots, samples were collected and submitted to the Purdue Plant and Pest Diagnostic Lab (PPDL) for diagnosis. In 2015, random placement of white ‘cottonball-like’ mounds of fungal mycelium on switchgrass leaf surfaces of a ‘Sunburst’ cultivar were observed during late elongation and early reproductive growth stages (Fig 1a). Official microscopic, morphological confirmation of the causal fungus, Beniowskia sphaeroidea (Fig 1b), a fungus not previously reported in Indiana, was provided by the Department of Agriculture-Animal Plant Health Inspection Service in Beltsville, MD. In 2016, unusual elongated flecking was submitted on switchgrass Figure 1b Figure 2b leaves (Fig 2a). Using transmission electron microscopy (TEM)(Fig 2b), PCR (Fig 2c) and Sanger sequencing, Switchgrass mosaic virus (SwMV) was detected for the first time in Indiana . In 2017, rusty-colored spore masses on switchgrass spikelets were identified using teliospore morphology, PCR amplification and Sanger sequencing as Tilletia maclaganii (head smut), a first report on switchgrass in Indiana.(Fig 3a,3b)

Impact Head smut (T. maclaganii) has been reported to be Figure 1a Figure 2a Figure 2c responsible for decreasing the yield of switchgrass (Thomsen et al., 2008). Further studies will be required to Beniowskia sphaeroidea Switchgrass mosaic virus verify the potential yield impact of Beniowskia and SwMV Signs of Beniowskia sphaeroidea on Panicum virgatum leaves included scattered to gregarious cream- White, streak-like leaf patterns on switchgrass foliage (Fig 2a) tested serologically with regard to use of switchgrass as an colored sporodochia, 0.5 to 3mm long, elongated along the veins and emerging from a central hyphal negative for the presence of Sugarcane mosaic virus, Panicum mosaic virus and all other herbaceous energy crop (HEC). attachment (Fig 1a). Sporodochia were often associated with dark streaks along the veins. In the in Agdia’s wheat virus screen. sporodochia, straight to apically coiled, septate, hyaline conidiophores (4-6µm wide) produced conidia on TEM examination of partially purified extracts from symptomatic switchgrass leaves To the best of our knowledge this is the first report of 1-2µm wide denticulate scars(Fig 1b) Conidia were globose, 10-12 X 9-11 µm, (n= 30), with flat basal scars. identified only spherical virus-like particles present (Fig 2b) which matched the size Tilletia maclaganii, Beniowskia sphaeroidea (Farr and Anastomoses were often observed in the sporodochial hyphae, conidiophores and conidia. The taxonomic (30nm) and shape of marafiviruses. These viruses contain empty virus shells penetrated Rossman 2018) and SwMV , on switchgrass in Indiana. placement of Beniowskia has long been debated. Sequences were generated for the large subunit (LS), by stain and intact particles that are not penetrated by stain. small subunit (SSU) (partial) and ITS regions and analyzed via BLASTn in GenBank. The highest similarity for This project was supported by Agriculture and Food the LS sequence was 94.69% to KX287253, Teratoramularia persicariae isolate CBS 195.27. The highest Reverse-transcriptase polymerase chain reaction (RT-PCR) was used to test symptomatic Research Initiative Competitive Grant 2011-68005-30411 similarity for the SSU sequence was 98.80% to EF137360, Capnobotryella renispora isolate CBS 214.90. The and asymptomatic switchgrass for the presence of virus. Expected PCR amplicons (638 from the USDA National Institute of Food and Agriculture ITS sequence also showed similarity to sequences of Teratoramularia, but the highest similarity was bp) (Fig 2c) were detected in fresh, symptomatic, infected (I) and dried infected (DI) leaf and by USDA-NPDN Funds. 91.29%. Based on these results Beniowskia sphaeroidia likely shares an evolutionary history with members tissue collected from different symptomatic switchgrass samples but were not detected of the Capnodiales but more work needs to be done to determine its precise phylogenetic placement. in healthy (H) leaf tissue. Purified PCR products were sequenced. Pairwise comparison of the protein sequence with other homologous sequences available in GenBank matched the switchgrass isolate from Illinois (JF727261.2) with the highest nucleotide References Tilletia maclaganii identity of 98.08%. To our knowledge this is the first report of SwMV in Indiana. Farr, D. F., and Rossman, A. Y. 2018. Fungal Databases, Syst. Microscopic examination of cross-sectioned, rusty-colored spikelets (Fig 3a) revealed ovaries replaced with Mycol. Microbiol. Lab., ARS, USDA. Retrieved 29 May 2018 fungal sori consistent with a smut fungus. Teliospores (N=50 per specimen) measured 16.5–29.6 (mean= from https://nt.ars-grin.gov/fungaldatabases/ 21.8± 0.4 µm) x 15.1–25.4 (mean= 20.1± 0.4 µm), were variable in shape from sub-globose to ovoid, Tilletia maclaganii Layton, C. N. and G. C. Bergstrom. 2011. Plant Dis. 95:1587. ellipsoidal, or partially irregular, and pale yellow to orange-brown or orange-yellow in color with a thick, https://doi.org/10.1094/PDIS-05-11-0401 finely verrucose cell wall (1.7–3.8 µm; mean thickness= 2.7± 0.1 µm) that appeared serrate in profile (Fig Thomsen, P. M., et al. 2008. Plant Health Prog. Online. 3b). Based on characteristic teliospore morphology, the fungus was identified as the head smut fungus, https://doi.org/10.1094/PHP-2008-0317-01-RS Tilletia maclaganii Clinton (Vánky, 2012). Vánky, K. 2012. Page 922 in: Smut Fungi of the World. American Phytopathological Society Press, St. Paul, MN. Morphological determination was confirmed by DNA analysis of the ITS region utilizing DNA extracted from [Google Scholar] teliospores from single spikelets and the primer-pair ITS5 and ITS4. Teliospores from each of the three Wang et al. 2019. Redefining Humicola sensu stricto and specimens yielded an identical ITS sequence (674 nucleotides; GenBank Tilletia_maclaganii_17-928 related genera in the Chaetomiaceae. Studies in Mycology MH256490, Tilletia_maclaganii_17-994 MH256491, Tilletia_maclaganii_17-1055 MH256492) and, following 93:65-153 a BLASTn search, shared 100% nucleotide identity (638/638 nucleotides) to that of the ITS sequence for T. maclaganii (JF745116) isolated from the switchgrass cultivar ‘Shelter’ in New York State (Layton and Bergstrom, 2011). Figure 3a Figure 3b