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In NorthAmerica, Fusarium graminearum is the main causal agent of Fusarium head blight (FHB), an important wheat disease in the region. In 2015, severe and widespread FHB epidemics occurred in wheat in Nebraska, USA. Symptomatic wheat spike samples (20 to 30 spikes/sample) at the hard dough growth stage were collected from 23 fields (1 sample/field) in 15 counties representing all the major wheat growing regions in the state (southeast, south central, southwest, and northwest). DNA was extracted from single-spored isolates grown on PDA using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). A portion of the translation elongation factor 1-[alpha] (EF-1[alpha]) gene from a subset of the isolates was amplified using primers EF-1f (CGACCACTGTGAGTACCA) and EF-1r (GTCAAGAACCCAGGCGTA) (Ward et al. 2008) and bidirectionally sequenced. The sequences, deposited in GenBank under accession numbers MH379136 to MH379138 and MH392474 to MH392477, were analyzed and assembled in MEGA7, and BLAST queries were performed in FusariumID (http://isolate.fusariumdb.org/guide.php) and Fusarium MLST (http://www.westerdijkinstitute.nl/fusarium/). Three isolates from Chase County in the southwest (isolates NE16-15Fb and NE19-15Fb) and Box Butte County (isolate NE21-15Fb) in the northwest had 100% similarity with the reference strain F. boothii NRRL 29105. The remainder were identified as F. graminearum based on similarity to F. graminearum NRRL 28336 ranging from 99.51% to 100%. To fulfill Koch's postulates, FHB-susceptible spring wheat cv. Wheaton was grown in the greenhouse in 15-cm-diameter pots, 12 plants per pot (experimental unit). Spikes were spray-inoculated at anthesis with spores of F. boothii isolates NE16-15Fb and NE19-15Fb and a single isolate of F. graminearum (NE20-15Fg) collected in the same survey in Kimball County in the southwest (ca. 1 X 105 spores/mL) or not inoculated, and enclosed in transparent Ziploc bags for 72 h. A randomized complete block design with five or six replications was used in two experiments. Symptoms (bleaching of spikeiets) appeared 3 days after inoculation (dai) in all three isolate treatments. At 21 dai, FHB severity (percent of bleached spikeiets on a spike) ranged from 18% to 87%. No symptoms developed in the check treatment. Mature grain was ground to flour and tested for deoxynivalenol (DON) and its acetylated derivatives 3-ADON and 15-ADON using gas chromatography/mass spectrometry (GC/MS). All three isolates had the 15-ADON chemotype. Single-spored cultures of the three isolates were recovered on PDA from symptomatic kernels obtained from both experiments. The three isolates produced fertile perithecia on carrot agar after incubation at room temperature for 7 weeks. Their colonies on PDA resembled typical F. graminearum colonies (Leslie and Summerell 2006). Measurements of ascospores (17.50-32.50 [micro]m X 3.75-7.50 urn) and macroconidia (36.25-67.50 [micro]m X 3.75-6.25 [micro]m) of the three isolates were similar to those reported for species in the F. graminearum clade (O'Donnell et al. 2004). F. boothii has been reported as the cause of FHB of wheat or Gibberella ear rot of maize in several countries including Mexico (Ceron-Bustamante et al. 2018) and South Africa (Boutigny et al. 2011). To our knowledge, this is the first report of F. boothii causing FHB of wheat in the United States. Research will be needed to determine its distribution, aggressiveness, and impact on yield in single or double infections with F. graminearum., https://apsjournals.apsnet.Org/doi/abs/10.1094/PDIS-04-18-0696-PDN Dr. Stephen N. Wegulo, Mr. Esteban Valverde-Bogantes, Mr. Carlos Bolanos-Carriel, Dr. Heather Hallen-Adams, Dr. Andreia Bianchini Huebner, Ms. Niki McMaster, Dr. David Garner Schmale [...]