First Report of Red Crown Rot of Soybean Caused by <i>Calonectria ilicicola</i> in Minnesota

Soybean plants (Glycine max L.) with symptoms and signs of red crown rot were observed in approximately 20% of plants in a 3 ha section of a large production field in Rock County, Minnesota in August 2025. The symptoms and signs included interveinal necrosis on the leaves, root rot, and reddish discoloration and raised red to brown perithecia on the lower stems and crowns. Two symptomatic plants were collected and transported to the Department of Plant Pathology at the University of Minnesota for diagnosis and analysis. Microscopic examination of the perithecia on both plants revealed asci and hyaline one-septate ascospores measuring 5 × 40 μm, consistent with the appearance of C. ilicicola (Padgett et al. 2015; Kleczewski et al. 2022). One isolate of the pathogen identified as noted below was obtained from the infected roots of each symptomatic plant and purified by single-spore and hyphal-tipping procedures. Genomic DNA was extracted from each isolate, and the nuclear ribosomal internal transcribed spacer (ITS), beta tubulin (BT), and elongation factor (EF) were sequenced using the ITS1F/ITS4, T1/Bt2B, and EF1-728F/EF1-986R primers, respectively (White et al. 1990, Gardes &amp; Bruns 1993, Crous et al. 2004, and Carbone &amp; Kohn 1999). The sequences for the amplified regions for both isolates had &gt;99.5% base pairs matching to multiple C. ilicicola accessions in GenBank using BLASTn, including the BT sequence for MK370992 (Kleczewski et al. 2019). Sequences from the two Minnesota isolates (Rock2 and Rock3) were deposited in GenBank as accessions PX715458 and PX715459 (ITS), PX733394 and PX733395 (EF), and PX733398 and PX733399 (BT). Koch’s postulates were completed by inoculating soybean variety MN1904 with an isolate of C. ilicicola from Minnesota. The isolate was grown for 5 weeks on potato dextrose agar (PDA) at 23 °C and macerated in a blender with an equal amount of sterile water. The mixture containing hyphae, chlamydospores, and agar was applied to the stems and upper root system of three plants growing in Pro-Mix® BKI (Premier Tech, Quakertown, PA) in each of 4 pots, 13 days post-sowing at the VC growth stage. Control plants (n=12) were grown in four other pots and mock-inoculated the same way with sterile PDA. Plants were watered daily and maintained in a greenhouse at 22 °C with a 14 hr photoperiod. Inoculated plants developed root rot, reddish discoloration of the stem near the soil line, foliar interveinal necrosis, and wilting 3 weeks post inoculation at the V3 growth stage. The symptomatic, inoculated plants were collected and the pathogen was re-isolated from two plants. These isolates were confirmed to be the same as the isolate used for inoculation via colony characteristics and sequencing of the EF region (as noted above). None of the mock-inoculated plants developed any of these symptoms, and isolation attempts from them did not yield any isolates with cultural or microscopic morphological characteristics similar to Calonectria spp. This inoculation experiment was repeated once with the same materials and methods, and the outcomes were the same. These results confirm for the first time that red crown rot caused by C. ilicicola occurs in Minnesota, approximately 600 km from the nearest confirmed location in the U.S. at that time in northwestern Illinois. The detection of red crown rot in Minnesota represents expansion of the known range of this disease and suggests it is a risk to soybean production in this state.