In August 2025, soybean (Glycine max) plants with red perithecia and interveinal chlorosis of the foliage consistent with red crown rot (RCR), caused by Calonectria ilicicola were observed at very low incidence and severity in a commercial field in northern Columbia County, Wisconsin. Symptomatic plants were submitted to the Plant Disease Diagnostics Clinic and Field Crops Pathology Lab at the University of Wisconsin-Madison for confirmation of RCR. Stems were cleaned in water and disinfested in a 0.825% NaOCl solution for 1.5 minutes, rinsed in sterile deionized water for 5 minutes, disinfested in 70% ethanol for 1 minute then rinsed in sterile deionized water. Disinfested stem pieces were blotted onto sterile filter paper and 10 perithecia were picked directly from a stem and placed into a 1.5 ml microcentrifuge tube containing 400 µl of sterile deionized water and ground with a sterile Kontes pestle. Two hundred microliters of the suspension was spread across Petri-plates containing water agar with a sterile cell spreader. Plates were incubated at room temperature in the laminar flow hood for 1.5 hrs before examination under a dissecting scope. Single ascospores were transferred to fresh Petri-plates of PDA amended with antibiotics, as above. After 5 days, fungal colonies with white to light yellow aerial hyphae, later turning reddish, were observed growing on the PDA. Cultures derived from single ascospores were grown in potato dextrose broth to obtain mycelia for DNA extraction. DNA was extracted using a FastDNA Spin Kit (MP Biomedicals, Santa Ana, CA). PCR and DNA sequencing were performed using primers ITS5 and ITS4 (White et al., 1990) to amplify portions of the internal transcribed spacer region of rDNA. Primers T1 (O’Donnell and Cignelik, 1997) and Bt2b (Glass and Donaldson, 1995) were used to amplify a fragment of the β-tubulin gene. Amplicons were sent for Sanger sequencing (Functional Biosciences, Madison, WI). Sequences were submitted to NCBI GenBank (accession numbers PX872521 for ITS and PX935143 for β-tubulin). DNA sequences showed 99.7 to100% identity to C. ilicicola for both ITS and β-tubulin genes (GenBank accession numbers MN245059 and MK189210, respectively). To fulfill Koch’s postulates, plugs from an actively growing culture of C. ilicicola isolate RCR4 were used to infest millet grain in Erlenmeyer flasks. After 3 weeks, the infested millet grain was used in controlled inoculation experiments where six seeds from three soybean cultivars (Dwight, Williams 82 and Sauk) were planted into 4 replicate, 12 oz Styrofoam cups containing infested potting mix or non-infested potting mix (non-treated controls). Plants were maintained in a grow room under grow lights (14 h light at 18℃/ 10 h dark at 16℃). After 21 days, the plants were removed from the cups, the roots were cleaned and evaluated. Inoculated plants exhibited symptoms typical of RCR infection with red/blackish discoloration at the base of the stems, and extensive root rot (Bish et al. 2025). Calonectria ilicicola was re-isolated from symptomatic plants and identified by DNA sequencing (GenBank accession numbers PX872746 (ITS) and PX935144 (β-tubulin). No symptoms of disease and no pathogens were identified on non-treated control plants. The pathogenicity experiment was repeated with similar results. Red crown rot has been confirmed in several Midwest states (Kleczewski et al., 2023). Additional research is needed to fully understand its distribution and to develop effective disease management strategies.
Groves et al. (Tue,) studied this question.