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

In July 2024, soybean (Glycine max) with symptoms of red crown rot, caused by Calonectria ilicicola, were observed in a commercial field with a history of corn-soybean rotation in Marion County, Missouri. Soybean were planted on May 12 and foliar symptoms including interveinal chlorosis and premature senescence, were visible on plants at the R3 growth stage with incidence estimated at 5% of the field. Reddish coloration was observed on the lower stem of symptomatic plants, along with reddish-orange globular perithecia measuring 300-500 µm in height and 250-350 µm in diameter, typical of C. ilicicola infection. Twelve symptomatic plants were collected, and stems were cleaned, followed by immersion in 70% ethanol for 30 seconds, 1.25% NaOCl for 5 minutes, and rinsing three times. Surface disinfected stems were dried at 24°C. After 24 hours, stems were split and placed on water agar (WA) amended with 1% penicillin-streptomycin solution (P4333; Sigma Aldrich). After 4 to 5 days, conidiophores characteristic of C. ilicicola, were observed. Conidia from nine stems were transferred to WA and serial transfers of conidia were made until pure single spore cultures of isolates were obtained and transferred to potato dextrose agar (PDA). On WA, the isolates produced hyaline mycelium that developed conidiophores and conidia as well as reddish brown chlamydospores. Single spore isolates transferred to PDA produced fluffy white mycelium that turned reddish. Three isolates were used for DNA extraction with the Zymo DNA extraction kit (ZD6005). Parts of the internal transcriber region (ITS) (ITS-F2: 5'-TTTACAACTCCCAAACCCCATGTGAAC-3'and ITS-R2: 5'-CTACCTGATTCGAGGTCAA CCAGAA-3') histone 3 (HIS3) (Crous et al. 2004), translation elongation 1α (EF1α) (Carbone and Kohn 1999; O'Donnell et al. 1998) and β-tubulin (TUB2) (Crous et al. 2004; O'Donnell and Cigelnik 1997) genes were amplified and their DNA sequenced. Sequences were processed in Geneious Prime 2024.0 and deposited at NCBI GenBank under the accession numbers PQ390253, PQ390254 and PQ390255 (ITS), PQ519580, PQ519581 and PQ519582 (HIS3), PQ507479, PQ507480 and PQ507481 (EF1α) and PV092563, PV092564 and PV092565 (TUB2). These DNA sequences show 100% identity to C. ilicicola sequences. To fulfill Koch's postulates, eight pots containing three plants (cultivar Williams 82) each were grown for two weeks in a growth chamber at 26°C with a 14-hour photoperiod and watered daily. Six pots were inoculated with 4 mm plugs of mycelium from the distal growing edge of the fungal isolate on PDA plates. The other two pots, treated with 4 mm plugs of PDA-only, served as negative controls. The planting medium at the base of each plant was gently removed, and inoculum was placed on the primary root, approximately 7 mm below the soil level. Five days after inoculation, plants started exhibiting reddish/blackish discoloration at the base of the stems, typical of red crown rot. Then, C. ilicicola was re-isolated from symptomatic plants three weeks post-inoculation and identified by conidial morphology. Together, these results confirmed the presence of C. ilicicola in Marion County, Missouri. Although C. ilicicola was first confirmed in the United States in 1965 on peanuts, it has gained renewed interest due to its presence in commercial soybean production, where symptoms can resemble sudden death syndrome. The pathogen poses a significant threat to >2,000,000 ha of soybean and approximately 8,000 ha of peanut grown in Missouri.