First Report of Calonectria ilicicola Causing Red Crown Rot of Soybean in Indiana

Soybean (Glycine max [L.] Merr.) samples from commercial fields in Decatur and Spencer counties, Indiana were submitted to the Purdue Plant and Pest Diagnostic Lab in August to October 2022. Plants exhibited whole-leaf to interveinal chlorosis of the foliage, red to dark brown external lesions on the crown spreading from the soil-line upward, and severe root rot. In the fields, patches of diseased plants were observed, with greater than 50% of the plants affected and yield loss up to 50%. Orange to red perithecia were present on the exterior of symptomatic stem tissue and ranged in size from 329 to 433 × 232 to 306 µm (n = 10). Stems were surface sterilized in 10% Clorox (0.825% NaOCl) for 1 min, then rinsed with sterile distilled water and dried. In a laminar flow hood, sections of symptomatic stem tissue were plated on using quarter-strength potato dextrose agar (QPDA) and incubated under fluorescent lights on a 12-hr light/dark cycle at 20°C. After 6 days, fungal colonies with fluffy aerial hyphae, which were white near the colony margins and orange to burnt-red near their center, grew uniformly from the stem tissue plated. Elongate, cylindrical hyaline conidia with zero to three septations measuring 45.5 to 73.8 × 4.4 to 6.7 µm (n = 22) grew in clusters from symptomatic stem tissue within the plate. Perithecia developed after 14 days. Falcate, hyaline ascospores with one to two septa measuring 29.4 to 54.7 × 4.6 to 6.8 (n = 23) µm developed within the perithecia. Calonectria ilicicola Boedijn & Reitsma was confirmed based on morphological characteristics (Padgett et al. 2015). Isolate PPDL 22-01457B was used for DNA extraction using the ZR Fungal/Bacterial DNA Miniprep kit (Zymo Research, Irvine, CA). The internal transcriber region (ITS), actin (ACT) and β-tubulin (TUB2) genes were amplified (Carbone and Kohn 1999; Glass and Donaldson 1995; O'Donnell and Cigelnik 1997; White et al. 1990). Amplicons were sent for Sanger sequencing (Genewiz, Inc., South Plainfield, NJ), submitted to Genbank, and assigned accession numbers ITS: OQ932995, Actin: OR484986, and β-tubulin: OR546281. Sequences were analyzed using the NCBI BLASTn tool with results showing 99.5 to 100% identical to C. ilicicola (GenBank accessions LC500063, OQ303403, CP085825, respectively). To perform Koch's Postulates, 90 soybean seeds (CP3620E) were planted in potting media (Berger, Saint-Modeste, Quebec, Canada) in a seed flat with 45 of the plants used as controls and grown under grow lights for 16hr light/8hr dark at 20℃. Individual seedling crowns were inoculated 3 days post-emergence with a 5 to 10 ml spore and hyphal suspension that was scraped from the surface of a 14-day old QPDA culture after adding 300 mL deionized (DI) to each plate grown at 20 to 22°C. The control plants received sterile-DI water. Plants were covered in a plastic bag for 72 h. Plant stems were sprayed with sterile-DI water once a day for seven days. Symptoms were observed after four days, but significant crown rot and lesions developed after two weeks before wilting and dying. Calonectria iliciola was isolated uniformly from symptomatic plants and identified morphologically. Control plants showed no symptoms. Inoculations were repeated 3 times with similar results. As of fall 2023, red crown rot has been confirmed in Adams and Rush counties in Indiana. Red crown rot has been confirmed in several Midwest states (Kleczewski et al. 2019, Neves et al. 2023), but the extent of its distribution and disease management strategies are still limited.