First Report of Charcoal Rot Caused by on Dry Bean ( L.) in Western Canada.

In 2021, several dry bean (Phaseolus vulgaris L.) plants at the mid-seed-fill growth stage displaying wilting, chlorosis of the leaves and reduced vigor were collected near the Pembina - Emerson Border of Manitoba, Canada and North Dakota, USA. When symptomatic plants were examined, gray to dark brown discoloration was observed on the lower stem and the roots. Afterwards, brown to black discoloration was noticed on stem and root sections. Root and lower stem pieces (1 to 2 cm) from affected plants were surface sterilized with 70 % ethanol, followed by 1% NaOCl, rinsed twice in sterilized water, air dried on sterilized filter papers, and placed on potato dextrose agar (PDA) amended with 1 mg/mL of streptomycin sulfate. The PDA plates were incubated at 28°C with 12 h light/12 h dark for 10 days. The growing hyphae were transferred using the hyphal tip method to new PDA plates. Growing cultures were initially hyaline and turned from light gray to dark brown or black with age. Abundant dark and spherical to oblong shaped sclerotia with an average diameter of 97.9 µm (range: 66.8 to 143.5 µm, n =30) formed on the pure cultures 7 days after incubation. Additional pure culture was obtained through an isolation of a single microsclerotium followed by a single hyphal tip transfer. One isolate was identified as Macrophomina phaseolina based on morphological characteristics (Smith and Wyllie 1999). The morphological identity was confirmed by sequencing the rDNA internal transcribed spacer (ITS) region with universal primers ITS1/ITS4 (White et al. 1990) and calmodulin (CAL) and translation elongation factor-1 alpha (TEF-1α) genes with MpTefF/MpTefR, and MpCalF/MpCalR primer sets (Santos et al. 2020), respectively. The online resource Basic Local Alignment Search Tool (BLAST; https://www.ncbi.nlm.nih.gov/BLAST) confirmed the fungus identity as 100% M. phaseolina. The sequences of the original isolate BF21-25 were deposited in GenBank with accession numbers OQ615297 (ITS), OR357630 (CAL), and OR363106 (TEF-1α). To confirm pathogenicity, bioassays were conducted under controlled conditions. Four seeds of cultivar 'Etna' were sown per pot, and five pots were used for inoculated (approx. 4 × 105 microsclerotia/pot) and control (mock-inoculated with sterile PDA medium) treatments. For the inoculum, 20 g of macerated 10 to 14-day old M. phaseolina culture grown on PDA medium was applied to each pot using an inoculum layering technique. Pots were kept in the greenhouse with 28/17°C day/night, 13 h light/11 h dark cycle, and 70% relative humidity and watered weekly. Disease symptoms similar to those observed in the field were visible on all inoculated plants at the mid-seed-fill growth stage. Mock-inoculated control plants didn't show any symptoms. The experiment was repeated twice with similar results. The pathogen was re-isolated from infected plants to confirm Koch's postulates and identified as M. phaseolina based on the morphology and sequences of ITS, CAL and TEF-1α regions. To our knowledge, this is the first report of charcoal rot caused by M. phaseolina on dry bean in Western Canada.