First Report of Northern Stem Canker Caused by Diaporthe caulivora on Soybean in Western Canada

Abstract

In 2017, soybean (Glycine max) plants (cv. 24-10RY, R1 growth stage) with dead brown leaves associated with light to dark reddish-brown lesions and necrosis of the lower half of the stem, especially at the base of branches, were collected at Carman, Manitoba. Reddish-brown discoloration was observed inside stems when split in half. Severely infected plants wilted and desiccated. These symptoms are typical of Northern stem canker (Fernandez et al. 1999). Small pieces of the diseased stems (1 to 2 cm) were surface sterilized in 0.5% NaOCl and then rinsed twice in sterilized water, air dried on sterilized filter papers, and plated on potato dextrose agar (PDA) amended with 100 mg/liter of streptomycin sulfate at room temperature with 12-h light/12-h dark for 3 days. The growing hyphae were transferred using the hyphal tip method to new PDA plates and incubated for 3 weeks at room temperature. Cultures produced white-to-brown colonies interspersed with cottony tufts of mycelium. Stromata were small and scattered irregularly on the plate (Fernandez et al. 1999). The identity of the recovered isolate (Carm15) was confirmed by sequencing the internal transcribed spacer (ITS) region and β-tubulin gene. The ITS region was amplified using the universal ITS1 and ITS4 primers (Glass and Donaldson 1995). The β-tubulin gene was amplified using primers T1 and T22 (O’Donnell and Cigelnik 1997). PCR products (∼650 bp for ITS; ∼1,500 bp for β-tubulin) were purified with the Wizard SV Gel and PCR Clean-Up System (Promega, U.S.A.) and sequenced (Macrogen, U.S.A.). Using BLASTN searches (Altschul et al. 1990), the ITS sequence of Carm15 (GenBank accession MH299927) matched the type sequence of Diaporthe caulivora strain CBS 127268 (accession NR_111845) from G. max in Croatia with identities = 546/546 (100%) and gaps = 0/546 (0%). The β-tubulin gene sequence of Carm15 (GenBank accession MH732622) matched the type sequence of D. caulivora strain CBS 127268 (accession KC344013) with identities = 703/703 (100%) and gaps = 0/703 (0%). To confirm the pathogenicity of Carm15, soybean plants (cv. 24-10RY) were grown in the greenhouse. The stems of 15 plants at the V3 stage were excised just below the fourth node. Mycelial plugs (9 mm in diameter) from a 1-week-old culture of Carm15 grown on PDA at 25°C were placed over stems previously wounded with a sterile scalpel (Vrandecic et al. 2005). Sterile PDA control plugs were placed over cut soybean stems of another 15 plants of the same cultivar. Plugs of both treatments were wrapped with Parafilm to avoid drying. The plants were incubated in a moist chamber for 3 days and then left to grow on a greenhouse bench at 24:16°C day/night temperature, 13:11-h light/dark cycle, 80% relative humidity, and daily watering. Symptoms similar to those observed in the field were visible on the stems of all inoculated soybean plants. Lesions extended from the inoculation sites after 27 days. No symptoms were observed on control plants. The pathogen was reisolated from infected stems (Carm15GH) and identified as D. caulivora as above; the ITS and β-tubulin sequences of Carm15GH were identical to those of Carm15. The experiment was repeated twice. To our knowledge, this is the first report of Northern stem canker on soybean in Western Canada. Documenting the occurrence of Northern stem canker in Western Canada is important because it can have a significant impact on soybean yield, potentially resulting in up to 50% loss (Fernandez et al. 1999).