First report of Stemphylium leaf spot of sugar beet caused by Stemphylium vesicarium in eastern North Dakota, USA

Abstract

In August 2022 and September 2024, sugar beet (Beta vulgaris L.) leaves with several moderately large, light tan to brown, oblong to irregular leaf spots were collected from fields in Pembina, Grand Forks, and Walsh counties in North Dakota. Leaf spots bordered by 1-2 mm healthy tissue were excised and incubated in humidity chambers for 48 h under fluorescent and UV-A light with a 12-h light/dark cycle at 21 ± 1°C. Stemphylium-like conidia suspensions were collected into 0.01% Tween 20 by gentle pipetting, and 120 µl of suspension was spread onto thin water agar amended with ampicillin (200 mg/liter). After 24 h, individual germinating conidia were transferred to clarified V8 agar amended with ampicillin. Two isolates were collected in 2022, and thirteen isolates were collected in 2024. All cultures produced dark, solitary, multiseptate conidia that were round to oblong, approximately 35 × 17 µm, and formed in irregular clusters, consistent with descriptions of Stemphylium vesicarium (Metheny et al. 2022; Khan et al. 2023) and S. beticola (Hanse et al. 2015; Woudenberg et al. 2017). Genomic DNA was extracted from fifteen isolates using the method described in Lien and Chanda (2026). The internal transcribed spacer region of rDNA was amplified with primers ITS1F/ITS4 (Gardes and Bruns 1993; White et al. 1990), calmodulin (cmdA) with CALDF1/CALDR1, and plasma membrane ATPase with ATPDF1/ATPDR1 (Lawrence et al. 2013) and sent for Sanger sequencing at MCLAB (San Francisco, CA). Sequences were submitted to GenBank (ITS: PZ290169; cmdA: PZ301221; ATPase: PZ301222), and BLASTn searches showed 100% identity with MH879836.1 for ITS (577/577 bp) and MK675696.1 for cmdA (699/699 bp), and 99.7% identity with JQ671770.1 for ATPase (1230/1234 bp), supporting identification as S. vesicarium. Sequence similarity to the S. beticola type-strain CBS 141024 was 98.3% for ITS (519/528 bp) and 90.5% for cmdA (632/698 bp). Pathogenicity tests were conducted on 8-10 leaf stage sugar beet germplasm line USDA F1042 using two 2024 isolates. Pure cultures were grown on CV8 for 7 days under fluorescent and UV-A light with a 12-h light/dark cycle, and conidia were collected in 0.2% malt extract solution (Stammler et al. 2014). Three leaves were marked on each plant, and one half of each marked leaf was lightly abraded with autoclaved fine white Ottawa sand to induce injury prior to inoculation. For each isolate, three plants were sprayed with 10 ml of spore suspension (1 × 10 3 conidia/ml). Three non-inoculated control plants were treated similarly and sprayed with 0.2% malt extract solution. Plants were moved to an environmental chamber maintained at >95% relative humidity and 20°C with a 14-h/10-h photoperiod for 7 days, then transferred to a greenhouse at 65 ± 10% relative humidity. The experiment was conducted twice. Leaf spot symptoms developed approximately 3 weeks after inoculation (WAI) on both injured and non-injured tissue (Supplementary Fig. S1) and were monitored until 5 WAI. No symptoms were observed on non-inoculated controls. Conidia, morphologically similar to the original isolates, were consistently reisolated after incubation of representative lesions in humidity chambers (Supplementary Fig. S2). These results expand the geographic distribution of S. vesicarium on sugar beet beyond Michigan and Minnesota. This study highlights the need for accurate diagnosis of leaf spot diseases and for developing effective disease management methods to minimize losses for growers.