First report of leaf spot of sugar beet caused by Stemphylium vesicarium (Wallr.) E.G. Simmons in Minnesota, USA

Authors: KHAN, MOHAMED - North Dakota State University; BHUIYAN, KHAN - North Dakota State University; Lakshman, Dilip; LUIS, DEL RIO MENDOZA - North Dakota State University; AZIZI, ABDOLBASET - North Dakota State University; AMEEN, GAZALA - South Dakota State University; SARWAR, ARSLAN - North Dakota State University

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/21/2023
Publication Date: 3/22/2023
Citation: Khan, M., Bhuiyan, K., Lakshman, D.K., Luis, D., Azizi, A., Ameen, G., Sarwar, A. 2023. First report of leaf spot of sugar beet caused by Stemphylium vesicarium (Wallr.) E.G. Simmons in Minnesota, USA. Plant Disease. https://doi.org/10.1094/PDIS-02-23-0256-PDN.
DOI: https://doi.org/10.1094/PDIS-02-23-0256-PDN

Interpretive Summary: Minnesota is the top sugar beet (Beta vulgaris L.) producing State in the United States. In August 2020, we discovered for the first time a new disease with oval to irregularly shaped light to dark brown color spots on leaves. A fungus was isolated from diseased leaves, which upon morphological and molecular analyses identified as Stemphylium vesicarium. The identity of the pathogen was confirmed by artificial inoculation on leaves of healthy sugar beet seedlings. Although the pathogen S. vesicarium was recently reported in sugar beet in Michigan (Metheny et al. 2022) in the United States, to our knowledge this is the first report of the S. vesicarium in Minnesota, USA. Our findings will help growers, plant pathologists, and extension workers to add strategic inputs to manage this foliar pathogen in sugar beet crops.

Technical Abstract: Sugar beet (Beta vulgaris L.) is a major crop in Minnesota (MN), USA. In July 2021, sugarbeet leaves with numerous tan to brown spots with white-bleached center and oval to irregularly shaped were collected near Foxhome (46.2774° N, 96.3100° W), MN from a single field with 15% disease incidence and 30% disease severity. Leaves were washed with tap water then surface disinfected in 1% NaOCl aqueous solution for 1 min. Samples were rinsed thrice with sterile distilled water and dried in a laminar air flow hood. A 2-cm leaf disc was plated on potato dextrose agar amended with streptomycin sulfate (200 mg/L) and incubated for four days at 25°C under 12-h light/dark cycle. Single spore cultures were obtained by suspending in sterile water spores harvested from a single colony. The suspension was streaked on a dish with V8 agar media and incubated as described. Five pure cultures were transferred to clarified V8 agar media for morphological feature observations. Colonies were uniform in appearance and developed light to olivaceous green mycelium. Conidia were dark brown to olivaceous green in color and measured 30 ' 18 µm (n=20). They were oblong to broadly oval shaped muriform, and multiseptated (1 to 5 septa). Hyphae were septate and pale brown. Conidiophores were short, septate, and light to dark brown in color. Based on the morphological characteristics, isolates were identified as Stemphylium vesicarium (Simmons 1969). Genomic DNA of all five isolates were extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). PCR amplification and sequencing of the internal transcribed spacer region (ITS1/ITS4), the largest subunit of RNA polymerase II (RPB-2 5F2/7cR) (O’Donnell et al. 2009), the plasma membrane ATPase (ATPD-F1/ATPD-R1) gene (Lawrence et al. 2013), glyceraldehyde-3-phosphate-dehydrogenase gene (GAPDH) (gpd1/gpd2) (Berbee et al. 1999), and beta-tubulin gene (Bt2a/Bt2b) (Glass and Donaldson 1995) were done using standard procedures. Sequences were submitted to GenBank under accession numbers OP584331 (ITS), OP589289 (RPB-2), OP589290 (ATPase), OP994239 (GAPDH) and OP382477 (beta-tubulin). The BLASTN search of the sequences showed 100% similarity with MT629829 (ITS) (525/525 bp), KC584471 (RPB-2) (859/859 bp), JQ671770 (ATPase) (794/794 bp), MK105974 (GAPDH) (519/519 bp) and MN410922 (beta-tubulin) (320/320 bp) reference sequences of S. vesicarium. Pathogenicity tests were done using four cv. Maribo MA 504 plants at the eight true leaf stage. S. vesicarium spore suspensions (1 ' 106/ml) were sprayed on three leaves from each plant. This trial was repeated with three replicates. A similar group of plants were sprayed with autoclaved distilled water to serve as a noninoculated control. All plants were incubated in the mist chamber for 5 days at 25oC, under daily 14/10 light-dark cycles, and >80% relative humidity, then transferred to the greenhouse. Fifteen days post-inoculation, all inoculated plants had developed multiple lesions with dark brown margins with a grayish center, whereas the noninoculated control plants were asymptomatic. The re-isolated fungus was morphologically similar to isolates retrieved from the field. S. vesicarium was reported on sugar beet in Michigan (Metheny et al. 2022). This is the first report of S. vesicarium causing disease on sugar beet in Minnesota. Stemphylium sp. is a major problem of sugarbeet in the Netherlands (Hanse et al. 2015). Efforts should be made to prevent introduction of susceptible beet cultivars so that the disease does not become widespread in the USA.