First Report of Leaf Spot of Sugar Beet Caused by Stemphylium vesicarium in Minnesota, USA.

Abstract

In July 2021, sugar beet (Beta vulgaris L.) leaves with numerous tan to brown spots with white-bleached center and oval to irregularly shaped were collected from a field in Minnesota (MN) (46.2774° N, 96.3100° W), 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 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 (ITS) region (ITS1/ITS4 primers), the largest subunit of RNA polymerase II (5F2/7cR primers) (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 β-tubulin gene (Bt2a/Bt2b primers) (Glass and Donaldson 1995) were done using standard procedures. Sequences were submitted to GenBank under accession numbers OP584331 (ITS), OP589289 (RPB2), OP589290 (ATPase), OP994239 (GAPDH) and OP382477 (β-tubulin). The BLASTN search of the sequences showed 100% similarity with MT629829 (ITS) (525/525 bp), KC584471 (RPB2) (859/859 bp), JQ671770 (ATPase) (794/794 bp), MK105974 (GAPDH) (519/519 bp) and MN410922 (β-tubulin) (320/320 bp) reference sequences of S. vesicarium. Pathogenicity tests were done using four cv. Maribo MA 504 plants. 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 non-inoculated control. All plants were incubated in the mist chamber for 5 days at 25°C, under daily 14/10 light-dark cycles, and >80% relative humidity, then transferred to the greenhouse kept at 23 ± 2°C and a 12-h photoperiod. Fifteen days post-inoculation, all inoculated plants had multiple lesions with dark brown margins with a grayish center, and non-inoculated 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 MN. Stemphylium sp. is a major problem of sugar beet 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.