First Report of Alternaria Leaf Spot Caused by Alternaria tenuissima on Sugar Beet (Beta vulgaris) in Minnesota, U.S.A.

Abstract

Minnesota is the top sugar beet (Beta vulgaris L.) producing state in the United States. In September 2018, foliar symptoms were first observed on older sugar beet leaves in about 3% of the cultivars in a coded variety trial in Renville (N 44.78, W –95.15), Minnesota. Symptoms were dark brown circular or irregular spots on healthy plants. Initially, the lesions were confined within distinct parallel veins of the leaves but eventually expanded and coalesced across veins. In severely affected plants, the entire symptomatic leaves died. Small square pieces (5 mm²) from the margin of lesions were excised with a sterile scalpel, surface sterilized with 70% ethanol for 1 min, rinsed thrice with sterile distilled water, air dried, transferred to potato dextrose agar, and incubated at 25°C with a 12-h photoperiod for 7 days. A colony that had white-brown velvety mycelia grew on the medium. Suspect isolates were further purified using a single-spore isolation method (Choi et al. 1999). Isolates were the same morphologically. Conidia were club-shaped, four to seven transverse septa, one to three longitudinal septa, and pale brown, with a beak-like apical cell, often in long chains or solitary. Conidia dimensions were 28 to 54 × 4.5 to 6 µm, and beaks when present were 2 to 4 µm (Kou et al. 2014). Based on the morphological characters, the fungus was tentatively identified as Alternaria species (Simmons 2007; Woudenberg et al. 2015). Fungal genomic DNA was extracted from two representative isolates using a Qiagen kit followed by PCR amplification with internal transcribed spacer (ITS) ITS 1/ITS 4 primers. The amplified PCR products were cleaned and sent for Sanger sequencing by GenScript (Piscataway, NJ). Isolates 1422F (GenBank accession MK611645) and 1424F (MK611649) had 98 and 100% sequence identity with Alternaria tenuissima (GenBank accessions MF373440 and MH374277), respectively (Ziedan et al. 2018). Pathogenicity testing was conducted by spraying a conidial suspension (5 × 10⁵ conidia/ml) onto 14-leaf growth stage sugar beet plants (Crystal Beet Seed proprietary material). Mock-inoculated plants were sprayed with autoclaved water as a control. Plants were kept at 25°C and 75 to 85% relative humidity in a humidity chamber for 7 days and then transferred to a greenhouse kept at 23 ± 2°C and a 12-h photoperiod. Plants were watered daily. The experiment was done twice with four replicates and 10 plants per replicate. Two weeks postinoculation, about 30% of the inoculated leaves were infected and started to display irregular to circular lesions as observed in the field. No symptoms were observed on the control plants. The fungus was reisolated from diseased leaf tissues as described above, and macroscopic and microscopic analysis indicated the similar white-brown colony morphology and conidial structure, respectively. Genomic DNA was extracted from a single isolate as described above. Molecular detection performed using the same primers further confirmed that the isolate (MN384262) was 100% similar to A. tenuissima (GenBank accession MK611651). A. tenuissima has the potential to become a problem if susceptible varieties are grown over large production areas. Alternaria leaf spot on sugar beet has been reported in California (French 1989) and Michigan (Rosenzweig et al. 2019), where the disease has been associated with economic loss since 2015. To our knowledge, this is the first report of A. tenuissima causing leaf spot disease on sugar beet in Minnesota, U.S.A.