First Report of Sclerotinia sclerotiorum Causing Stem Rot on Soybean (Glycine max) in Ethiopia

Abstract

Sclerotinia sclerotiorum causes a devastating disease on soybean (Sclerotinia stem rot) and attacks over 500 other hosts (Grau and Hartman 2015). In October 2018, research plots at Jimma Agricultural Research Center, Ethiopia, were evaluated for soybean diseases. A sample of 100 randomly selected plants of soybean line T44-15-T110-16SH1 were evaluated for Sclerotinia stem rot in a research plot that was 4 × 2.4 m with 60 cm between the four rows and 5 cm between plants within a row. All but 16 stems had stem rot symptoms with an average plant severity rating of 3.5 (SE = 0.18) based on an adapted 1 (1 to 10% of the stem affected) to 5 (91 to 100% of the stem affected) rating scale (Little and Hills 1978). Five infected stems were selected and sent to the USDA-ARS Soybean Disease and Pest Research Laboratory, Urbana, IL. Three sclerotia removed from each of three infected stems ranged from 4 to 18 mm long and 1 to 2 mm wide. Sclerotia were placed on potato dextrose agar (PDA) in 9.8-cm-diameter Petri plates and incubated at 24°C with a 16-h photoperiod for 4 days. The white, appressed mycelia grew from the sclerotia and covered the entire plate after 48 h, with sclerotia forming on the edge of the plate by 4 days. To confirm pathogenicity, a mycelial plug was removed from the margin of a 2-day-old colony of one of the isolates, was obtained by pressing the large end of a 200-μl pipette tip into the culture, and was placed on top of a cut stem above the second trifoliolate of four 3-week-old plants of soybean cultivar Williams 82. Plants were then incubated in a moist chamber for 48 h prior to being placed in a greenhouse held at 22°C with a 16-h photoperiod. Necrotic lesions and white mycelia appeared on the stems 4 days postinoculation. The pathogen was reisolated and cultured on PDA. After 4 days, the reisolated cultures with sclerotia appeared morphologically to be S. sclerotiorum. For further confirmation, 8-mm-diameter mycelial plugs were excised from the three representative cultures. DNA was released from the mycelia by disruption in Lysing Matrix A and CLS-Y solution, as provided by the FastDNA Spin Kit (MP Biomedicals, Solon, OH). Disruption was accomplished in a FastPrep-24 lemniscate homogenizer (MP Biomedicals) for 40 s at a speed of 6 m/s. DNA was extracted as instructed by the manufacturer. The resulting eluates were diluted 10-fold with 5 mM Tris, pH 8, containing 1 mM NaCl. Five-microliter subsamples were subjected to PCR using ITS (ITS4, 5′-TCCTCCGCTTATTGATATGC-3′; ITS1, 5′- TCCGTAGGTGAACCTGCGG-3′) primers, which produced 477-bp amplicons that were purified using the QIAquick PCR Cleanup Kit (Qiagen, Germantown, MD) and delivered to a core facility for Illumina sequencing (Roy J. Carver Biotechnology Center, University of Illinois), using the same primers. The top BLAST hit for the ITS region (GenBank accession MK882510) had 99.79% identity to S. sclerotiorum (MF776031.1; Alaska). In Africa, S. sclerotiorum has been found on soybean in Nigeria (Akem and Dashiell 1992) and South Africa and on common bean in Ethiopia (Allen 1995). To our knowledge, this is the first report of Sclerotinia stem rot on soybean in Ethiopia. Sclerotinia stem rot could become a major limiting factor, because soybean production based on harvested hectares has increased from 3,807 ha in 1997 to 39,021 ha in 2017 (FAO 2019).