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First Report of Paramyrothecium roridum Causing Myrothecium Leaf Spot on Soybean in Africa

Haudenshield, J. S., Pawlowski, M., Miranda, C., Hartman, G. L.
Plant disease 2018 v.102 no.12 pp. 2638
DNA, DNA primers, Myrothecium roridum, boxes (containers), conidia, cultivars, culture media, fungi, hosts, internal transcribed spacers, leaf spot, leaves, mycelium, pH, pathogenicity, pathogens, polymerase chain reaction, sodium chloride, sodium hypochlorite, soybeans, sporodochia, Ghana
Myrothecium leaf spot, caused by Paramyrothecium roridum (Tode) L. Lombard & Crous (Basionym Myrothecium roridum), has been reported on a wide range of plants (Farr and Rossman 2018) including soybeans in the United States (Miller and Roy 1982). To our knowledge, this fungus has not been reported on soybeans grown in Africa. In November 2016, collections of leaves with leaf spots symptoms from three different fields near Tamale, Ghana, in the Northern Region were sent for identification to the USDA-ARS Soybean Disease and Pest Laboratory, Urbana, IL. Leaf spots had dark edges and somewhat lighter centers ranging from 0.3 to 1 cm in diameter. In the leaf spots were scattered dark stoma, each 40 to 70 µm wide (n = 50), observed on collected samples. Infected leaf tissue was cut into 2- to 3-mm pieces and surface disinfested with 0.5% NaOCl for 1 min, followed by three rinses with sterile deionized water. Tissue samples were plated on potato dextrose agar (PDA) and incubated at 25°C in the dark for 10 days. The isolates were white with dark green to black stroma. The conidia developed from sporodochial conidiomata and were aseptate, hyaline and 5 (to 7.6) to 10 µm × 1 (to 2.2) to 3 µm (n = 50). Morphologically, the fungus appeared to be P. roridum (Lombard et al. 2016). Pathogenicity was completed by inoculating detached leaflets of soybean cultivar Williams 82 with 1-cm² plugs cut from the margins of 3-day-old PDA cultures. Detached leaves were incubated inside clamshell containers and incubated with a 12-h light/dark cycle for 7 days. Symptoms appeared at 3 days postinoculation as necrotic brown spots with a dark border and a chlorotic halo. Dark green clusters of conidiomata were inside the necrotic lesion. The pathogen was reisolated and cultured for 7 days on PDA and appeared morphologically to be the same fungus. To further confirm the pathogen, mycelia of less than 1 cm² was excised from a representative culture. 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 LSU (LR5, 5′-TCCTGAGGGAAACTTCG-3′; LSU1Fd, 5′-GRATCAGGTAGGRATACCCG-3′) and ITS (ITS4, 5′-TCCTCCGCTTATTGATATGC-3′; ITS5, 5′-GGAAGTAAAAGTCGTAACAAGG-3′) primers, which produced amplicons (900 and 577 bp) that were purified using the Qiaquick PCR Cleanup Kit (Qiagen, Germantown, MD) and delivered to a core facility for Sanger sequencing, using the same primers. The two top-scoring BLAST hits for the LSU region (GenBank accession MH379146) had 96% coverage and 98% identity to P. roridum (M. roridum). The three top BLAST hits for the ITS1 region (GenBank accession MH379145) had 100% coverage and 99% identity for P. roridum, Myrothecium sp., and P. roridum (M. roridum). This is the first confirmed report of P. roridum causing leaf spot on soybean in Africa, although the fungus occurs on other hosts in Africa. The distribution of the pathogen on soybean in the United States is not known.