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First Report of Stem Disease of Soybean (Glycine max) Caused by Diaporthe gulyae in North Dakota

Mathew, F. M., Gulya, T. J., Jordahl, J. G., Markell, S. G.
Plant disease 2018 v.102 no.1 pp. 240
DNA, Diaporthe gulyae, Glycine max, Helianthus annuus, acreage, conidia, crop yield, culture media, cutting, greenhouses, internal transcribed spacers, mortality, mycelium, pathogenicity, pathogens, plant pathology, pycnidia, sequence analysis, soybeans, stem cankers, stems, sterilizing, North Dakota
The planted soybean (Glycine max L.) acreage in North Dakota increased approximately six-fold in the last two decades to over 6 million acres in 2016. In September 2012, soybean plants exhibiting reddish-brown stem cankers (∼60 mm length) were observed in a production field in Grand Forks county (49°11′N; 98°09′W). Incidence of infected stems was estimated in excess of 95% in parts of the field. Ten plants exhibiting symptoms were randomly sampled and brought to the Department of Plant Pathology at NDSU to identify the causal pathogen. Isolations were made from the canker margins by cutting, surface sterilizing, and plating small (5 mm) symptomatic stem pieces onto potato dextrose agar (PDA). After incubation at 25°C for 14 days, tan to gray colonies with interspersed or aerial mycelium were purified by hyphal-tip transfer to fresh PDA plates. Two pathogens were identified: Diaporthe caulivora (Athow and Caldwell) Santos, Vrandečić and Phillips (isolation frequency = 100% of stems) and an unknown Diaporthe sp. (isolate ND1) producing black pycnidia with ostiolate beaks and alpha conidia (isolation frequency = 10% of stems). DNA was extracted from the mycelium of ND1 and sequenced using the ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R primers (Carbone and Kohn 1999). BLAST analysis of the ITS and EF1-α sequences (accession nos. KY670608 and KY670609) showed 100% identity to Phomopsis sp. AJY-2011a strain T12505G (D. gulyae Shivas, Thompson and Young; accession no. JN645803 and JF431299). Isolate ND1 and the D. caulivora isolate (isolate ND2) were recovered from the same plant, and both were tested for pathogenicity on soybean cv. RG200RR using cut-seedling assay (Li et al. 2010). Succinctly, the stem of 2-week-old soybean plants (10 plants per isolate and control) were wounded below the first trifoliate node and a Diaporthe-infested mycelial plug was secured on the wound using Parafilm. The pots were placed on the greenhouse benches at 22°C under a 16-h light/dark cycle. Ten days after inoculation, dark brown lesions with nongridling cankers (mean length ∼40 mm) were observed on all 10 plants inoculated with D. caulivora. However, reddish-brown lesions (mean length ∼25 mm) without cankers extending upward from the inoculation site and causing host mortality were observed on all plants inoculated with ND1. Ten plants inoculated with sterile PDA plugs expressed no symptoms nor was any Diaporthe reisolated. To complete Koch’s postulates, D. gulyae was isolated from the soybean plants previously inoculated with ND1 and pathogen identity was confirmed via morphology and EF1-α gene sequencing (Carbone and Kohn 1999). To our knowledge, this is the first report of D. gulyae causing a stem disease on soybean in North Dakota. The importance of D. gulyae on soybean yield is unknown. However, D. gulyae is one of the two primary causal agents of Phomopsis stem canker of sunflower (Helianthus annuus L.) in the northern Great Plains (Mathew et al. 2015). Thus, the identification of D. gulyae on soybeans in North Dakota, which produces ∼40% of the U.S. sunflower crop annually, is alarming. It is possible that the increasing production of soybeans in traditional sunflower growing areas in the state could serve as a reservoir for D. gulyae and exacerbate Phomopsis stem canker on sunflower.