First Report of Didymella americana on Lima Bean (Phaseolus lunatus) in Delaware and Maryland

Abstract

Delaware and Maryland have a long history of commercial production of lima bean (Phaseolus lunatus) for the processing market. In 2015, we observed lima bean plants that had irregular tan lesions with reddish borders in two fields in Sussex County, DE. Lesions ranged from 0.4 to 2.0 cm in diameter. Approximately 10% of leaves were symptomatic, and severity ranged between 5 and 10%. Symptomatic leaves (n = 6 to 10) were collected in September and October from each field for isolation and evaluation. Similar symptoms were observed on lima bean plants at the University of Maryland Lower Eastern Shore Research and Education Center in 2018, and additional symptomatic leaves (n = 6) were collected. Isolations were conducted from six diseased leaves per field (n = 18) by placing 2- to 4-mm sections of leaf on potato dextrose agar (PDA). All lesions resulted in isolates that appeared similar in morphology. Hyphae from the representative colonies were placed on water agar, and after 24 h, hyphal tips were excised. The hyphal tip procedure was repeated once to obtain pure cultures. Six isolates from Delaware and one from Maryland were stored at –80°C until use. Morphological evaluation of isolates grown on oat agar (OA), malt extract agar (MEA), and PDA revealed conidial morphology and dimensions consistent with previously published reports of Didymella americana (Aveskamp et al. 2010; Gorny et al. 2016). Two isolates, De15-002 and Md18-06, were selected for molecular analysis. DNA was extracted from colonies grown on OA; the internal transcribed spacer (ITS) region was amplified with primers ITS1/ITS4, and the partial actin locus region was amplified with primers act-512/act-783. Basic local alignment searches compared sequences to the ITS (GenBank accession no. FJ426972) and partial actin (FJ426870) sequences of D. americana reference strain CBS 185.85 (Chen et al. 2017; Gorny et al. 2016). The ITS sequences of isolates De15-002 and Md18-06 (MN698269 and MN944417, respectively) shared a 100% similarity with the reference strain. The partial actin sequences of isolates De15-002 (MN820723) and Md18-06 (MN820722) were also identical and differed from the reference sequence by 1 bp (99% similarity). To fulfill Koch’s postulates, four 3-week-old lima bean plants (cv. C-Elite) were sprayed with a conidial suspension (3 × 10⁵ conidia/ml) from 30-day-old cultures grown on MEA or with distilled water (control). Following 28 days of incubation at approximately 20°C, lesions were observed on all plants that were inoculated with isolates De15-001, De15-002, De15-004, De15-005, De15-007, De15-009, and Md18-06, but not on the water-inoculated control plants. Reisolations (n = 4 per isolate) onto water agar and MEA were examined after 7 days to confirm morphology similar to the original isolates. Based on the morphological and molecular characteristics, the pathogen was identified as D. americana. This is the first report of D. americana on lima bean in the Mid-Atlantic region of the United States. Lima bean is the most widely grown vegetable crop in Delaware, and in 2018, the state led production nationally. A new disease on the crop is important to the agricultural economy of Delaware, and the yield loss potential of this pathogen needs assessment in the region. This species has only been reported in 2016 on lima bean, in New York (Gorny et al. 2016).