First report of Diaporthe ambigua causing Phomopsis stem canker on sunflower in Argentina

Phomopsis stem canker, a disease emerging on sunflower (Helianthus annuus) in response to environmental change, has already been described in Argentina with Diaporthe helianthi as the main aetiological agent (Ridao et al., 1994). During a survey (2020-2021) in the Argentinian sunflower-growing region of Pampeane, 208 sunflower plants with canker symptoms were collected from 75 cultivated fields (Fig. 1). Necrotic triangular dark leaf lesions invaded the stem, forming a conspicuous brown to black canker at the corresponding nodes. The infected stems were surface sterilised with 70% (v/v) ethanol for 30 seconds and in 1% (w/v) sodium hypochlorite for one minute. Tissue fragments were excised aseptically from the internal leading lesion's edge, transferred to potato dextrose agar (PDA) with a sterilised soybean stem fragment, and incubated at 25°C for 14 days for anamorph development and 40 days for teleomorph maturation (12-hour photoperiod, UV light, 345–400 nm). Diaporthe ambigua was identified based on the following features. Colonies on PDA, flat, floccose, white, presenting coriaceous and dark stromatic patches arranged in an annular pattern; reverse pale, dark under the stromata. Mycelium consisting of a dense dirty white to cream-coloured layer supporting floccose hyphal tufts. Perithecia globose, solitary with a long neck (Fig. 2). Asci unitunicate, cylindrical-clavate with a refractive apical ring, 8-spored, 42–46 × 6–7 μm in size. Paraphyses unbranched, constricted at the septa, tapering towards the apex with a rounded tip, extending above the asci. Ascospores bicellular, hyaline, smooth, 11–13 × 2–3 μm with four guttules. Pycnidia, globose, solitary or in groups. Conidiophores simple, composed of a globose basal cell and an integrated conidiogenous cell. Alpha conidia ellipsoidal, with slight central constriction and two conspicuous guttules, obtuse apex and papillose base, smooth walled (6-7 × 2–3 μm) (Fig. 3). Beta conidia not seen. The internal transcribed spacer (ITS) and translation elongation factor 1-α (EF1-α) were amplified and sequenced using primers ITS1/ITS4 (White et al., 1990) and EF1-728F/EF1-986 (Carbone et al., 1999) respectively. The sequences were deposited in the GenBank database, ITS (Accession No. ON099487) and EF1-α (ON146034). After BLASTn analysis, the Argentinian isolate showed more than 99% identity with D. ambigua (NR_119434.1 and KC343736.1). To investigate pathogenicity, two experiments (15 plants each) were conducted in the greenhouse. A susceptible sunflower hybrid (ACA 869 DM) was inoculated at the V8 stage using the stem wounding method described by Mathew et al. (2018). After 14 days, all inoculated plants showed cankers with necrosis and wilting, while control plants remained symptom free. Diaporthe ambigua was reisolated consistently from artificially infected plants using the methods above. Diaporthe ambigua was isolated from sunflower in Italy (Gomes et al., 2013) but to our knowledge, this is the first report of D. ambigua in Argentina. This work used the genomics facility in UGB, IABIMO, CICVyA, INTA. Funding from Instituto Nacional de Tecnologia Agropecuaria (INTA, I.90; I.114), Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, PICT2019-2932) and Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET).