First report of Pyrenophora avenicola causing leaf spot of oat in India

Oat (Avena sativa) is an allohexaploid cereal grown worldwide primarily for food and fodder. During autumn 2022, leaf spot and blight symptoms were observed on oat (cv. OL 1861) in Ludhiana, Punjab, India. Further surveillance of 60 fields, each at least one acre, in the state of Punjab revealed the presence of the disease in 52 fields. Disease severity, estimated as the diseased proportion of the surface of 10–20 infected leaves, ranged from 10 to 50% (mean = 34%). Initial symptoms appeared as brownish-black lesions, about 2–7 mm in diameter, gradually enlarging with straw-coloured centres, oval to elliptical in shape with regular margins (Fig. 1). As the disease progressed, the small spots coalesced to form blight on the leaves. The fungus was isolated from diseased leaves using standard isolation and pure culture procedures. On potato dextrose agar, colonies exhibited light grey to greyish black mycelial growth (Fig. 2) with regular margins, covering a 90 mm Petri plate after eight days' incubation. Conidiophores were produced in single or clusters, 280 × 3.4-5.8 μm (n = 30). Conidia were cylindrical to slightly curved ranging from 22.4-68.5 × 8.2-14.6 μm, with 1–6 distoseptate and flat to inconspicuous hila (n = 50) (Fig. 3). Characteristics of the pathogen were consistent with the genus Pyrenophora according to the descriptions of Helminthosporium by Alcorn (1988). Genomic DNA was isolated using a CTAB protocol as described by Rogers and Bendich (1994) and the internal transcribed spacer region (ITS) and glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) were amplified by PCR using the ITS1/ITS4 (White et al. 1990) and GPD1/GPD2 (Berbee et al. 1999) primer pairs, respectively. The amplicons were purified, cloned and sequenced, and the sequences were deposited in GenBank (ITS: PP732531.1; GAPDH: PP763574.1). BLASTn analysis of the ITS showed the highest identity to Drechslera avenae and Pyrenophora avenicola, while GAPDH exhibited most homology with P. avenicola and P. chaetomioides. A phylogenetic tree with concatenated ITS and GAPDH sequences of P. avenicola from the current study (PAU isolate) and other closely related species was constructed using the maximum likelihood approach in MEGA v11.0. This analysis confirmed P. avenicola as the causal organism associated with oat leaf spot (Fig. 4). Pathogenicity was confirmed by spray inoculation of 17- to 20-day-old oat plants (cv. OL 1861) with a conidial suspension (1×106 conidia/ml + 0.1% Tween-20) under controlled conditions (>90% relative humidity, 28 ±2°C). Additionally, plants were sprayed with distilled water and maintained as controls. Seven days after inoculation, initial symptoms of oval to oblong, dark spots appeared on inoculated plants, while control plants remained symptomless. Later, spots were developed, which resembled the typical leaf symptoms observed under field conditions. Koch's postulates were fulfilled by re-isolation and morphological identification of the pathogen. To our knowledge, this is the first report of P. avenicola causing leaf spot on oat in India. As oat is an economically important crop, such disease may cause damage if neglected.