Alternaria malorum, a new causal agent of cowpea leaf spot disease in Iran

Abstract

Cowpea (Vigna unguiculata, Fabaceae) is an annual herbaceous legume cultivated for seed and fodder. During a survey in May 2022, cowpea leaves with leaf spot symptoms were collected from a field in which c. 40% of the plants were diseased (Karaj City, Alborz province, Iran). Leaf spots began as small circular water-soaked lesions at the leaf centre and margins which expanded and on occasion, coalesced. The infected tissue became pale brown and necrotic. Isolation of the causal pathogen was performed using the high humidity blotter method. After five days, growing fungi were transferred with a fine sterile needle to 2% water agar. Pure cultures were obtained using the hyphal tip method on synthetic nutrient-poor agar (SNA) and potato carrot agar culture media. Fungal colonies were incubated at 25°C with an 8 hour light/16 hour dark photoperiod for seven days. A typical colony (ALT1) reached 58 mm in diameter after seven days incubation on SNA culture medium. The colony was olive-brown to dark brown at the centre, with a white margin. Aerial mycelia were sparse. Mycelia were septate, pale brown and smooth–walled. Sporulation was abundant on SNA, mostly from the aerial hyphae. Conidiophores arose singly from the aerial mycelia, and were brown, erect, simple, short, smooth, frequently reduced to conidiogenous cells with a slight swelling at the apices, 7–30 × 3–5 μm. Conidiogenous loci were 1–3 per conidiogenous cell, and brown. Ramoconidia were 0–3 septate, ellipsoid to cylindrical, smooth, brown, 15–32 × 3–5 μm, with a short beak (n = 50). Conidia were subcylindrical, ellipsoid or fusiform, brown, smooth, in long acropetal chains, simple or with lateral branches and 8–11 × 3–3.5 μm (n = 50) (Figure 1). Morphological and cultural characteristics of the recovered isolate (ALT1) were similar to the description of Alternaria malorum provided by Goetz & Dugan (2006) and Crous et al. (2009). Molecular characterisation of isolate ALT1 was done by PCR amplification and sequencing of the internal transcribed spacer (ITS) region using the ITS1 and ITS4 primers (White et al., 1990). The obtained sequence was deposited in GenBank (Accession No. OP006224) and a BLAST search showed it had 100% identity to isolates of A. malorum (MW335156, LR134074, FJ839611 and KY077556). Maximum likelihood analysis was performed by heuristic search with Mega X software. In the generated phylogenetic tree (Figure 2), isolate ALT1 was separated from closely related Alternaria species and other fungal genera, and clustered in the same group as A. malorum isolates. The molecular analysis confirmed the morphological identification of the pathogen. Pathogenicity testing was performed by spraying a conidial suspension (106 conidia/ml) of isolate ALT1 on healthy and surface-sterilised cowpea leaves without wounding, and was replicated three times. Sterile water was sprayed on the leaves of control plants. All plants were covered with plastic bags to maintain high humidity and placed on a glasshouse bench at 25°C with a 12 hour photoperiod until the development of disease symptoms. Leaf spots appeared twelve days after inoculation (Figure 3) and resembled those observed in the field. The pathogen was re-isolated from the newly generated leaf spots, fulfilling Koch's postulates. In Iran, Alternaria malorum was first reported by Asgari et al. (2004) as Cladosporium malorum from the leaves of the Hordeum vulgare in East Azarbaijan province. Alternaria malorum has also been reported as a fungus associated with declining Persian oak trees (Alidadi et al., 2018). Bagherabadi & Zafari (2022) also reported A. malorum as the causal agent of bark canker in walnut trees. Several diseases are known to infect cowpea, including anthracnose, Cercospora leaf spot, damping off, Fusarium wilt, Sclerotium stem, root and crown rot, Septoria leaf spot, and scab (Dugje et al., 2009). This is the first report of Alternaria malorum as a causal agent of cowpea leaf spot disease in Iran and globally. We are thankful to the University of Tehran for supporting this research.