First report of Alternaria alternata causing Alternaria leaf spot of cotton in Pakistan

Cotton (Gossypium L.), known as “white gold,” is a globally important fiber and food crop. Pakistan is the world’s fourth largest cotton producer, with cultivation concentrated along the Indus River in of Punjab and Sindh. Cotton occupies 14% of Pakistan’s cultivated area (1). Fungal pathogens remain the most serious challenge for growers. In 2022–2023, similar disease symptoms were observed on upland cotton leaves at maturity in Multan and Rahimyar Khan districts of Southern Punjab. Symptoms begin as small circular lesions on the upper leaf surface, enlarging into necrotic dark-brown concentric rings up to 1.5 cm. Disease incidence was 20–25%. Forty diseased samples were collected, surface disinfected with 2% NaOCl for 1 min, rinsed thrice with sterile distilled water, and plated on Potato Dextrose Agar (PDA). Plates were incubated at 25 ± 2°C in dark, then purified. A total of 28 single-spored isolates were obtained for morphological characterization. Colonies on PDA were olivaceous brown to black after 7–8 days of incubation. Conidiophores were septate, light to olive golden brown with conidial scars. Conidia occurred singly or in short chains, obpyriform to obclavate. They began small, oval, brown with septa, later enlarging into longitudinal conidia with a beak. These features matched Alternaria alternata (Fr.) Keissler (2). Mycelia from 10-day-old cultures of four arbitrarily selected isolates (GHLS01, GHLS04, GHLS12, and GHLS17) were collected for DNA extraction using the Qiagen DNeasy Kit. PCR of ITS rDNA with ITS1/ITS4 (3) and GAPDH with gpd1/gpd2 (4) were performed. Sequences were deposited in GenBank (PV747746.1–PV747749.1 ITS; PV759324.1–PV759327.1 GAPDH). BLASTn analysis showed 99–100% sequence identity with isolates of A. alternata. Pathogenicity was confirmed by attached leaf assay. The experiment used 40-day-old Upland cotton plants in a greenhouse maintained at 25–30°C with ≥90% relative humidity, conducive for disease development. A total of 36 selected and tagged leaves from 12 plants were inoculated with the four isolates (GHLS01, GHLS04, GHLS12, and GHLS17). Mycelial plugs (5 mm) from 10-day-old PDA cultures were ground in sterile water to 10⁶ conidia/ml and applied to leaves by using spraying inoculation method. Control plants (n = 3) were untreated; the experiment was repeated twice. After 11–12 days, small brown spots expanded into concentric rings; controls remained asymptomatic. The pathogen was re-isolated and found morphologically and molecularly identical to A. alternata, thus satisfying Koch’s postulates. Alternaria alternata has previously been reported on upland cotton in New Mexico, (5) and Arizona (6). To our knowledge, this is the first report of A. alternata causing Alternaria leaf spot of cotton in Pakistan. This finding highlights a potential emerging threat to cotton production in southern Punjab and underscores the need for continued disease surveillance and management strategies.