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Eggplant (Solanum melongena L.) is a popular vegetable that is grown in both tropical and subtropical regions all year long. The crop is cultivated on small family farms and is a good source of income for resource-limited farmers in Pakistan. In early May 2019, leaf spots on eggplant (cv. Bemisaal) were observed in an experimental field (31°26'14.0"N 73°04'23.4"E) at the University of Agriculture, Faisalabad, Pakistan. Early symptoms were small, circular, brown, necrotic spots uniformly distributed on leaves. The spots gradually enlarged and coalesced into large, nearly circular or irregularly shaped spots that could be up to 3 cm in length. The center of the spots was light tan, surrounded by a dark brown ring, a chlorotic halo, and tended to split in the later developmental stages. Disease incidence was approximately 35% in the infected field. The causal agent of this disease was isolated consistently by plating surface sterilized (1% NaOCl) sections of symptomatic leaf tissue onto potato dextrose agar (PDA). After 6 days incubation at 25°C with a 12-h photoperiod, fungal colonies had round margins and the cottony mycelia were dark olivaceous with a mean diameter of 7.5 cm. For conidial production, the fungus was grown on potato carrot agar (PCA) and V8 agar media under a 16-h/8-h light/ dark photoperiod at 25°C. Conidiophores were septate, light to olive golden brown with a conidial scar, from which conidia were produced. Conidia were borne singly or in short chains and were obpyriform to obclavate, measured 29 ± 4.8 × 13.25 ± 2.78 μm (n=30) with zero to three longitudinal and two to six transversal septa. The morphological characters matched those of Alternaria alternata (Fr.) Keisel (Simmons et al. 2007). DNA was extracted using the DNAzol reagent (Thermo Fisher Scientific MA, USA). For molecular identification, internal transcribed spacer (ITS) region between ITS1 and ITS2, actin gene (β-Actin), translation elongation factor (TEF-1α) gene, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of two representative isolates (JLUAF1 and JLUAF2) were amplified with primers ITS1/ITS4 (White et al. 1990), β-Actin 512 F/783 R, EF1-728F/-986R (Carbone et al. 1999), and gpd1/gpd2 (Berbee et al. 1999), respectively. The sequences were deposited in GenBank (accession nos. MT228734.1 and MT228735.1 for ITS; MT260151.1 and MT260152.1 for β-Actin, MT260163.1 and MT260164.1, for TEF-1a, and MT260157.1 and MT260158.1 for GAPDH). BLASTn analysis of these sequences showed 100% identity with the sequences of A. alternata for ITS rDNA, β-Actin, TEF-1α, and GAPDH, respectively. Based on the morphological characters and DNA sequences, the leaf spot isolates of eggplant were identified as A. alternata. To confirm the pathogenicity on eggplant, six-week old healthy potted eggplants of cv. Bemisaal were sprayed at the true leaf stage with conidial suspensions of A. alternata (106 conidia/ml; obtained from 1-week-old cultures) amended with 0.1% (vol/vol) of Tween 20 until runoff (1.5 to 2 ml per plant) using an atomizer in the greenhouse. Three plants were inoculated with each of the two isolates (JLUAF1 and JLUAF2), whereas three control plants were sprayed with sterile distilled water amended with 0.1% Tween 20. The plants were incubated at 25 ± 2°C in a greenhouse, and the experiment was conducted twice. After 10 days of inoculation, each isolate induced leaf spots which were similar to typical spots observed in the field, whereas the control plants remained symptomless. The fungus was re-isolated from symptomatic tissues. Re-isolated fungal cultures were morphologically and molecularly identical to A. alternata, thus fulfilling Koch's postulates. Previously, A. alternata has been reported to cause leaf spots on eggplant in India (Raina et al. 2018). To our knowledge, this is the first report of A. alternata causing leaf spot on eggplant in Pakistan. The disease could represent a threat for eggplant crops due to its increasing cultivation. It is important to develop disease management strategies for Alternaria alternata causing leaf spot of Eggplant in Pakistan. |
