First Report of Leaf Spot of Pumpkin Caused by Curvularia hawaiiensis in Pakistan

Pumpkin (Cucurbita pepo)is a well-known edible plant with many medicinal properties. In Pakistan, this highly consumable crop has a great impact in the local markets. In October 2018, pumpkin plants (cv. Autumn Gold), growing on the premises of University of Agriculture, Faisalabad (31°26?14.3?N, 73°04?28.6?E)suffered a disease outbreak that resulted in severe leaf necrosis occurring on more than 51 percent of all plants in the 2-ha experimental field. Leaves displayed circular necrotic lesions, minor leaf tip necrosis, and leaf spotting (6 to 9 mm in diameter)covering approximately 20 to 30 percent of the leaf blade. To isolate the causal agent, the margins of infected leaves were cut into small pieces and surface sterilized with 0.1 percent HgCl2 for 1 min followed by 70 percent ethanol for 30 s and rinsed twice in sterile distilled water. The samples were blotted dry on sterilized filter paper, placed on the Petri plates containing potato dextrose agar medium, and incubated for 1 week at 25 ± 1°C with 12-h light/dark photoperiod. Initially, mycelium showed light-brown edges and dark brown pigmentation at the center of the Petri plate. Fungal colonies also formed two to three concentric rings on the reverse of the plate. Conidia were characterized as solitary, obovoid to fusiform, round at both ends, three to six distoseptate (16.53 to 27.50 × 6.31 to 7.87 µm). Conidiophores were simple or branched, multiseptate, straight to flexuous, smooth to verruculose, and cylindrical. The morphological characteristics were typical of Curvularia hawaiiensis (Kusai et al. 2016; Manamgoda et al. 2012). Sequence identification was performed using the D1 to D3 domains of the large subunit (LSU)and the internal transcribed spacer region (ITS)of the nuclear rDNA, and the fragments of translation elongation factor 1-? (tef1)and partial DNA-directed RNA polymerase II second largest subunit (rpb2)genes, which were amplified using primers LROR-F/LR5-R (Schoch et al. 2012), ITS1F/ITS4R (White et al. 1990), EF1-983F/EF1-2218R, and 5F2/7CR (O'Donnell et al. 2007), respectively. The sequences of the ITS (615 bp), rpb2 (771 bp), tef1 (918 bp), and LSU (912 bp)gene regions were deposited in GenBank with accession numbers MN053866, MN067868, MN067869, and MN055715, respectively. A BLASTn search of the derived sequences resulted in 99 percent nucleotide similarity of the ITS and rpb2 sequences and 100 percent similarity of tef1 and LSU genes with the corresponding sequences of C. hawaiiensis (GenBank accession nos. MH864413, HG779167, KM196587, and JN941532, respectively). Leaves of 3- to 4-week-old pumpkin plants (cv. Autumn Gold)were inoculated by spraying a conidial suspension of 106 conidia/ml prepared from 14-day-old culture of C. hawaiiensis. Plants were incubated at 25 ± 1°C and covered with plastic bags for the maintenance of relative humidity. After 8 to 10 days, plants showed the characteristic symptoms the same as the original, and no symptoms were observed on control plants sprayed with distilled water. The fungal pathogen (C. hawaiiensis)was successfully reisolated from the symptomatic tissues of the inoculated plants, compared with the original, and found identical, fulfilling the Koch's postulates. To the best of our knowledge, this is the first report of leaf spot of pumpkin caused by C. hawaiiensis in Pakistan and in the world. The identification of this pathogen is critical to ensuring maintained productivity of an important vegetable crop in Pakistan and should lead to the development of management strategies to combat the disease.