First Report of Alternaria alternata Causing Fruit Rot of Grapes in Pakistan

In July 2016, a severe fruit rot was observed on table grapes (Vitis vinifera) from five different fruit packing houses in the Rawalpindi district (33°38′19.2″N, 73°01′45.0″E) of Punjab province, Pakistan. During the survey, dark gray to black mycelium was observed on the surface of the berries and rapidly expanded to entire bunches. After 3 to 5 days of postharvest storage at ambient temperature, prevalence of fruit rot was 100% in all locations of Rawalpindi district. A total of 200 symptomatic berries were surface disinfested with 1% sodium hypochlorite for 2 min, rinsed three times with sterile distilled water, and then dried on a filter. Excised pieces of sterile tissue (4 mm²) at lesion margins were transferred to potato dextrose agar (PDA) and incubated at 25 ± 2°C for 3 to 5 days. Fungal cultures derived from the isolates gave rise to colonies that were dark olivaceous green with light brown margins, velvety, appressed, and cottony at the center. Conidiophores were septate, bent or sometimes branched, 25.6 to 55.4 μm long and 2.5 to 3.6 μm wide. Conidia were borne singly or in short chains and were obpyriform to obclavate, measuring 9.5 to 55.5 × 6.1 to 25.6 μm with zero to three longitudinal and two to six transverse septa. Morphological characters matched those of Alternaria alternata (Fr.) Keissl (Simmons 2007). For molecular identification, ITS1, 5.8S; ITS2, RNA polymerase II (RPB2); and endopolygalacturonase (endoPG) gene of two representative isolates (Alt 01 and Alt 05) were amplified with primers ITS1/ITS4 (White et al. 1990), fRPB2-7cF/fRpb2-7cR (Liu et al. 1999), and PG3/PG2b (Andrew et al. 2009), respectively. The amplicons were sequenced and deposited in GenBank (accession nos. MG763130, MF785102, MG773211, MG773212, MG786192, and MG786193). A BLAST search of the GenBank database revealed close identity (98 to 100%) with the sequences of various A. alternata isolates (accession nos. KU182490 and MF141012 for ITS gene, KX938348 and KX938349 for RPB2 gene, and KY969535 and KY969535 for endoPG gene) of A. alternata in NCBI. To confirm pathogenicity, 10-µl aliquots of conidial suspension (10⁶ spores/ml) were pipetted onto three nonwounded and four wounded asymptomatic grape berries per isolate. The berries were incubated at 25 ± 2°C in sterile moist chambers, and the experiment was conducted twice. After 3 days, dark gray to black mycelium, similar in appearance to the original isolates, developed on both wounded and nonwounded inoculated berries, whereas control berries inoculated with sterile distilled water remained symptomless. The morphology of the fungus isolated on PDA was identical to that of the original cultures. A. alternata has been reported to cause postharvest fruit rot on grapes in South Africa (Swart and Holz 1991) and Slovakia (Kakalikova et al. 2009). To our knowledge, this is the first report of A. alternata causing fruit rot of grapes in Pakistan, where the disease poses a significant threat to sustainability of grape producers.