In June 2015 & 2016, a postharvest survey of table grapes (Vitis vinifera) cv. King's Ruby, was carried out in five different commercial fruit markets of Rawalpindi (33°38'19.2″N, 73°01'45.0″E) district, Punjab Province. Symptoms appeared as brownish lesions with black sporulation on grapes berries. The incidence of these symptoms on bunches ranged from 12 to 17% at all sites. Symptomatic tissue pieces were surface-sterilized with 0.1% sodium hypochlorite (NaOCl) for 30 seconds, rinsed three times with sterile distilled water, dried on filter paper for 45 seconds, and incubated on potato dextrose agar (PDA) at 25°C. After 3 days, dark brown to black mycelium were formed on PDA media. A total of 24 isolates were examined morphologically. The apex of the conidiophore was observed to be radiate. Vesicles were found to be spherical and covered with irregular metulae and phialides. Conidia were globose or subglobose measured (3.14 μm ± 2.24 in averaged diameter: n=50), dark brown to black, with roughened cell walls. The conidiophores were also smooth-walled, hyaline, and became melanized toward the vesicle. These characteristics of the fungus were similar to those described for Aspergillus niger van Tiegh (de Hoog et al. 2000). For molecular identification, the internal transcribed spacer (ITS) region, beta-tubulin (Bt) gene and partial RNA polymerase II largest subunit (RPB2) gene of representative isolate (Asp.n02) was amplified using primers ITS1/ITS4, BT2a/BT2b and RPB2-6F/RPB2-7R respectively (White et al., 1990; Glass & Donaldson, 1995; Liu et al. 1999). Sequences were deposited in GenBank (ITS, MN658871; Bt2, MT117924; and RPB2, MT318289). Based on BLAST analysis, sequences of the ITS region, Bt2 genes, and RPB2 gene showed 99 to 100% similarity of isolate Asp.n02 to Aspergillus niger (Accession Nos. MK307680.1, MN195121.1, MF078661.1 for ITS gene, MN567299.1, MK451029.1, MK451020.1 for Bt2 gene, and MK450788.1, MK450790.1 for RPB2 gene). To complete Koch's postulates, 10-µl aliquots of spore suspensions (106 spores/ml) of isolate: Asp.n 02 was pipetted onto three non-wounded and four wounded (5 mm diam) asymptomatic grape berries cv. King's Ruby (seven berries per isolate), Sterile distilled water was applied to asymptomatic berries similaries to serve as a negative control (Ghuffar et al. 2018; Jayawardena et al. 2018). Berries were incubated at 25 ± 2°C in sterile moisture chambers, and the experiment was conducted twice. Brownish lesions leading to black sporulation similar to the original symptoms were observed on both wounded and non-wounded inoculated berries after 3 days, whereas no symptoms were recorded on the negative control. The morphology of the fungus that was re-isolated from each of the inoculated berries was identical to that of the original cultures. Aspergillus niger was reported previously in Europe and Israel causing mycotoxin (Ochratoxin A) OTA production on Table grapes (Bau et al. 2006). To our knowledge, this is the first report of Aspergillus niger causing black rot of grapes in Pakistan. This finding will help to plan effective disease management strategies against the black rot of grapes in Pakistan.
Keywords: Aspergillus niger; ITS region; RPB2; beta tubulin.