First report of fruit rot of pomegranate caused by Aspergillus aculeatus from Bangladesh

Pomegranate (Punica granatum) is cultivated in Bangladesh but domestic production is supplemented by imports from China, India and South Africa. Common postharvest pathogens of pomegranate fruit include Alternaria alternata, Aspergillus niger, Botrytis cinerea and Penicillium spp. (Munhuweyi et al., 2016). In March 2021, imported pomegranate fruit with post-harvest rots were collected from a local market in Gazipur, Bangladesh (23° 59′ 20.4504'' N, 90° 25′ 5.4012'' E). Water-soaked spots initially developed and later, dark brown fungal spores were observed on the surface of the fruits (Figure 1). The disease incidence was approximately 15% of the consignment in the market store. Epidermal tissues of diseased fruit were plated on potato dextrose agar medium at 25°C. The fungus grew rapidly and produced brown to black colonies after three days' incubation (Figure 2a). The conidial head was radiated, vesicles (n = 25) were globose, mean 25.78-50.40 μm, and covered with uniseriate phialides. The stipe of the conidiophore (n = 25) was hyaline to pale with a mean width and length of 12.14-88.2 μm. Conidia (n = 25) were dark brown, transparent and ellipsoidal with a mean diameter of 3.1-5.54 μm (Figure 2b) (Rahman & Zakaria, 2020). To further ascertain the identity of the isolate, total genomic DNA was extracted using a Wizard DNA purification kit (Promega Corporation, USA) following the manufacturer's protocol. The ITS rDNA gene region was amplified using the primer pair ITS1/ITS4 (White et al., 1990), and the amplified PCR product was sequenced (GenBank Accession No. OP585638). A BLAST search in the GenBank database revealed that the isolate had 99% identity with sequences of Aspergillus aculeatus (OQ085123 and OP787511). Phylogenetic analysis showed that the ITS sequence data formed a well-supported clade with a support value of 87% (Figure 3). From this analysis, together with the morphological and molecular evidence, the isolate was identified as A. aculeatus. A pathogenicity test was conducted twice by inserting 40 μL of a conidial suspension (5 ×106 spores/mL) of the fungal isolate on six wounded pomegranate fruits. Sterile water was applied to six wounded fruits as a control. The fruits were placed at 25°C in plastic containers to maintain a humid environment. Disease symptoms resembling those observed in the market appeared two days after inoculation. Control fruits did not develop symptoms. Koch's postulates were fulfilled by re-isolating A. aculeatus from the inoculated fruits. Aspergillus aculeatus has been reported previously causing post-harvest rot on citrus and grapes (Adisa & Fajola, 1982; Leong et al., 2004; Ghezel et al., 2011). This study demonstrates the presence of A. aculeatus in pomegranate imported from an unknown source. Although it is unclear if A. aculeatus has become established in Bangladesh, it nevertheless represents a potential threat to the production of pomegranate in recently developed orchards in the country. This is the first report of the infection of Aspergillus aculeatus in pomegranate fruits in Bangladesh and worldwide.