First Report of Pomegranate Fruit Rot Caused by Botryosphaeria dothidea in Anhui Province of China

Abstract

Pomegranate (Punica granatum L.) is an important commercial fruit rich in antioxidants grown worldwide and China has the biggest planting area in the world. In spring 2019, symptoms of fruit rot were observed for the first time in some orchards located in Huaiyuan County of Anhui Province, one of the main pomegranate producing areas in China. Disease symptoms on fruit were characterized by enlarging and merging coarse irregular spots that coalesced and became large decay lesions on the fruit surface. Infected fruits developed internal rot, became discolor and dropped. Aggregated pycnidia were found in a black stroma on fallen mummified fruit. For isolation of the pathogen, more than twenty infected pomegranate fruit samples were collected from an orchard. After surface cleaning with sterile water to clear away some dust and other microbes, dried and disinfected with 70% ethanol for 1 minute, rinsed twice with sterile water, small pieces with diseased and healthy tissue parts were placed on water agar plates (WA). Small piece of every fungal isolate grown on the WA was transferred onto PDA and incubated at 28 °C for 5 days. All fungal colonies on potato dextrose agar (PDA) plates gradually changed from gray to dark grey with olive green back and abundant aerial mycelia, bearing pycnidia with fusiform, hyaline and 16.2 to 20.1× 3.8 to 5.4 μm (n = 50) in size. Twenty isolates with the similar cultural characteristics were stored for further work. The internal transcribed spacer (ITS) region, β-tubulin gene (TUB) and translation elongation factor 1-α (EF 1-α) gene were amplified and sequenced from three typical isolates using the universal primer pairs ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass et al. 1995) and EF1-728F/EF1-986R (Carbone et al. 1999), respectively. The BLAST results of ITS (MN453421), TUB (MN515421), and EF 1-α (MN515422) sequences showed 99.64%, 99.07% and 98.89% identity with the B. dothidea sequences available in the GenBank database (KP708996, MH724212 and KP183206), respectively. To complete Koch's postulates, healthy fresh pomegranate fruits were disinfected with 70% ethanol solution for one minute and rinsed with distilled water. Fruits were allowed to dry and inoculated on the wounded surface using a mycelial plug of 5 mm in diameter excised from the edges of actively growing fungal colonies, fruits were inoculated with PDA plugs served as negative control. Three fruits were used for each treatment and control. These fruits were kept under high humidity (95% RH) using sterile distilled water in artificial incubator and the experiment was repeated twice. Decay lesions resembling natural infections were observed on fruits after 5-7 days of inoculation. Re-isolation of the pathogen, which was also identified based on the above morphological and molecular analysis as B. dothidea, produced the same fungus as the one used for inoculating the fruit, thus completing the Koch's postulates. Therefore, the pathogen of this pomegranate fruit rot in China is B. dothidea. Botryosphaeria pomegranate rot caused by B. dothidea in Florida has been reported previously and was hypothesized that this fungus could be transmitted by leaf-footed bug (Leptoglossus clypealis, Michailides, 2014). To our knowledge, this is the first report of B. dothidea causing fruit rot on pomegranate in China.