First Report of Black Rot on Persimmon Fruit Caused by Diaporthe eres in China.

Abstract

The fruit of persimmon (Diospyros kaki L.) is popular with consumers for its delicious taste and is widely grown in China. In September 2022, fruit rot symptoms were found on persimmons grown at the Tobacco Research Institute of Chinese Academy of Agricultural Sciences (120°26'43.879″E, 36°7'59.794″N) in Qingdao City, Shandong Province, China. Diseases incidence on fruit is 70%. Typical symptoms were small, black spots, which gradually expanded, and became rot lesions, causing fruit rot and serious losses. To isolate the pathogen, small sections (approximately 2 × 2 mm) of symptomatic tissues from five persimmon fruits were surface-sterilized in 5% NaClO, followed by 75% ethanol, rinsed with sterile distilled water, and then incubated on potato dextrose agar (PDA) plates incubated at 28℃ for 5 days. By single spore isolation method, seven dominant fungal isolates developed with similar colonies. They were initially white and gradually turned to gray after 7 d. The alpha conidia were unicellular, colorless, elliptic or fusiform, and 5.0 to 8.0 μm × 2.5 to 3.5 μm in size. Beta conidia were unicellular, colorless, filiform, often hooked at one end and 18 to 28 μm × 1.3 to 1.5 μm in size. These morphological features were consistent with Diaporthe spp (Udayanga et al. 2014). To confirm identity of the causal fungus, the internal transcribed spacer (ITS) rDNA, translation elongation factor 1-a (TEF1-a), b-tubulin (TUB), and histone H3 (HIS) genes of three representative isolates (De2, De3 and DeR2) were amplified and sequenced using primers ITS1/ITS4, EF1-728F/EF1-986R, Bt2a/Bt2b and CYLH3F/H3-1b as described by Chaisiri et al. (2021). The resulting sequences were deposited in GenBank (accession nos. OP963195, OP963386 and OP963389 for ITS; OP968958, OP968959 and OP9689600 for EF1-α; OP978141, OP978142 and OP978143 for TUB; OP968955, OP96896 and OP968957 for HIS), and showed 99-100% similarity with D. eres by NCBI blast. Phylogenetic analysis was conducted using MEGA-X software, with the NJ method. In the phylogenetic tree, the combined sequences attributed the three isolates to the D. eres. On the basis of morphological and molecular characteristics, the pathogen was identified as D. eres. The strain De2 was preserved in the Agricultural Culture Collection of China (preservation number: ACCC 35482). Pathogenicity tests were performed by wound inoculation on healthy persimmon fruit. These were surface sterilized with 75% ethanol for 1 min, rinsed in sterile water three times, and allowed to dry. A 5-mm agar disc with mycelium of the causal pathogen was placed on the surface of fruit, which was wounded with a sterile needle in advance, with the sterile PDA plugs used as control. Three persimmons were used for each treatment, repeat three times. All the treated fruits were incubated in the sterilized transparent plastic cans at 28°C and 90% RH. After 3 days inoculation, the first symptom of rot lesion appeared on the fruit with mycelial plugs, whereas the control fruit remained symptomless. The same causal pathogens were re-isolated from the lesions and identified by morphologyand confirmed to fulfill Koch's postulates. This is the first report of D. eres causing fruit rot on persimmon. This finding showed that Diaporthe eres is a causal agent of persimmon fruit rot in China.