Fruit Rot on Persimmon Caused by Neopestalotiopsis saprophytica and Neopestalotiopsis ellipsospora in Guangxi, China

Persimmon (Diospyros kaki Thunb.) is widely cultivated in China. On October 15, 2019, about 10% of persimmon fruits showed fruit rot in the orchards of Guilin, Guangxi, China (24°45' N, 110°24' E), which could cause more than 15% of yield losses. The initial symptoms of fruit rot exhibited irregular brown to black spots (range from 2 to 4 cm in diameter), the areas surrounding the blackened spots would be soft and rotten, and three diseased fruit samples were collected from three orchards, respectively. Tissues (5×5 mm) were cut from infected margins, surface-disinfected in 75% ethanol for 10 s, 2% NaClO for 2 min, rinsed three times in sterilized distilled water, and incubated on potato dextrose agar (PDA) at 25°C under 12/12 h light/darkness for a week. Forty-one tissues yielded morphologically similar cultures, and three representative isolates LPG1-1, LPG1-2, and YSG-1 were selected from three samples for further study, respectively. Their colonies showed wavy edges, white surfaces, and dense aerial hyphae on PDA after two weeks. Conidia were fusiform, straight to slightly curved, and 4-septate; basal cells were conical, hyaline, thin, and verruculose with two or three long and hyaline apical appendages and one short apical appendage; three median cells of LPG1-1 with length 14.06 to 17.69 μm (n=100), and LPG1-2 with length 14.03 to 17.61 μm (n=100) were dark brown to olivaceous, while three median cells of YSG-1 with length 12.54 to 15.58 μm (n=100) were dark brown. The conidial sizes of LPG1-1, LPG1-2, and YSG-1 were 17.41 to 27.68 × 4.63 to 8.55 μm (n=100), 18.06 to 27.41 × 4.33 to 8.21 μm (n=100), and 16.58 to 27.73 × 4.99 to 8.39 μm (n=100), respectively. The morphological characteristics were consistent with Neopestalotiopsis spp. (Maharachchikumbura et al. 2012; Maharachchikumbura et al. 2014). Primer pairs ITS4/ITS5, BT2a/BT2b, and EF1-526F/EF-1567R were used to amplify internal transcribed spacer (ITS), beta-tubulin (TUB2), and translation elongation factor 1 alpha (TEF1-α), respectively (Shu et al., 2020). All DNA fragments were sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). Sequences have been deposited in GenBank (ITS: OM349120 to OM349122, TUB2: OM688188 to OM688190, TEF1-α: OM688191 to OM688193). Based on BLASTn analysis of ITS, TUB2, and TEF1-α sequences, the LPG1-1 and LPG1-2 showed over 99% similarity to N. saprophytica, and YSG-1 showed over 99% similarity to N. ellipsospora. Phylogenetic analysis of the three isolates was performed with MEGA10 (version 10.0) based on sequences of ITS, TUB2, and TEF1-α using maximum parsimony analysis. The results revealed that LPG1-1 and LPG1-2 were clustered with N. saprophytica, and YSG-1 was clustered with N. ellipsospora. Pathogenicity tests of three isolates were conducted on 72 healthy persimmon fruits with and without wounds, and 9 fruits are for each treatment. The wound was made by a sterilized needle. Fruits were pre-processed with 75% ethanol for 10 s, 1% NaClO for 2 min and rinsed three times in sterile water. Conidial suspensions (10 µL, 106 conidia/mL in 0.1% sterile Tween 20) were inoculated on each site (4 sites/fruit). Control group was treated with 0.1% sterile Tween 20. All inoculated sites were covered with wet cotton. The inoculated fruits were placed in a plastic box to maintain humidity at 28℃. After 5 days, all wounded fruits showed fruit rot, whereas unwounded and control fruits remained asymptomatic, there were significant differences (P<0.05) in aggressiveness between N. saprophytica (average lesion diameter 13.1 mm) and N. ellipsospora (average lesion diameter 14.9 mm). Koch's postulates were fulfilled by re-isolating the causal agents from inoculated fruits. N. ellipsospora was previously reported as an endophyte in D. montana in southern India (Reddy et al. 2016). N. saprophytica could cause leaf spot of Erythropalum scandens and Magnolia sp., and fruit rot of Litsea rotundifolia in China and leaf spot of Elaeis guineensis in Malaysia (Yang et al. 2021, Ismail et al. 2017). To our knowledge, this is the first report of N. ellipsospora and N. saprophytica causing fruit rot on persimmon in the world. The results will provide a foundation for controlling fruit rot caused by pestalotioid fungi on persimmon.