First Report of Neopestalotiopsis rosae Causing Postharvest Fruit Rot of Sweet Persimmon in Mengzi, China

Sweet persimmon (Diospyros kaki L.) is a deciduous perennial tree widely cultivated across Asia, prized for its nutritious and commercially valuable fruit. Between September and October 2024, postharvest fruits of Diospyros kaki in fields located in Mengzi City (23°22'N, 103°23'E), Yunnan Province, southwestern China, developed symptoms of black decay spots accompanied by white mycelium and black dots. The disease incidence was recorded at 25 to 40% in natural conditions (n=100). The 22 infected fruits were surface-disinfected with 75% ethanol for 30 s, followed by 1% NaClO for 2 min, and then rinsed three times with sterile water. Forty diseased tissues were put on the medium of potato dextrose agar (PDA) and incubated at 25±1°C in darkness for 7 days. Fungal hyphal tips were subsequently transferred to fresh PDA plates for purification. Twenty fungal isolates were obtained via single-spore isolation. All isolates exhibited similar morphological characteristics. Three representative isolates (YNDK05, YNDK09, and YNDK14) were preserved in 15% glycerol at -80°C in the Laboratory of Plant Disease and Pest Biology, Honghe University. After 7 days, the colonies were circular and up to 85 mm in diameter on PDA plates. The aerial mycelium appeared white initially and gradually darkened in color, which was velvety to fluffy in texture, forming concentric zonation. The globose, solitary, semi-immersed, black conidiomata formed superficially, scattered over PDA medium after incubation for 14 days, and contained slimy black conidial mass. Conidiophores were cylindrical, hyaline, septate, with a slightly flexuous and swollen apex. Conidia were fusoid or ellipsoidal, and had four septa and five cells with an average dimension of 27.86 × 8.27 μm ‌(n=50). Basal and apical cells were hyaline, while three middle cells were brown with darker septa. The apical cells had two to three appendages (10.17 to 28.33 μm in length), and the basal cell had a single appendage (6.27 to 16.71 μm in length). And the three isolates described above were used for molecular identification. Fragments of the internal transcribed spacer (ITS), partial translation elongation factor (TEF), and β-tubulin (TUB2) gene regions were amplified with primer pairs ITS4/5, EF1-728F/EF-2 and Bt2a /2b, respectively (White et al. 1990, Glass et al.1995, Carbone et al. 1999). The sequences were uploaded to GenBank (YNDK05: PX497391, PX739024, PX739021; YNDK09: PX497392, PX739025, PX739022; YNDK14: PX497393, PX739026, PX739023). The ITS, TEF, and TUB sequences showed similarities of 99.63%, 99.79%, and 99.32% to the type strain of Neopestalotiopsis rosae (CBS 101057) in BLASTn searches. A phylogenetic tree was constructed by combining the ITS, TEF, and TUB regions of three isolates using PhyloSuite, thereby confirming the results. Pathogenicity tests were performed using the three fungal isolates. Nine mature fruits of sweet persimmon were wound-inoculated at each of 2 sites per fruit with 200 μl of conidia suspension of these isolates (×106 conidia/ml) for 14 days. Degreasing cotton dipped in sterile water was used to raise the humidity in preservation boxes. The boxes were incubated at 25°C for 14 days under a 12:12 h light/dark photoperiod. Three persimmons as controls were treated only with sterile distilled water in the same way. The test was performed thrice. Within 2 days of inoculation, typical sunken lesions and rot appeared, expanding to diameters of 1.6 to 3.3 cm after 5 days. The same pathogen was re-isolated from the diseased tissues on day 15 post-inoculation, confirming Koch's postulates. No symptoms were observed on control fruits. Morphological and phylogenetic analyses identified the fungal pathogen as Neopestalotiopsis rosae (Maharachchikumbura et al., 2012, 2014). To our knowledge, this is the first report of Neopestalotiopsis rosae causing postharvest fruit rot on sweet persimmon in Yunnan, China. The disease will affect quality and taste of persimmon, so it is critical to deploy appropriate management strategies to limit the fungus spread.