First Report of Rugonectria rugulosa Causing Dieback of Prunus salicina "Banbianhong" in China.

Abstract

Banbianhong (Prunus salicina) is a valuable fruit crop cultivated in the southwestern regions of China for its high quality fruit and substantial economic benefits (Wang et al. 2021). In July 2023, dieback disease was observed in Banbianhong in Suijiang County, Zhaotong, Yunnan Province, with an incidence of up to 10%. The disease caused brownish nodules in the cortex of the infected plant, leading to dry cracking, and exposing grayish-brown xylem, which caused the plant to wither and die. Upon longitudinal cutting of twigs, lesions were evident and characterized by browning and necrosis of the cauline bundle and pith within the tissue. The diseased tissues were sectioned into 2 mm2 pieces and surface disinfected with 70% ethanol and 1% NaClO and rinsed with double distilled water. The tissue fragments were plated onto potato dextrose agar (PDA), and incubated for 7 days at 28°C. A total of six isolates were obtained, and two isolates (WG2-1 and WG2-3) were selected for morphological, molecular identification and pathogenicity testing. On PDA, the colony diameter of WG2-1 and WG2-3 were recorded as 5.50±0.56 mm/day and 6.08±0.37 mm/day, respectively. The microconidia of both isolates were zero-septate, smooth, greenish, subglobose to ellipsoidal, and measured 5 to 10 × 1.5 to 3 µm (n = 50) in size. However, macroconidia were not observed. The internal transcribed spacer rDNA (ITS) and β-tubulin 2 (TUB2) genes were amplified using the primer pairs ITS1/4 (White et al. 1989) and Bt2a/2b (Glass and Donaldson 1995). BLAST analysis of the obtained ITS sequences (accession Nos. PP581792) and TUB2 sequences (accession Nos. PP2815212) sequences showed 100% identity with Rugonectria rugulosa (accession nos. MG991753 for ITS and KM232007 for TUB2). Based on these characteristics, the isolates have been identified as R. rugulosa and have been deposited in the Agricultural Environment and Resource Research Institute plant pathogen lab at Yunnan Academy of Agricultural Sciences. In this study, Pathogenicity tests were conducted on one-year-old Prunus salicina "Banbianhong" twigs to complete Koch's postulates. The twigs were wounded using a 1-mm sterile corking borer and 14-day-old mycelium plugs of isolate (WG2-1) were inoculated and covered with sealing films. Sterile PDA plugs were placed into the wounds of control twigs. Eighteen healthy twigs were inoculated with isolates or PDA plugs (n = 6 twigs/treatment, the size of the twigs: 8 cm length and diameter about 0.7 cm, with three replications) and were placed in a closed sterile tray with the wet filter papers in a thermostatic incubator (type, HYC-260) at 28°C, respectively. The twigs inoculated with the isolates exhibited brown lesions site of inoculation, whereas the controls remained asymptomatic. R. rugulosa was successfully reisolated from the lesions but not from these control wounds and was identified by the methods described above. R. rugulosa has been reported to be associated with the die-back disease of Falcataria moluccana in China (Wang et al. 2019). The occurrence and spread of R. rugulosa in growing areas of Prunus salicina "Banbianhong" in Suijiang county have caused severe economic losses. To our knowledge, this is the first report of the die-back disease of Prunus salicina "Banbianhong" caused by R. rugulosa. These results confirm the pathogenicity of R. rugulosa in Prunus salicina "Banbianhong" and provide valuable insights for developing disease management and prevention strategies.