First Report of Causing Soft Rot on Pear Fruit in China.

Pear (Pyrus pyrifolia) is an economically important fruit and widely planted in China. In September 2023, soft rot disease was observed on more than 20% of pear fruits (cv. Jinghua) in a plantation of approximately 1.0 ha of Shilin County, Kunming City, Yunnan Province, China. The initial symptoms manifested as water-soaked lesions. Then, they rapidly expanded and covered the whole fruit, which was soft and necrotic. At severe stage, infected fruits turned brown and dropped prematurely. To isolate and identify the causal pathogen, two diseased fruits were collected and pieces of rotted fruit tissue were cut into small fragments (5×5 mm), disinfested in 75% ethanol (30 s) and 2% sodium hypochlorite (60 s), and rinsed three times with sterile distilled water. The sterilized sections were soaked in 2 ml of sterile water and shaken for 3 min in a vortex oscillator. The suspension was streaked on Luria-Bertani (LB) agar medium. After incubation at 28°C for 36 h, single colonies were restreaked three times to obtain purified isolations. Thirteen bacterial strains with similar morphology were isolated from different fruits (seven strains in one fruit and six in the other), and their colonies were yellowish white, round, and convex with smooth surfaces on the LB agar plates. Representative strains YNPA1 and YNPA2, isolated from different fruits, were selected for further analyses. The two strains were Gram-negative, tested negative for citrate and catalase, but positive for sucrose and glucose. The 16S rRNA gene of these strains (GenBank accession nos. PP917741, PP917742) was amplified using primer pair 27F/1492R and sequenced. BlastN searches revealed that the obtained sequences shared >99% identity with Pectobacterium aroidearum type strain (GenBank accession no. NR_159926). A multilocus sequence analysis based on concatenated sequences of five housekeeping genes was conducted, of which gyrA (product sizes: 987bp, GenBank accession no. PP928290, PP928291), icdA (589bp, PP928292, PP928293), mdh (547bp, PP928288, PP928289), proA (734bp, PP928294, PP928295), and rpoS genes (892bp, PP928296, PP928297) was amplified, using primer pair gyrA1/gyrA4, icdA400F/icdA977R, mdh86F/mdh628R, proAF1/proAR1, rpoS1/rpoS2, respectively (Ma et al. 2007; Waleron et al. 2008). The reconstructed maximum likelihood tree (Tamura-Nei model; with 1,000 bootstrap replicates) showed that strains YNPA1 and YNPA2 clustered with strains of P. aroidearum. Pathogenicity tests were performed on three healthy pear fruits by injecting 10 μl of bacterial suspensions of P. aroidearum strains YNPA1 and YNPA2 (108 CFU/ml), respectively; another three healthy control pear fruits were injected with 10 μl of sterile water. All tested fruits were covered with plastic bags and incubated at 28°C and 80% humidity in a growth chamber. After 3 days, all inoculated pear fruits showed soft rot symptoms resembling those observed in the plantation, while control fruits remained symptom-free. Bacteria were successfully reisolated from the symptomatic tissues and identified as P. aroidearum by PCR and sequencing of the 16S rRNA gene as described above. P. aroidearum has been reported as a pathogen of Syngonium podophyllum (Xu et al. 2020) and Prunus persica (Liang et al. 2022) in China. The current study expands the known host range of P. aroidearum and helps raise attention for controlling pathogen's spread.