First Report of <i>Botryosphaeria dothidea</i> Causing Hawthorn Ring Rot Disease in Tianjin, China

Hawthorn (Crataegus pinnatifida), a significant medicinal and edible tree and a key fruit crop in Tianjin, was newly affected by ring rot in October 2023 and 2024 at a Tianjin orchard (39.059880°N, 117.044400°E). In a 0.3-hectare experimental planting, fruit rot incidence reached 53.6% (15 of 28 trees), causing about 30% yield loss at packing. Symptoms started as reddish-brown circular lesions that turned brown, formed concentric rings, and led to soft rot and fruit decay. To identify the causal pathogens, two typically symptomatic fruits were collected from each of 10 trees, totaling 20 samples. Infected fruit were disinfected with 1% sodium hypochlorite for 1 min, followed by 75% ethanol for 30 s, and washed three times with steile water. Air-dried tissues pieces (5 mm × 5 mm) taken from lesion margins were plated on potato dextrose agar (PDA) and incubated at 28°C and 80% relative humidity. Twelve morphologically similar strains were isolated, all showing initially appeared white, and gradually turned dark gray with dense aerial mycelia. Mycelium producing abundant conidia appeared transparent, branched, and septate after 21 days. Conidia were fusiform, hyaline, aseptate, measuring 15.06 - 22.92 μm × 7.22 - 8.34 μm (n = 50). Based on these features, the isolates were preliminarily identified as Botryosphaeria dothidea (Yan et al. 2013). Strain SZLW5 was selected for molecular identification. The ITS region, EF1-α, and TUB2 genes were amplified using primers ITS1/ITS4, EF1-728F/EF-986R, and Bt2a/Bt2b, respectively (Carbone and Kohn 1999; Zheng et al. 2020). Sequences were deposited in GenBank with accession nos. PP257689 (ITS), PP297567(EF1-α) and PP297568 (TUB2). BLASTn analysis of three sequenced genes showed 99.6 to 100% identity with the ex-type strain of B. dothidea CMW 7780. A multi-gene phylogenetic tree constructed using Maximum Likelihood in MEGA 7.0 confirmed that SZLW5 clustered with B.dothidea. Thus, morphological and molecular data identified the pathogen as B. dothidea. The pathogenicity of strain SZLW5 in hawthorn was tested using Koch's postulates. Sterilized hawthorn fruits were inoculated with 5-mm colony discs of SZLW5 at 28°C and 80% humidity for 7 days. The negative control used PDA plugs of the same size. Dark brown lesions, matching field symptoms, appeared on inoculated fruits, while controls showed no symptoms. Pathogens were isolated and purified from lesions, and the strain was confirmed as SZLW5 based on morphology and molecular biology. In this study, the pathogen of hawthorn ring rot in Tianjin was isolated and purified. Through morphological comparison, molecular biology and pathogenicity identification, the pathogen was identified as B. dothidea. B. dothidea is a widely distributed plant pathogenic fungus which can cause fruit rot, branch wilt and other diseases (Garibaldi et al. 2012; Wang et al. 2021; Wang et al. 2025; Zheng et al. 2020), resulting in serious economic losses to crop production. To our knowledge, this study represented for the first time that the pathogen causing ring rot on C. pinnatifida is B. dothidea in Tianjin, China, providing a reference for the breeding of resistant varieties and the exploration of prevention and control strategies for hawthorn ring rot.