First Report of Botryosphaeria dothidea Causing Fruit Rot on Hawthorn (Crataegus pinnatifida) in China.

Abstract

Hawthorn (Crataegus pinnatifida) is an important economic fruit and Chinese medicinal plant, which is widely distributed in the northern China. In early July 2024, a fruit rot disease was observed on the young fruits of hawthorn in a park of Shouguang, Shandong Province, China (36°53'42.16″N, 118°47'22.4″E). Forty plants were surveyed (from approximately 120 trees), which had 15 to 30% of fruits infected. Diseased fruits displayed brown, sunken, approximately circular lesions. Small sections (3-4 mm) cut from the edges between the diseased and healthy tissue from 20 diseased fruits were surface sterilized by using 75% ethanol for 30 s, and rinsed thrice with sterile distilled water. The diseased tissues were then dried and plated onto potato dextrose agar (PDA) and incubated at 25°C in the dark. Cultures were purified by hyphal tipping. In total, 12 colonies were isolated from the necrotic tissues, and all of them showed similar morphological characteristics. The colonies were initially white, gradually turning gray-green to dark gray after 5 days. Conidia were hyaline, aseptate, ellipsoidal to obovoid, 20.1-29.3 × 5.0-8.3 μm, with an average of 24.2 × 6.6 μm (n = 30). These morphological traits suggested that the pathogen shares similarities with the Botryosphaeria dothidea (Zhang, et al., 2021). For accurate identification, three representative isolates (SGSZ-01 to SGSZ-03) were selected for molecular identification by amplifying and sequencing the internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (TEF1-α), and β-tubulin (TUB2) with the primers ITS1/ITS4, EF1-728F/986R, and Bt2a/Bt2b (Carbone and Kohn 1999; Jia et al. 2019), respectively. GenBank accession numbers were PQ197601, PQ586368, and PQ586369 for ITS, PQ201924, PQ588676, and PQ588677 for TEF1-α, and PQ201925, PQ588678, and PQ588679 for TUB2, and gene sequencing showed 99.2 to 100% identity with the ex-type strain of B. dothidea (CMW 8000). Results from the maximum likelihood phylogenetic analysis, based on the three concatenated gene sequences, placed our isolates in a clade with B. dothidea. The pathogenicity of SGSZ-01 was conducted using a wound inoculation method on 11-week-old fruits of 10-year-old hawthorn trees both on living plants and detached fruits. All fruits were wounded with a sterilized needle and inoculated by placing 5 mm² agar plugs of 5-day-old cultures grown on PDA. Control fruits were treated with agar plugs. All the attached fruits were enclosed in a plastic bag with a wet cotton ball for 5 days. The same number of detached inoculated fruits were placed in Petri dishes on a piece of wet filter paper and incubated at 25°C. The pathogenicity test was conducted three times independently. Necrotic lesions (4.5 ± 0.3 mm in diameter) resembling natural infections were observed on fruits 7 days after inoculation, and no symptoms appeared on the control fruits. The pathogen was reisolated from the lesions and was identified by the methods described above. No pathogens were isolated from control fruits. Therefore, the pathogen of hawthorn fruit rot in China is B. dothidea. B. dothidea has previously been reported causing fruit rot on mango, plum, pomegranate fruit, and apple in China (Feng et al. 2023; Gu et al. 2020; Tang et al. 2012; Yuan et al. 2024). To our knowledge, this is the first report of B. dothidea causing fruit rot disease on C. pinnatifida. The information on identification of this fungus may be helpful to the control and prevention of the disease.