First Report of Botryosphaeria dothidea Causing Fruit Soft Rot on Pyracantha fortuneana in China.

Abstract

Pyracantha fortuneana (Maxim.) H. L. Li is mainly distributed in the southern and southwestern regions of China, locally called "Jiu Bing Liang". Recently, it has gained attention for its antioxidant and anticancer properties, positioning it as a promising functional food in China (Yao et al. 2020). On October 10, 2024, fruit soft rot of P. fortuneana was observed on the campus of Qujing Normal University (103.75°E, 25.52°N), Qujing City, Yunnan Province, with an incidence of 20%. The infected fruits exhibited shrinkage and soft rot, accompanied by a change in color from red to black. Small segments (0.2 × 0.5 cm) were excised from the junction of disease-healthy tissue of infected fruits, soaked in 75% ethanol for 4 minutes, rinsed in sterile water three times, and cultured on potato dextrose agar (PDA) at 28°C in the dark for 24 hours. A thin layer of white mycelium emerged around the tissue, which was subcultured onto fresh PDA medium. After 3 days, the colony center turned yellowish-gray, and the mycelium covered the entire 9 cm diameter of the medium after four days. After 10 days, the colony had turned completely blue-black. Eight strains with similar morphological characteristics were isolated, and one representative isolate (BHA) was used for morphological and molecular characterization. To observe conidia, culture conditions were modified by alternating temperatures (24°C and 28°C), exposure to alternating ultraviolet and visible light, and cultivation on nutrient-poor agar (SNA). Despite extending the observation period to 45 days, no conidia were detected. Molecular identification was performed using specific primers: ITS1/ITS4, EF1-728F/EF1-986R, and Bt2a/Bt2b to amplify the internal transcribed spacer (ITS1-5.8S-ITS2) region, the translation elongation factor (EF1-α) gene, and the beta-tubulin (β-tubulin) gene, respectively (Marsberg et al. 2017; Slippers et al. 2004). The sequences were deposited in GenBank under accession numbers PQ895675 (ITS), PV082023 (EF1-α), and PV068288 (β-tubulin). A BLAST search of GenBank showed that the ITS, EF1-α, and β-tubulin sequences of this isolate were similar to those of Botryosphaeria dothidea KY393137 (identity = 511/511; 100%), MN548726 (identity = 269/269; 100%), and MK511445 (identity = 434/434; 100%) respectively. A maximum-likelihood and Bayesian posterior probability-based phylogenetic analyses using PhyloSuite version 1.2.3 with the concatenated sequences (ITS, EF1-α, β-tubulin) placed BHA in the clade of B. dothidea. Based on the multigene phylogeny and morphology, BHA isolate was identified as B. dothidea. Pathogenicity was confirmed by inoculating PDA plugs containing mycelium onto both wounded and unwounded fruits of P. fortuneana. After seven days, symptoms developed exclusively on wounded fruits. The pathogen was reisolated from the infected tissues and reconfirmed as B. dothidea through morphological and molecular analyses, fulfilling Koch's postulates. To our knowledge, this is the first report of B. dothidea causing soft rot of P. fortuneana fruits in China. This study highlights the need to monitor and manage B. dothidea infections in P. fortuneana orchards to protect its economic and functional value in China.