First report of Botryosphaeria dothidea causing fruit rot on Chinese olive (Canarium album Raeusch.) in Guangdong province of China.

ABSTRACT

Chinese olive (Canarium album Raeusch.) is a traditional Chinese medicinal plant, mainly cultivated in Guangdong and Fujian provinces in China (Lai et al. 2022). In October 2023, Chinese olive fruit spots were observed in all the Chinese olive orchards surveyed in Chaozhou city (23.75°N, 116.67°E) of Guangdong, with an incidence up to 15%. Early disease symptoms on fruits appeared as circular or irregular, dark brown to black spots with yellowish lesions, and later the spots slowly coalesced to form large necrotic areas, which seriously affected the fruit marketability. To isolate the causal agent, small pieces (~0.3 mm2) of fruit tissue were excised from the lesion margins, and surface-disinfested with 75% (v/v) ethanol for 1 min, followed by 1% NaClO for 3 min, and rinsed three times with sterile water. The pieces were then placed on potato-dextrose-agar (PDA) and incubated at 27°C. Ultimately, four fungal isolates were obtained with similar morphology phenotypes, colonies initially appeared white with irregular margins and after 4-6 days turned dark gray gradually with dense aerial myceliu. Microscopy revealed conidia were single-celled, hyaline, aseptate, fusiform to subclavate, and measured 18.1-22.5 µm × 6.4-9.3 µm (19.8 × 7.4 m on average, n = 30), which were consistent with those descriptions of Botryosphaeria dothidea (Vasic et al. 2013; Zhang et al. 2023). To further identity the isolates, partial sequences of ribosomal transcribed spacer (ITS), translation elongation factor 1-α (TEF1-α), and ß-tubulin (TUB2) genes were amplified using primers ITS1/ITS5, TEF-F/R, TUB2-F/R, respectively (Xu et al., 2023; Hong et al. 2006). The sequences of four isolates were identical, and those of representative strain GDCZ-1 were deposited in GenBank (ITS, OR584295; TEF1-α, OR685157; TUB2, OR685158). Using Neighbor-Joining algorithm, phylogenetic tree based on concatenated sequences of ITS, TEF1-α, and TUB2 showed that GDCZ-1 clustered with B. dothidea. To fulfill Koch's postulates, pathogenicity tests were performed on healthy Chinese olive fruits using the needle-prick inoculation method. The fruits were wounded with a sterile needle at the equatorial area (depth of 3-4 mm), and inoculated with 10 µL of spore suspension (106 /mL). The control fruits were inoculated with sterile water. Inoculated fruits were placed in sterile plastic containers to maintain high relative humidity (almost 100%) and incubated at 27°C. After 4 days, the inoculated fruits showed similar symptoms with those observed in the field infected fruits, while the control remained asymptomatic. Pathogen re-isolated from the inoculated fruits showed identical morphological characteristics to the original isolate GDCZ-1. As far as we know, fruit rot caused by Alternaria alternata has been recently reported on C. album in China (Shao et al. 2024). To our knowledge, this is the first report of B. dothidea causing fruit rot disease on C. album in Guangdong. Our report will provide crucial information for studying the epidemiology and management of this disease.