First report of Colletotrichum plurivorum and Colletotrichum chrysophilum causing fruit anthracnose on Chinese olive ( Canarium album ) in Guangdong, China

Chinese olive (Canarium album) is a popular edible-medicinal plant widely cultivated in southern China and increasingly threatened by fungal diseases (Zhuang et al. 2024). Between April and June 2025, Chinese olive plants in a roughly 300‑hectare conventional orchard in Jieyang, Guangdong, China (23°28′39.14″N, 116°0′46.97″E), which had been managed for sustainable Chinese olive production for more than ten years, developed anthracnose on fruits, with a disease incidence of 43% (n = 100 plants). Symptoms initially appear as small, circular to irregular, grayish-brown spots on the fruit surface, particularly at the apex, with slightly raised, sharply defined margins. The spots gradually enlarge and become sunken, developing a grayish-white center and dark brown margins. Lesions often coalesce, eventually leading to partial or complete fruit rot, followed by fruit drop. To identify the causal agent, 20 symptomatic fruits from 20 trees were collected. Small pieces (approx. 2 × 2 mm) were excised from the necrotic lesion edges of the symptomatic fruits and surface-disinfected in 75% ethanol for 10 s and 2% sodium hypochlorite (NaClO) for 1 min and washed three times in sterile water. The pieces were placed on potato dextrose agar (PDA), incubated in the dark at 28°C for 5 days, then subcultured and incubated for another 7 days. A total of 20 morphologically similar colonies were obtained, with 100% isolation frequency. Of the 20 isolates, FA-1, FA-2, FA-3 were selected as representative strains for intensive study. Colonies on PDA were initially white and turned light grey with dense aerial mycelium after 7 days. Off-white mycelia were observed for all isolates. The conidia of isolates FA-1, FA-2, and FA-3 were hyaline, aseptate, straight cylindrical with rounded ends, and measured 12.0–18.0 × 4.5–5.7 μm, 12.5–18.5 × 4.4–6.0 μm, and 12.6–18.5 × 4.5–5.5 μm (n = 50 each), respectively. Genomic DNA was extracted from the three representative isolates. The internal transcribed spacer (ITS) region, actin (ACT) gene, and β-tubulin (TUB2) gene were amplified and sequenced using primer pairs ITS1/ITS4, ACT-512F/ACT-783R, and Bt2a/Bt2b, respectively (Weir et al. 2012). The sequences obtained from the FA-1, FA-2, and FA-3 isolates have been deposited in NCBI GenBank (ITS, PZ493294, PZ493296 and PZ493316; ACT, PZ543437, PZ538385 and PZ511098; TUB2, PZ511099, PZ534558, and PZ511100). BLAST analysis revealed that the ITS, ACT, and TUB2 sequences of FA-2 and FA-3 shared 99.4%-100% identity with those of Colletotrichum chrysophilum (GenBank accession nos. JX010187 [ITS], JX009437 [ACT], JX010398 [TUB2]), while those of FA-1 showed 100% identity with C. plurivorum (OM007966 [ITS], OM884112 [ACT], OM662280 [TUB2]). A phylogenetic tree was constructed using the maximum likelihood method based on the concatenated ITS, ACT, and TUB2 sequences. Phylogenetic analysis placed isolate FA-1 in the same clade as C. plurivorum, while FA-2 and FA-3 clustered with C. chrysophilum in a distinct clade. Based on morphological characteristics and phylogenetic analysis, isolate FA-1 was identified as C. plurivorum, while isolates FA-2 and FA-3 were identified as C. chrysophilum. Pathogenicity of isolates FA-1, FA-2, and FA-3 was tested on healthy fruits of Canarium album. Fruits were surface-disinfected with 75% ethanol, rinsed with sterile water, and air-dried. A conidial suspension of each isolate (1 × 10⁶ conidia/mL) was sprayed onto the fruit surface. Control fruits were sprayed with sterile water. The inoculated fruits were placed in a greenhouse at 28°C and 80% relative humidity. After 4 days, the inoculated fruits developed anthracnose symptoms similar to those observed in the field, whereas the control fruits remained symptomless. The fungi were reisolated from the lesions and identified as C. plurivorum and C. chrysophilum based on morphological characteristics and DNA sequencing, thus fulfilling Koch's postulates. Previous studies have shown that C. gloeosporioides causes fruit anthracnose on Canarium album in Fujian, China (Zhang et al. 2002). To our knowledge, this is the first report of C. plurivorum and C. chrysophilum causing fruit anthracnose on Canarium album in China. The disease causes severe fruit rot and premature fruit drop, significantly reducing fruit quality and overall yield. This finding provides valuable information for developing effective control strategies against this emerging disease.