First Report of <i>Phyllosticta capitalensis</i> Causing Leaf Blotch on Chinese Olive (<i>Canarium album</i>) in Guangdong Province of China

Chinese olive (Canarium album) is a well-known plant used both as food and medicine, extensively cultivated in southern China (Kuo et al., 2016). Its production is challenged by various fungal diseases. From July to October 2023, Chinese olive plants in an approximately 400 hectare conventional hillside orchard in Chaozhou, Guangdong province, China (23°39'45.36″N, 116°37'45.48″E), where sustainable Chinese olive farming has been practiced for over a decade, exhibited leaf blotch symptoms with a disease incidence of 40% (n = 100 plants investigated). Symptoms include yellow or brown leaf blotches, followed by drying, curling, and eventual leaf drop. To identify the causal agent, seven symptomatic leaves from seven trees were collected. The symptomatic leaves were cut into pieces of about 0.3 mm2 at the necrotic lesion edges and surface disinfected. The pieces were placed on potato dextrose agar (PDA), incubated in the dark at 28°C for 5 days, then subcultured for 7 days. Four fungal isolates were obtained with an isolation frequency of 57%. Genomic DNA was extracted from the isolates. Morphological analysis and internal transcribed spacer (ITS) region sequencing revealed no observable differences among isolates. The representative isolate (LB-1) cultured on PDA was initially white, turning light grey with dense aerial mycelium after 7 days. The transparent hyphae displayed intricate branching patterns and multiple cellular structures. The conidia were single-celled, hyaline, fusiform structures measuring 3.6-7.8 × 1.17-2.83 μm (n = 50). Morphologically, the fungal isolates resembled Phyllosticta capitalensis (Wikee et al. 2013). Primers ITS1/ITS5, ACT-512F/ACT-783R, and EF1/EF2 were used to amplify the ITS region, the actin (ACT) gene, and the transcription elongation factor (TEF), respectively (Wang et al. 2023). The sequences obtained from the LB-1 isolate have been deposited in NCBI GenBank (accession nos. PP922193; PP960207 and PP960206). A phylogenetic tree was constructed based on aligned sequences (Wang et al. 2023). BLAST analysis showed LB-1 isolate shares 99.4%-100% similarity with sequences KY855645, KY855590, and KY855919 from P. capitalensis CPC27084. Based on morphological and molecular analysis, isolate LB-1 was identified as P. capitalensis. Pathogenicity assays of the LB-1 isolate were conducted on 2-year-old Canarium album seedlings. A 10⁶ spores/mL LB-1 suspension was applied to 3 mm spots on the leaves of four plants for inoculation. Controls leaves was treated with sterile water. The inoculated plants were placed in a greenhouse at 28°C with 80% humidity. The experiment was repeated 3 times. Leaves inoculated with LB-1 isolates showed blotch symptoms similar to those in the field, the controls remained healthy. Reisolation of P. capitalensis was performed confirmed by morphology and DNA sequencing, fulfilling Koch's postulates. Previous studies have shown that P. capitalensis causes fungal diseases on fruit trees such as Citrus lalifolia and Mangifera indica (Wikee et al. 2013). To our knowledge, this is the first report of Phyllosticta capitalensis causing leaf blotch on Chinese olive in China. Preliminary observations suggest it may be present in other olive-producing regions of Guangdong. It causes significant leaf wilting and shedding, potentially reduces fruit quality, and lowers overall yield. This discovery will offer valuable insights for creating effective control measures against this recently emerged severe disease.

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