Occurrence of Leaf Spot on Elaeocarpus decipiens Caused by Alternaria alternata, a New Disease in China

Elaeocarpus decipiens is widely cultivated as an ornamental tree of commercial importance in southern China. During March 2018 to March 2021, leaf spot disease was observed in about 40% of E. decipiens on the campus of Jiangnan University in Wuxi, Jiangsu, China (31.48°N, 120.46°E). Leaf symptoms began as small, light brown lesions that enlarged, turned olive brown in color and then became necrotic. Ten symptomatic leaves were collected from five different trees on the Jiangnan University campus and surface sterilized with 75% ethanol for 30 seconds, followed by 1% sodium hypochlorite for 1 minute, and rinsed three times with sterile distilled water before being cultured onto potato dextrose agar and incubated in the dark at 25°C for 5 days. Five purified fungal isolates were obtained by the single spore isolation method. Emergent fungal colonies were olive-green in color with 1 to 3 mm white margins and abundant aerial hyphae. Conidia were borne in chains or singly and were obclavate or obpyriform and measured 6.5 to 17.4 × 21.3 to 32.8 μm (n=50) with one to seven transverse septa and zero to three longitudinal septa. Based on morphological characteristics, the pathogen was identified as Alternaria spp.(Simmons 2007). Three representative isolates, At1, At2 and At3, were selected for molecular identification, total genomic DNA of the fungus isolates were extracted with Plant/Fungi DNA Isolation Kit (Sigma-Aldrich, Ontario, Canada). Plasma membrane ATPase (ATP) gene, chitin synthase (CHS) gene and translation elongation factor 1-alpha (EF1) gene were amplified with primers ATPDF1/ATPDR1, CHS-79F/CHS-345R (Lawrence et al. 2013) and EF1-728F/EF1-986R (Carbone and Kohn 1999). The amplification results of the three isolate genes were consistent, and we deposited the results of the ATP (MN046377), CHS (MN046378) and EF1 (MN046379) sequences of At1 in the NCBI GeneBank. The ATPase gene from the representative isolate At1 shared 99.83% similarity to A. alternata causing leaf Spot of Codonopsis pilosula in China (OM362504, Shi et al. 2022), the CHS gene shared 100% similarity to A. alternata causing brown leaf spot on Paris polyphylla var. chinensis in China (MK391053, Fu et al. 2019), and the EF1 gene shared 100% similarity to A. alternata CBS 916.96 ex-type on Arachis hypogaea in India (KC584634). A phylogenetic tree constructed with the EF1 gene using the neighbor-joining algorithm in MEGA 11 software with 1,000 bootstrap replicates revealed that the examined isolate, At1, belongs to the fungus A. alternata. For pathogenicity tests, 10 leaves of five healthy plants were sprayed with spore suspensions (1 × 107 conidia/ml) of the 10-day-old isolates (At1, At2 and At3, respectively). As a control, five plants were sprayed with sterile distilled water. After inoculation, use the bags to moisturize for 48 hours. Pathogenicity tests were conducted three times. Fourteen days after inoculation, olive brown necrotic lesions developed on inoculated leaves while control leaves remained symptomless. The pathogen was reisolated from infected leaves and confirmed as A. alternata based on morphological characteristics and molecular markers. To date, A. alternata has been reported to cause leaf spot disease on many plants inculuding Ficus religiosa (Du et al. 2022), Tilia miqueliana (Yue et al. 2023), Ligustrum japonicum (Fang et al. 2023) and so on. To our knowledge, this is the first report of the occurrence of A. alternata causing leaf spot on E. decipiens in China. The increasing area of E. decipiens cultivation and global climate change have led to an increase in the incidence of E. decipiens diseases, which should be taken into account by forest conservationists.