First Report of <i>Alternaria alternata</i> Causing Fruit Black Spot on <i>Elaeagnus angustifolia</i> in Xinjiang, China

Elaeagnus angustifolia L. is a multipurpose woody species in China’s arid and semi-arid northwest (75°-125°E, 35°-48°N), valued for its use in saline-alkali ecological restoration and Uyghur medicine (Abulizi et al. 2024; Zhu et al. 2019). Since 2023, severe black spot disease (incidence of 50-100%) has affected about 5000 hectares of plantations in Kashgar (77°28´-79°05´E, 38°25´-39°22´N). Symptoms begin as pale-yellow speckles during ‌fruit coloring‌, progressing to reddish-brown speckles that initially appear at the fruit’s waist or shoulder, which subsequently expand into circular or irregular dark brown lesions with slightly raised and wrinkled surfaces. Black powdery mold (indicative of pathogenic fungal conidia) becomes visible in the later stages. Using a random sampling method, thirty diseased fruits with typical black spot symptoms (lesions but not rotten) were collected from ten small plantation areas (1 m2) for pathogen isolation. After surface disinfection in 75% ethanol for 2 min, the fruits were rinsed five times in sterile distilled water and then air-dried in a sterilized atmosphere. The internal tissue of the lesion (approximately 5 × 5 mm2) is excised and inoculated onto potato dextrose agar (PDA) medium, then incubated in the dark at 25 °C for 5 days. The isolated strains were purified using hyphal tip cutting methods. Based on morphology observation, the same isolate EA01 was isolated from different samples, which form gray to dark brown colonies on PDA medium. Microscopic observation revealed that the conidia are arranged in chains (5-10 conidia per chain), with the conidia being inversely club-shaped or oval, containing 3-6 transverse septa and 1-3 longitudinal septa, and measuring approximately 21-42.4×4-5.9 μm. These characteristics align with the description of Alternaria (Woudenberg et al. 2015). Further molecular identification was conducted by sequencing the gene fragments of ITS, CAL, and GAPDH (Hu et al. 2024). All the sequences were deposited in GenBank with accession nos. PV413026 (ITS), PX308855 (CAL), PX308856 (GAPDH), respectively. Phylogenetic analysis using the three combined sequences indicated EA01 clusters within the clade of Alternaria alternata. Pathogenicity tests were conducted on healthy fruits at both coloring and mature stages, separately, in a sterile incubator at 25 °C with 85-90% relative humidity. Purified conidia suspension (105 conidia/mL) of EA01 was inoculated onto healthy fruit epidermis (wound and non-wound inoculation). The fruits inoculated with sterile water served as negative controls. Each test was performed in triplicate, with each replication consisting of five fruits. Ten days after inoculation, the inoculated site turned black, and a visible mold layer developed, accompanied by signs of soft rot on the fruit. The observed disease symptoms were consistent with those in the field. The control group exhibited no symptoms. The same pathogen was reisolated from the diseased fruits. To our knowledge, this is the first report of A. alternata causing fruit black spot on E. angustifolia L. in China, providing a theoretical basis for disease control and plant protection.