First Report of Alternaria alternata Causing Leaf Spot on Schefflera heptaphylla in China

Schefflera heptaphylla is a traditional Chinese medicinal plant distributed in southern China. The aerial parts of this plant have been used for the treatment of inflammation, rheumatism, fever, and traumatic bleeding. (Liu et al. 2019). In July 2024, leaf spot symptoms were found on leaves of S. heptaphylla plants in Yangshuo County (24.78°N, 110.49°E), Guilin City, Guangxi Province, China. Further investigation revealed that the disease incidence was approximately 70% in a 3-hectare nursery from a company located in Guangxi, Guilin, resulting in substantial economic losses. Early symptoms were small yellow spots on leaves. Later, the spots gradually expanded and turned into necrotic tissues with a clear yellow halo and a white center. To isolate the causal pathogen, 10 leaves with typical symptoms were collected from severely diseased plants. Tissue segments (8 mm x 8 mm) at the lesion margins were excised and surface disinfected in 75% ethanol for 60 s, rinsed three times with sterile water, transferred to PDA plates, and incubated at 25℃ in the dark for 3 days. Grayish fungal colonies appeared on the PDA; pure cultures were produced by transferring hyphal tips to new PDA plates. The single spore isolation method was used to obtain pure cultures by diluting collected spores, spreading the suspension on a plate, selecting a germinated spore under a stereomicroscope, and transferring its hyphal tip to a new PDA plate. After 7 days, the fungal colonies were olive-green with white margins, black pigment, and abundant aerial hyphae. Conidia were solitary, and conidiophores were unbranched. The conidia were light brown to brown, obclavate, with one to five transverse septa, and were 19.5 to 43.9 μm in length and 7.1 to 17.2 μm in width, characteristic of the Alternaria genus (Simmons 2007). Three representative strains, YJM22072701, YJM22072705, and YJM22072706, were selected from the 18 isolates for subsequent testing. DNA was extracted from the fungal colonies using a Plant Genomic DNA Kit for TIANGEN (DP305-02). The ITS gene was amplified and sequenced to confirm the fungal isolates belonged to the genus Alternaria, followed by PCR amplification for 3 housekeeping genes: alternaria major allergen (Alt a 1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation elongation factor 1-alpha (TEF) (Peever et al. 2005; Hong et al. 2005). The obtained sequences were submitted to GenBank under accessions PV243627-PV243629, PV536048-PV536050, PV536051-PV536053, and PV536054-PV536056. Gene sequences were aligned, concatenated, and used to generate a phylogenetic tree using the neighbor-joining method in MEGA11 (Tamura et al. 2021). Phylogenetic analysis revealed that the isolate was clustered with A. alternata strain CBS 102598. (Hu et al. 2023). The fungus associated with leaf spot on S. heptaphylla was therefore identified as A. alternata. For pathogenicity testing, twelve S. heptaphylla plants were inoculated by placing A. alternata mycelial plugs onto wounds created with a sterile needle on six asymptomatic leaves per plant. Another twelve plants were wounded similarly and inoculated with sterile PDA plugs as negative controls. The experiment was conducted three times. All plants were maintained in a greenhouse incubator at 28℃ and 85% relative humidity. After 7 days, brown spots with white centers appeared at the inoculation sites on all inoculated leaves, while negative controls remained asymptomatic. The fungus that was reisolated from lesions exhibiting identical morphology to the original isolate, fulfilling Koch’s postulates. To our knowledge, this is the first confirmed report of leaf spot caused by A. alternata on S. heptaphylla in China. This study enhances our understanding of the pathogenic leaf spot on S. heptaphylla and lays the groundwork for developing targeted management strategies.