First Report of Alternaria alternata Causing Black Leaf Spot Disease on Campsis grandiflora in China

Campsis grandiflora, a garden ornamental vine valued for its seamless integration of exceptional ornamental value and vigorous growth, is increasingly cultivated across China. From September 2022 to September 2024, a severe foliar spot disease was consistently observed in a plantation in Tianjin, initially affecting approximately 28.3% (17 of 60 plants) in 2022. Due to the highly contagious nature of the pathogen, the incidence progressively increased over the following two years and had exceeded 50% of the plant population by September 2024. Small brown spots initially appeared on the underside of the leaf surface and expanded from irregular dark brown lesions to large black lesions across the leaf veins and petiole, often leading to rapid defoliation and plant decline. Consequently, identifying the causative agent was essential for disease management. 24 symptomatic tissues from six infected plants were surface-sterilized with 1% sodium hypochlorite for 1 min, followed by 70% ethanol for 30 s, and washed three times with sterile water. Air-dried tissue pieces (4 mm×4 mm) taken from lesion margins were plated on PDA and incubated at 23°C and 85% relative humidity. All five isolates (TJLXH01–05) formed colonies with concentric rings, light brown aerial mycelia, and dark reverse pigmentation after 5 days. The mycelium was septate and branched, with an average width of 4.36 ± 1.12 μm. After 18 days, the conidiophores were transparent, branched, and septate, bearing abundant conidia. Conidia were light brown, ellipsoid to obclavate, with 1–4 transverse and 0–3 longitudinal septa, measuring 13.3–53.1 × 8.3–14.3 μm (n = 56). Based on these morphological characteristics, the isolated strains were initially classified as Alternaria (Simmons. 2007). For molecular identification, the ITS, RPB2, SSU, LSU, and Alt a 1 genes of TJLXH01-05 were amplified and sequenced (Schoch et al. 2012; Giuseppe et al. 2024; Suo et al. 2024). BLAST analysis of TJLXH03 sequences (GenBank PX369439, PX556777, PZ103522, PZ103521, PP297564) showed 99% identity with A. alternata. A multi-gene phylogenetic tree constructed with Maximum Likelihood Estimate method grouped TJLXH03 in a well-supported clade with reference strains of A. alternata. Combined morphological and molecular data identified the pathogen as A. alternata (Trifinopoulos et al. 2016). Pathogenicity of strain TJLXH03 in C. grandiflora was confirmed using Koch’s postulates. A total of 12 leaves (6 pairs of opposite leaves, 2 pairs per plant) from 3 plants were wounded three times with a sterile needle and subsequently inoculated with a spore suspension of TJLXH03 (3×10⁶ conidia/mL). Negative control leaves from the same leaf pairs were similarly wounded but mock-inoculated. After 5 days at 23 °C and 85% relative humidity, inoculated leaves developed black lesions identical to the original symptoms, while controls remained asymptomatic. The pathogen was re-isolated and purified from black spots, and the strain was confirmed as TJLXH03 based on morphology and molecular biology. In this study, the pathogen of C. grandiflora black leaf spot in Tianjin was isolated and purified. Through morphological comparison, molecular biology and pathogenicity identification, the pathogen was identified as A. alternata, which is an important pathogen that causes black spot in crops (Pedrotti et al. 2022; Zhirui et al. 2023; Siliang et al. 2023.). To our knowledge, this is the first report of Alternaria alternata causing black leaf spot on Campsis grandiflora in China. Given the ubiquitous nature and high infectivity of A. alternata, this first report highlights a potential cross-infection risk to neighboring plants and underscores the importance of integrated disease management strategies.