First Report of Botryosphaeria dothidea Causing Leaf Spot on Atraphaxis bracteata in China

Atraphaxis bracteata is an important sand-fixing shrub in desert regions. In August 2024, an epidemic of leaf spot disease on A. bracteata was observed in Shapotou District, Zhongwei City, Ningxia, China (37°27′ N, 104°57′ E, altitude approx. 1250 m). Disease surveys, conducted on 100 randomly selected plants, revealed a high incidence of ~75%. Infected plants initially developed small yellow spots on leaves, which subsequently expanded into irregular necrotic lesions with yellow margins and dark brown centers. Severe infections led to premature leaf drop. Tissue sections from the lesion margins were surface-sterilized with 75% ethanol for 30 s, soaked in 1% NaClO solution for 1 min, rinsed three times with sterile distilled water, air-dried, and then placed on Potato Dextrose Agar (PDA) at 25°C for 3-7 days. A Botryosphaeria-like fungus was consistently isolated from all samples. Pure cultures were obtained via single-spore isolation (Cai et al., 2009), where individual spores from a diluted suspension were germinated on agar plates. Colony morphology was observed after 5 days of incubation, and spore morphology was examined after 14 days. Colonies transitioned from white to greyish-black, reaching 85-90 mm diameter. Hyphae were septate, branched, hyaline initially, turning dark brown with age. Pycnidia produced brown, elliptical, aseptate conidia (9-18) × (2-8) μm; mean ± SD = 15.2 ± 1.8 × 6.3 ± 0.9 μm; n = 30. The partial internal transcribed spacer region (ITS1), the partial β-tubulin gene (TUB2), and the partial translation elongation factor (TEF1-α) gene were amplified from pure cultures of isolates YFZ1 and YFZ2 (Espinoza-Lozano et al., 2023). The primer pairs used were ITS1 / ITS4 (White et al., 1990), Bt2a / Bt2b (Glass et al., 1995), and EF1-728F / EF1-986R (Carbone et al., 1999), respectively. Sequences were deposited in GenBank (YFZ1: ITS PV926689, TUB2 PX434322, TEF1-α PX434323; YFZ2: ITS PV926690, TUB2 PX434324, TEF1-α PX434325). BLASTn analysis revealed that the ITS, tub2, and tef1 sequences of both isolates shared over 99% homology with the corresponding sequences of the Botryosphaeria dothidea ex-type strain (e.g., CBS 110302 and CMW8000). A maximum likelihood phylogenetic tree was reconstructed based on the concatenated ITS-TEF1-TUB2 sequences using MEGA v.7 under the K2+G evolutionary model (selected by ModelTest) with 1000 bootstrap replicates. The analysis confirmed that both isolates YFZ1 and YFZ2 clustered within a well-supported clade comprising reference strains of Botryosphaeria dothidea, with Macrophomina phaseolina CBS 227.33 (ex-type strain) designated as the outgroup. Pathogenicity tests were conducted by spraying healthy A. bracteata leaves inoculated with 10 μL of spore suspension (106 /mL). Control plants were sprayed with sterile water. All potted plants were individually covered with transparent polyethylene bags to maintain high humidity and incubated in a greenhouse at 23-29 °C. Fourteen days post-inoculation, typical lesions consistent with field symptoms appeared on treated leaves, while control leaves remained asymptomatic. Fungal isolates re-isolated from the diseased leaves exhibited identical morphological characteristics and molecular identification results to the original inoculum strains, fulfilling Koch's postulates. This study provides the first confirmation of B. dothidea’s pathogenicity on A. bracteata in China, aiding diagnosis and management of this emerging disease.