Occurrence of Diplodia seriata causing leaf blight on Aucuba japonica in China.

Abstract

Aucuba japonica is widely planted in China for landscaping purposes, often used for decoration in gardens and parks. In October 2023, a leaf blight on A. japonica was observed in Meicheng Park of Nanyang City (32°59'21″ N, 112°32'54″ E), Henan province. Subsequently, surveys were conducted in different sections of the park, a plant diseases incidence rate of 52% (n = 100). Initial symptoms included black spots and enlarged to leaf blight lesions on leaves. These lesions subsequently spread throughout the plant, causing defoliation, and aesthetic damage. Twenty diseased leaves from 15 plants were collected, and sections between the symptomatic and healthy tissues were cut into 3 × 3 mm2 pieces to isolate the pathogen. The disinfestation process involved surface sterilization with 75% ethanol solution for 30 s, followed by 1% NaClO solution for 1 min., then rinsing with sterile water, and placing on potato dextrose agar (PDA) plates, incubating at 28°C for 5 days under a photoperiodic condition of 12-hour light and 12-hour dark. After isolation, purified fungal isolates (n = 28) were obtained from 33 samples plated. These fungal isolates showed similar morphological phenotypes. The three isolates (DS2, DS8, and DS28) from different park areas were selected for subsequent identification. The colonies were gray to dark brown with abundant aerial mycelium on the surface. For sporulation, isolates were cultivated on 2% water agar bearing autoclaved poplar twigs at 25°C for 21 days (Phillips et al. 2007). Immature conidia were oblong to ovoid with rounded ends, aseptate, and became dark brown at maturity, measuring 25.1 to 21.3 × 11.9 to 7.7 µm (n = 100 per isolate). These morphological characteristics were consistent with the genus Diplodia (Bhat et al. 2023), which may lead to canker, dieback, fruit rot, and leaf spots on economically important forest and horticultural species. For further molecular identification, the genomic DNA of three strains was extracted. The rDNA internal transcribed spacer (ITS) region and β-tubulin (tub) and translation elongation factor 1-alpha (tef1) genes were amplified using primers ITS1/ITS4, Bt2a/Bt2b, and EF1-728F/EF1-986R, respectively (Díaz et al. 2019). The ITS (PP957738 to PP957740), tub (PP960547 to PP960549), and tef1 (PP960550 to PP960552) sequences were submitted to GenBank. A phylogenetic tree of concatenated markers sequences (ITS, tub, and tef1) and ex-type strains sequences was constructed using MEGA software (version 11) with the neighbor-joining method. The three strains formed a clade with the strains CBS114791 and CBS113508 of D. seriata in the phylogenetic trees. Molecular analyses supported the identification of the strains as Diplodia seriata. To verify pathogenicity, conidial suspensions (106 conidia ml-1of isolate DS2) were sprayed onto the wound-free leaves of five A. japonica of three-month-old seedlings. In contrast, another five plants were sprayed with sterile water as a mock-inoculated control. Thirty days post-inoculation, lesions consistent with those observed in the field were present on the inoculated plants, while no symptoms appeared on the mock-inoculated controls. The entire inoculation experiments were repeated thrice. Isolates recovered from the symptomatic plants were identified as D. seriata based on morphological and ITS, tub, and tef1 sequence analyses, fulfilling Koch's postulates. Gummosis of Prunus persica and cankers on Juglans regia and Malus domestica caused by D. seriata have been reported in China (Wang et al. 2011; Zhang et al. 2017; Sha et al. 2023). This is the first report of D. seriata causing leaf blight on A. japonica. Leaf blight seriously affects the aesthetic landscape, and this finding provides valuable insights into the control and spread of D. seriata.