First report of leaf spot caused by Corynespora cassiicola on Ligustrum lucidum in China

Abstract

Ligustrum lucidum, commonly known as Chinese privet, is an ornamental tree widely cultivated in China. In August 2024, a severe leaf spot disease was observed on Ligustrum lucidum in and around Dushan Forest Park (33°04′13″N, 112°35′11″E) in Nanyang, China. Disease incidence reached 59% among 100 surveyed trees, reducing the ornamental value of affected plants. The lesions were initially small, scattered, and pale gray on the adaxial leaf surface, mostly circular to irregular in shape. On the abaxial surface, the corresponding lesions were slightly sunken and light brown. As the disease progressed, adjacent lesions enlarged and coalesced, and the necrotic tissue occasionally detached, producing shot-hole-like perforations. For pathogen isolation, ten symptomatic leaves were randomly collected. Small tissue segments were excised from the margin between healthy and diseased tissues, surface sterilized in 75% ethanol for 30 s, followed by 1% NaClO for 1 min, rinsed twice in sterile water, and plated on potato dextrose agar (PDA) supplemented with 50 μg/ml streptomycin. Plates were incubated at 25°C in darkness. In total, 16 isolates with similar morphological characteristics were obtained. Three representative isolates (DYNZ09, DYNZ17, and DYNZ20) sampled at separate sites were selected for further investigation. Colonies grew slowly on PDA at 25°C, with a growth rate of 3 mm per day. Conidia were light brown and obclavate to cylindrical, measuring 16.3–95.0 × 4.5–10.7 μm (n = 100). These morphological features were consistent with descriptions of Corynespora cassiicola. For molecular identification, genomic DNA of three representative isolates was extracted using the CTAB method. The rDNA internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1-α), and β-tubulin genes (tub) were amplified using primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The obtained sequences were deposited in GenBank (ITS: PX488446–PX488448; tef1-α: PX549115–PX549117; tub: PX549119–PX549121). BLAST analysis showed 99.1-100% identity with sequences of C. cassiicola. Phylogenetic analysis based on concatenated ITS, tef1-α, and tub sequences using the maximum likelihood method (MEGA12) (Kumar et al. 2024) demonstrated that the isolates clustered with C. cassiicola strains. Pathogenicity assays were performed by spraying a conidial suspension (106 conidia ml-1) onto healthy leaves of L. lucidum seedlings (60–80 cm in height). Control plants were treated with sterile water. Inoculated seedlings were maintained in chambers at 28°C and 90–95% relative humidity under a 12 h light/12 h dark photoperiod. At 14 dpi, inoculated seedlings developed foliar symptoms, which were identical to the field observations. Control plants remained symptomless. C. cassiicola was consistently reisolated from diseased tissues, thereby fulfilling Koch’s postulates. C. cassiicola is a well-known pathogen with a broad host range and has been reported to cause diseases on soybean (Lu et al. 2021). C. cassiicola has been reported on other Ligustrum species, including L. quihoui (Yu et al. 2023) and L. × vicaryi (Wang et al. 2013). However, these reports involved different host species. To our knowledge, this is the first report of leaf spot caused by C. cassiicola on L. lucidum in China. This finding provides a basis for accurate diagnosis and future management of Corynespora leaf spot on L. lucidum.