First report of Colletotrichum truncatum causing leaf spot on Orychophragmus violaceus in China

Abstract

Orychophragmus violaceus (L.) O. E. Schulz has important ecological, ornamental and edible value, and is also used as a traditional medicinal plant. In September 2025, severe leaf spot symptoms were observed on O. violaceus in a plot of about 30 m2 in Jingzhou City, Hubei Province, China (30°20′11″N, 112°14′29″E), with a field disease incidence of approximately 30%. Lesions on infected leaves were circular to elliptical, with centers appearing grayish-white to light brown, and were sometimes surrounded by dark brown halos. Symptomatic leaf samples were collected and surface-disinfected as follows: immersion in 75% ethanol for 30 s, treatment with 1% sodium hypochlorite solution for 2 min, and rinsing three times with sterile distilled water. Small tissue pieces (5 mm) excised from lesion margins were placed on potato dextrose agar (PDA) and incubated in darkness at 25 °C for 3–5 days. Colonies developed from the tissues were transferred to fresh PDA plates and sub-cultured three times for purification. All pure cultures showed consistent colony morphology, so three representative isolates (YzU F 26001, YzU F 26002, and YzU F 26003) were selected for further identification. On PDA, the colonies were circular, white to smoke gray, with light brownish tan margins. Conidia were falcate, hyaline, smooth walled, measured 22.1–25.5 μm × 2.97–3.3 μm (n = 50). Appressoria were light brown, elliptical to subglobose. Setae were dark brown, acicular, septate, and radiating outward from the acervuli. Morphological characteristics of the fungus were similar to those of Colletotrichum truncatum (Schwein.) Andrus & W.D. Moore (Damm et al. 2009). For molecular identification, four genomic regions of the fungus were amplified as previously reported (Aktaruzzaman et al. 2018), including the internal transcribed spacer (ITS) region of ribosomal DNA, and the glyceraldehyde 3 phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS 1), and actin (ACT) genes. The three isolates had identical sequences in the above regions. Sequences of isolate YzU F 26002 were deposited in GenBank with accession numbers PZ213298 for ITS, PZ293201 for GAPDH, PZ293202 for CHS 1, and PZ293200 for ACT. Based on multi-locus phylogenetic analysis, the three isolates clustered with C. truncatum. Therefore, based on morphological and molecular data, the fungus was identified as C. truncatum. For the pathogenicity test, three living plants of O. violaceus were sprayed with 15 mL conidial suspension (10⁵ conidia/mL) of isolate YzU F 26002, and maintained in a climate chamber (25°C, high humidity, 12 h light/dark cycle). Control plants were treated with sterile water. After 7 days, pathogen-inoculated leaves developed necrotic spots, similar with the symptoms of field observation, whereas no symptoms were observed on control plants. The fungus was re isolated from symptomatic tissues and confirmed as C. truncatum by morphology and ACT sequence, fulfilling Koch’s postulates. The pathogenicity test was repeated three times with similar results. Several fungal pathogens have been reported on O. violaceus, such as Alternaria japonica and A. brassicae causing leaf spot, and Erysiphe cruciferarum causing powdery mildew (Qian et al. 2022; Sein et al. 2020; Tian et al. 2024). To our knowledge, this is the first report of C. truncatum causing leaf spot on O. violaceus worldwide. The findings provide a basis for developing future management strategies for the disease.