First Report of Leaf Blight on Justicia gendarussa Caused by Paramyrothecium nigrum in China

Justicia gendarussa is a traditional medicinal plant used to treat bone fractures, traumatic injuries, and rheumatic pain (Chandra and Lo 2021), and is widely cultivated as a medicinal and ornamental subshrub in southern China. It has been reported to be infected by Puccinia thwaitesii causing leaf rust (Motokura et al. 2005), and by a 16SrXIV-A subgroup phytoplasma linked to witches’ broom symptoms (Tiwari et al. 2016). In October 2025, an undocumented leaf blight disease was observed on J. gendarussa at a medicinal botanical garden in Nanning, Guangxi, China (108°17'17" E, 22°50'47" N). A field survey was conducted in a 200‑m² plot containing approximately 500 plants, using a five‑point sampling method with 10 plants examined per point (50 plants in total). The survey revealed a disease incidence of 30%–50%. Disease severity was rated on a 0–5 scale (0 = no symptoms; 1 = 0.1–10.0%; 2 = 10.1–25.0%; 3 = 25.1–50.0%; 4 = 50.1–75.0%; 5 = 75.1–100.0% leaf area affected), with the majority of infections falling into grades 1–3 (mild to moderate severity). The disease symptoms initially appeared as irregular, yellowish-brown to grayish-black necrotic lesions at the tips or margins of leaves, sharply demarcated from adjacent healthy tissue. As the disease progressed, dieback extended from the leaf tips to half or the entire leaf, leading to defoliation. Numerous black sporodochia were observed on the back surface of the lesions. Tissues (5 mm × 5 mm) were excised from the lesion margins of ten diseased leaves randomly collected from ten different J. gendarussa plants. Tissues were surface-sterilized in 3% sodium hypochlorite for 30 s, followed by 75% ethanol for 60 s, rinsed three times with sterile distilled water, and then placed onto potato dextrose agar (PDA). After incubation at 28 °C for 5 days, mycelium from the colony edges was transferred to fresh PDA plates for purification. Ten fungal isolates were successfully purified, all of which exhibited consistent morphological characteristics. Five isolates (XBG1-5) were randomly selected for ITS sequence verification, which revealed 100% sequence identity among them (GenBank accession nos. PZ493500, PZ583828- PZ583831), and XBG1 was used for further study. On PDA at 28 °C for 10 days, colonies reached 72–75 mm in diameter, were white to off-white, with a dense velvety to cottony texture. Numerous black sporodochia, oval to irregular in outline, 50–800 μm in diameter, superficial and stromatic, developed on the medium surface in concentric rings, each with a distinct white setose fringe surrounding an olivaceous-green to black slimy conidial mass. Setae arising from the stroma were hyaline, thin-walled, 1–3-septate, 60–100 × 2.0–3.0 μm. Conidiophores consisted of a stipe and penicillately branched conidiogenous apparatus, terminating in whorls of 3–6 phialidic conidiogenous cells (8.5–12.5 × 1.0–2.0 μm). Conidia were hyaline, aseptate, smooth-walled, cylindrical to ellipsoidal, measuring 5.0–6.5×1.0–2.5 μm (x̄ = 6.0×1.5 μm, n=50), and contained 2–5 oil guttules. The colony reverse was pale luteous. Subsequently, multilocus phylogenetic analysis of isolate XBG1 was performed based on Lombard et al. (2016) using four additional loci—beta-tubulin (tub2), calmodulin (cmdA), RNA polymerase II second largest subunit (rpb2), and translation elongation factor 1-alpha (tef1). BLASTn searches of the sequences deposited in GenBank (tub2: PZ498315; cmdA: PZ498316; rpb2: PZ498317; tef1: PZ595201) showed 99.19–100% identity to the ex-type strain P. nigrum CBS 116537. Additional sequences of Paramyrothecium and related species were retrieved from GenBank based on Matić et al. (2019) and Sun et al. (2025). The five loci were aligned and concatenated into a 2,916-bp dataset with gene regions of ITS 1–568, tub2 569–940, cmdA 941–1,624, rpb2 1,625–2,336, and tef1 2,337–2,916. The dataset was partitioned by gene, with the best-fitting substitution models determined under AIC: ITS = K2+G+I, tub2 = TN93+G, cmdA = T92+G+I, rpb2 = K2+G+I, and tef1 = TN93+G+I. A maximum likelihood tree was constructed using IQ-TREE with 1,000 ultrafast bootstrap replicates (≥ 75% support considered significant). The tree placed isolate XBG1 in a well-supported clade with all P. nigrum reference strains and in a subclade with the ex-type CBS 116537 (98% bootstrap). Thus, based on morphological and multi-locus phylogenetic data, isolate XBG1 was identified as P. nigrum. For pathogenicity testing, ten detached leaves and six whole J. gendarussa plants per treatment were surface‑disinfested with 75% ethanol for 60 s, rinsed three times with sterile water, and then inoculated with either a conidial suspension (1 × 10⁶ conidia/mL) or sterile water (negative control). After incubation under humid conditions at 26 °C (14 h light/10 h dark) for 7 days, all conidial‑inoculated leaves and plants showed typical field symptoms (yellowish‑brown lesions with black sporodochia on the underside), while sterile‑water controls remained asymptomatic. The fungus was re‑isolated from lesion margins of inoculated plants and confirmed as P. nigrum by morphological and molecular characterization. The pathogenicity test was repeated three times independently with consistent results, fulfilling Koch’s postulates. P. nigrum has been reported on a wide range of hosts, including Citrullus lanatus in Iran (Atashi Khalilabad and Fotouhifar 2023) and Lactuca sativa, Valerianella olitoria, Ocimum basilicum, and Rudbeckia hirta in Italy (Matić et al. 2019). To our knowledge, this is the first report of leaf blight on J. gendarussa caused by P. nigrum. This finding indicates a potential threat to J. gendarussa production and further research is needed on its epidemiology and management.