First report of leaf spot disease caused by Bipolaris setariae on Cenchrus fungigraminus in China
Cenchrus fungigraminus is an important grass used for forage, ecological restoration and bioenergy (Zheng et al. 2023). In September 2025, leaf spot symptoms were observed on C. fungigraminus in Jiangsu, China (33°2′43.12″N, 120°41′16.76″E) with disease incidence reaching 60% based on all leaves of 100 plants surveyed in five random 5×5 m plots. Initially, symptoms appeared as small (2-5 mm) round, ovoid or linear lesions with pale brown to nut brown centers, pale-yellow to brownish margins, which gradually expanded into irregular necrotic lesions, leading to withering, blight and collapse. Leaf tissues from affected C. fungigraminus plants were surface sterilized with 75% ethanol for 30 s, followed by 2% NaOCl for 1 min, rinsed with sterile distilled water and plated on PDA plates. After incubation at 28℃ and 80% relative humidity for 5 to 7 days, uniform fungal colonies were observed. Hyphal tips from colony margins were inoculated onto fresh PDA plates according to a previously purification method (Li et al. 2019). Five morphologically identical isolates (PG1-5) were selected for further characterization. Colonies had a 7 cm diameter after incubation for 8 days with loosely distributed aerial hyphae, initially white, later turning to gray and black after 10 to 15 days at 28°C. Conidiophores were predominantly aggregated in clusters 3-5 or solitary, exhibiting pale olive to brown pigmentation and distinct septation, occasional branching and knee-like flexures at the apex, ranging from 60 to 200μm×3-10μm (n=50). Conidia were dark brown, cylindrical to fusoid, typically curved, rarely straight, and 50-100μm×8-20μm (n=50), with 5–9 distoseptate pseudosepta. The morphological characteristics were consistent with previously reported Bipolaris species (Adhikari et al. 2021). Therefore, the five isolates PG1-5 were identified as the same species. Given morphological identity, PG1 was chosen as representative to confirm identification. ITS (White et al. 1990), LSU (Vilgalys 1990), and gapdh (Berbee et al. 1999) genes were amplified and sequenced using the primers ITS1/ITS4, LROR/LR5 and Gpd1/Gpd, respectively. The sequences of ITS, LSU and gapdh were deposited in GenBank under the following accession numbers PX642338, PX642339, and PX864569. Phylogenetic analysis of concatenated multilocus tree was performed using IQ-TREE with ModelFinder and 1000 ultrafast bootstrap replicates revealed that PG1 clustered with B. setariae. To fulfill Koch’s postulates, 5 mm mycelial plugs from 8 day old PG1 culture were placed on slightly wounded leaves of 15 C. fungigraminus plants, and 10 plants served as negative controls (sterile PDA plugs), incubating at 28°C and 90% relative humidity for 7 to 10 days.The experiment was independently repeated three times with consistent results. All 15 inoculated plants exhibited identical leaf spot symptoms to previously described, whereas control plants were asymptomatic. The fungi were reisolated from symptomatic leaves and identical to B. setariae corroborated by morphology (conidiophore and conidia) and ITS + gapdh sequences. B. setariae was previously reported as the causal agent of leaf spot in Guinea grass, maize and sugarcane (Adhikari et al. 2021). To our knowledge, this is the first report of leaf spot on C. fungigraminus caused by B. setariae in China. This finding will thereby provide fundamental reference for disease monitoring, prevention and host germplasm protection of this economically valuable grass.