First report of leaf spot disease on sugarcane caused by Exserohilum rostratum in China

Sugarcane (Saccharum spp. hybrids) is an important industrial crop cultivated in tropical and subtropical regions worldwide, primarily for sugar and ethanol production. In November 2024, a new sugarcane leaf spot disease was found in the sugarcane breeding base of South China Agricultural University and other sugarcane planting areas in Guangdong Province of China. The average incidence of this disease was 5% to 10% in the whole province, and it reached 10% to 15% in individual fields. In the early stages, symptoms manifested as spots featuring yellow halos and streaks. These subsequently developed into multiple lesions that coalesced progressively, ultimately causing extensive necrosis and leaf withering. Three symptomatic leaf samples were collected from different plants of the sugarcane cultivar GT55 for pathogen isolation. Leaf segments (approx. 0.5 cm) from lesion margins were surface-sterilized by sequential treatment with 75% ethanol (30 s) and 0.1% HgCl₂ (3 min), rinsed thoroughly with sterile water, blot-dried on sterile filter paper, and plated on PDA for final incubation at 25 °C in complete darkness. Eight pure cultures were obtained by monosporic isolation. Five morphologically similar strains were selected for pathogenicity testing of leaves in vitro. Each was inoculated onto surface-wounded, detached sugarcane leaves and incubated at 28°C and 80% relative humidity for 6 days. Strain PL2428 was identified as the most pathogenic. On PDA plates, the colonies of PL2428 appeared white initially and turned dark gray after 5 days of incubation at 25 °C in complete darkness. Conidia were dark brown, with straight, curved, oval and spindle shapes, with a size of 43.5 to 120 × 4.5 to 20 μm (n = 50) and six to 12 septa. The basal septum of conidia was darker and thicker than the other septa and bore a distinctly protruding, hilum. These morphological features align with the typical features of Exserohilum spp. (Hernández-Restrepo et al. 2018) (Fig. 1). Molecular identification was performed by partial sequences of ITS (ITS4 and ITS5 primers) (White et al. 1990), GAPDH gene (GPD1 and GPD2 primers) (Berbee et al. 1999) and TEF1-α gene (EF1-983 and EF1-2281 primers) (Rehner and Buckley 2005). Sequences were deposited in GenBank (ITS: PV981959, GAPDH: PX088164, TEF1-α: PX088170). In the BLAST analysis, the ITS, GAPDH and TEF1-α sequences all showed >99% identity with GenBank sequences of Exserohilum rostratum strains USJCC-0093 and CBS 188.68. The maximum-likelihood phylogenetic tree, constructed from the concatenated sequences of the three genes, clustered strain PL2428 with E. rostratum isolates (Fig. 2). In pathogenicity tests, leaves of six healthy potted sugarcane seedlings (five- to six-leaf stage) were needle-wounded and inoculated with a conidial suspension (10 6 conidia/mL) until runoff, whereas controls received sterile distilled water. All inoculated seedlings were kept in a greenhouse maintained at 28 °C and 80% to 90% relative humidity. While all plants inoculated with sterile water showed no symptoms 14 days post-inoculation, all plants inoculated with the conidial suspension displayed the same spot symptoms as observed in the field. E. rostratum was re-isolated from the symptomatic leaves, thus confirming Koch's postulates. To our knowledge, this is the first report of leaf spot disease caused by E. rostratum on sugarcane in China. Identifying the causal agent of this disease is fundamental to formulating effective control measures.