First report of Bipolaris setariae causing sugarcane brown stripe in the United States

Sugarcane is Louisiana’s most valuable agricultural row crop, contributing more than $4 billion annually to the state’s economy. Since 2016, the dominant variety ‘L01-299’ has been frequently affected by epidemics of brown stripe, particularly in spring as plants resprout following winter freezes. The ‘L01-299’ variety occupies 54% of Louisiana's nearly 220,000 ha sugarcane industry, and brown stripe affects nearly every field. Leaf symptoms initially appear as spindle-shaped brown lesions. Often, chlorotic halos surround the spindle-shaped lesions following leaf veins, expanding more pronouncedly longitudinally and slightly widening laterally, eventually coalescing, turning necrotic, and curling leaf margins. Leaves of ‘L01-299’ showing typical brown stripe symptoms were collected from commercial farms, and lesion margins were surface sterilized in 70% ethanol for 1 min, followed by 0.5% sodium hypochlorite for 1 min, and two rinses in sterile distilled water, and placed on water agar to minimize contamination. Once mycelial growth was observed, agar plugs were transferred to PDA for isolation and culture. Colonies on PDA were irregular, raised, dark gray with lighter margins. Conidia were gray to dark brown, spindle-shaped, straight or slightly curved, showing 4 to 9 distosepta, measuring 32.2 to 89.3 × 8.5 to 15.6 μm (mean 65.6 x 13.1, n=30). Four representative single-spore isolates were obtained, and genomic DNA was extracted using Qiagen’s DNeasy Plant Mini Kit following the manufacturer protocol for fungi. The internal transcribed spacer region (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the translation elongation factor 1-α genes (TEF1- α) were amplified for four representative isolates using primer pairs ITS1/ITS4 (White et al., 1990), gpd1/gpd2 (Berbee et al., 1999) and EF1-983/EF1-2218R (Manamgoda et al., 2012). The sequences for ITS (PZ327421-PZ327424), GAPDH (PZ357598-PZ357601), and TEF1-α (PZ357602-PZ357605) for each isolate were deposited in GenBank. Maximum-likelihood phylogenetic analysis placed our isolates in strongly supported (99%) clade with isolates previously identified as Bipolaris setariae. BLASTn analysis showed ITS sequences shared 100% identity with ex-type strain B. setariae CBS 141.31 (accession HE792936.1), and TEF1- α and GAPDH sequences also shared 100% similarity with available B. setariae sequences (accessions: ON614795.1 and PX088161.1, respectively). One representative isolate was inoculated onto healthy 2-month-old ‘L01-299’ sugarcane plants (n=5) using a 10 6 conidia/ml suspension, and plants were kept under dew chamber conditions for 48 h to fulfil Koch’s postulates (Li et al. 2023). A control set of plants was kept under the same conditions but were mock-inoculated with sterile distilled water (n=5). Plants were monitored daily for 11 days, with initial lesions appearing 48 h after inoculation and later developing into typical necrotic lesions. All inoculated plants developed typical brown stripe symptoms, whereas mock inoculated plants remained healthy. Reisolation of B. setariae was done in all inoculated plants, fulfilling Koch’s postulates. These results show that the same species responsible for major brown stripe epidemics in China causes the disease in Louisiana. Identifying B. setariae as the causal agent is pivotal for future studies developing management strategies for this important fungal disease in Louisiana sugarcane fields.