A validated SYBR-based RT-qPCR assay reveals seasonal dynamics and within-stool heterogeneity of sugarcane yellow leaf virus in sugarcane

Sugarcane yellow leaf virus (SCYLV) occurs in all sugarcane producing regions of the world and can reduce yield up to 50%. In Louisiana, USA, infections are asymptomatic, making reliable detection paramount to avoid unintentional disease spread. While reverse-transcription polymerase chain reaction (RT-PCR) assays are available, they may lack sensitivity. This study aimed to develop a reliable and sensitive assay appropriate for state certification and routine virus detection. Using an RNA transcript encoding the coat protein of SCYLV, a one-step, SYBR green-based quantitative RT-PCR (RT-qPCR) assay was developed that can detect as few as seven viral copies from three SCYLV genotypes. Monthly sampling conducted from April to August in 2024 (cultivar LCP 85–384) and April to September in 2025 (cultivar L 01–299) showed that SCYLV is consistently detectable in the top visible dewlap leaf (TVD) in both cultivars. In LCP 85–384, virus titer varied in April (p = 0.002), but in L 01–299, variation occurred in June (p < 0.001), contributing to significant differences in titer across months. Current Louisiana certification protocols call for 50 leaves collected per 5–10 acres, likely underestimating incidence as this study revealed uneven SCYLV distribution across canes in newly infected stools. Those stools exhibited non-ubiquitous virus distribution (56.1 ± 7.0%; F₁,₃₀ = 20.2, p < 0.001) between canes compared to plants from already infected planting material (least squares mean = 95.4 ± 5.3%). Stools that were originally SCYLV-negative and infected post planting displayed a significantly lower viral titer than stools planted with SCYLV-positive material (p = 0.002). This study provides a sensitive RT-qPCR assay for SCYLV detection further validated using field collected samples, confirms the TVD is adequate tissue for detection throughout the growing season, and emphasizes the criticality of thorough collection methods for reliable detection of this economically important virus.