The Australian sugar industry is currently the world's third-largest supplier of raw sugar, producing 30-35 Mt of cane per year, equating to 4 to 4.5 Mt of raw sugar (Australian Sugar Milling Council 2023). However, major diseases, such as ratoon stunting, leaf scald, smut, chlorotic streak, mosaic, Fiji leaf gall, pachymetra root rot, pineapple sett rot, and red rot, can cause significant losses to sugarcane productivity if not manage properly. Accurate pathogen detection at the early stage of infection is important for the prevention of disease spread, and mitigation of yield losses. Recently, significant advancement has been achieved in the diagnosis of sugarcane pathogens. Next-generation sequencing (NGS), high-throughput metagenomic sequencing, and quantitative polymerase chain reaction (qPCR) are the standard molecular techniques currently used by the Australian sugarcane industry to diagnose the causal agents of endemic and exotic diseases. Although accurate and robust, existing diagnostic methods contain a multi-step sample processing stage starting with nucleic acid extraction and purification, which is time consuming, labour intensive, expensive, and requires skilled personnel and centralized laboratory facilities. This highly involved sample preparation process also makes these methods inappropriate to use in resource-poor conditions. Here, we introduce a novel single-step method for in situ nucleic acid isolation and quantification from diverse sample types including leaves, meristematic tissues, and xylem sap of sugarcane plants (Figure 1). The method integrates the benefits of conventional isothermal amplification-based quantification and naked-eye detection where positive results are detected by colour change (i.e., pink to greenish yellow) in the reaction medium. Key outputs The method has sensitively detected Leifsonia xyli subsp. xyli and Xanthomonas albilineans, the causal pathogens of ratoon stunting and leaf scald diseases of sugarcane, respectively. This method has the potential to be integrated within a handheld device (Strachan et al. 2023) to detect other sugarcane pathogens for use by the growers in the field.