Amplification-Free Dna−Gold Affinity-Based Assay for Detecting Leifsonia Xyli Subsp. Xyli the Causal Agent of Sugarcane Ratoon Stunting Disease

Abstract

Sugarcane ratoon stunting (RSD), a prevalent disease caused by the bacterium Leifsonia xyli subsp. xyli (Lxx), poses a significant threat to sugarcane crops globally. RSD is challenging to diagnose due to invisible symptoms leading to its spread in commercial plantations. Existing molecular detection methods for Lxx are costly, require sophisticated equipment, are labor intensive, and entail prolonged sample-to-result times. To resolve these issues, an innovative electrochemical (EC) nano-biosensor approach was developed for detecting Lxx DNA in sugarcane xylem sap. The method involved three key steps: (i) boiling lysis-based DNA isolation from sugarcane sap; (ii) magnetic purification of target sequences directly from the lysate using magnetic bead-bound capture probes; and (iii) EC detection of the target. The detection sensitivity of the method was 10 cells/µL, and is highly reproducible (Standard deviation, SD = <5%, for n = 3) over a broad linear dynamic range (10 nM–1 fM or 105-100 copies/µL, r = 0.99). Furthermore, the method was validated across infected sugarcane cultivars with known RSD disease ratings (susceptible, intermediately resistant, and moderately resistant) collected from an inoculated field trial. The detected Lxx levels were subsequently correlated with the cultivar disease ratings. Additionally, EC method results were strongly correlated with those from qPCR assays with the same samples (r = 0.84, n=10, P < 0.001). The novel Lxx EC biosensor method holds promise as a commercially viable and DNA isolation/purification-free alternative to current Lxx diagnostics, a crucial step in developing a handheld on-farm Lxx detection and quantification device for RSD diagnostics.

Keywords: Nucleic acid isolation, Plant pathogen diagnostic, Sugarcane, Biosensing, Affinity interaction, Resistance screening