A novel Leifsonia xyli subsp. xyli quantitative LAMP-based diagnostic correlated with sugarcane ratoon stunting disease rating

Context: Ratoon stunting disease (RSD), caused by Leifsonia xyli subsp. xyli (Lxx), poses a significant economic threat to sugarcane (Saccharum hybrid) worldwide. RSD is hard to manage due to its elusive visible symptomology and disease rating of cultivars is subjective. Aims: We aimed to develop a sensitive, rapid, and quantitative Lxx diagnostic method able to correlate Lxx titre and disease resistance rating of sugarcane cultivars. Methods: A Lxx diagnostic method was developed using heat lysis-based reagent-free DNA isolation from xylem sap followed by loop-mediated isothermal amplification (LAMP)-based colorimetric and fluorescence quantification within a single microcentrifuge tube. Bacterial titre was then statistically correlated with industry-agreed disease resistance ratings for key sugarcane cultivars. Key results: The diagnostic was highly sensitive (1 cell/μL) and reproducible (%s.d. = <5%, for n = 3), and showed excellent linear dynamic range (i.e. 10 pM-1 aM or 107-100 copies/μL, r = 0.99) for quantitative Lxx detection. LAMP quantifications were completely concordant with quantitative polymerase chain reaction quantification from the same samples. Additionally, a strong correlation was determined between the detected quantitative bacterial titres and known cultivar disease resistance ratings (r = 0.82, n = 10, P < 0.001). Conclusion: The novel LAMP-based Lxx diagnostic was validated as a fast, simple, and relatively cost-effective means of RSD resistance rating, making it a reliable contribution towards RSD management. Implications: The development of this diagnostic tool provides a practical solution for accurately measuring Lxx titre and assessing disease resistance in sugarcane plants, aiding in effective risk management of RSD spread, and mitigating its economic impact on sugarcane crops worldwide.