Naked eye evaluation and quantitative detection of the sugarcane leaf scald pathogen, Xanthomonas albilineans, in sugarcane xylem sap

Abstract

Sugarcane leaf scald caused by the bacterium Xanthomonas albilineans is a major disease of sugarcane worldwide. Whereas erratic symptoms make phenotypic detection challenging, molecular methods require expensive instruments and labour, and longer sample-to-answer times. We report a novel method for detection of X. albilineans DNA in sugarcane xylem sap. The method involves (i) boiling lysis-based DNA extraction from sugarcane sap; (ii) magnetic purification of target sequences directly from the lysate through use of magnetic bead-bound capture probes; and (iii) DNA sandwich hybridisation platform for HRP/TMB/H2O2 reaction-based naked eye visualisation and electrochemical detection of the target. The method is sensitive (limit of detection 100 fM) and reproducible (relative standard deviation <7%) with linear dynamic range 100 fM-1 nM (R2 = 0.99). The method was tested on a range of sugarcane cultivars of known resistance ratings (susceptible, intermediate resistant, and resistant) for leaf scald disease from an inoculated field trial. Detection levels agreed with the resistance rating of cultivars tested. In addition, qPCR results strongly correlated with our assay (r = 0.91-0.99, P < 0.01) and cultivar resistance rating. We believe that our assay could be useful for rapid screening as well as sensitive quantification of target pathogen DNA in infected sugarcane plants.