RSD diagnosis and varietal resistance screening in sugarcane using the EB-EIA technique

A new serological assay for detection of the RSD pathogen Clavibacter xyli subsp. xyli is described. In the evaporative-binding enzyme-linked immunoassay (EB-EIA), xylem extract from a piece of sugarcane stalk is dried onto the surface of microplate wells before processing with an enzyme-linked immunoassay procedure. EB-EIA can detect down to 5 X 105 bacterial cells/mL in xylem extract from infected cane, and colour intensity was highly correlated with bacterial population. The sensitivity of the test was optimised by increasing the proportion of coating buffer compared with xylem extract to 16:1 compared with the initial ratio of 4:1. In 1992-93, EB-EIA was used to diagnose the RSD status of 12,439 samples from throughout the Australian sugar industry. All positive and doubtful positive samples and 20% of negative samples were examined for C. xyli subsp. xyli with the phase-contrast microscope. EB-EIA detected 96% of the samples rated as positive with the phase-contrast microscope, and there was 99% agreement between the two techniques for samples rated negative by EB-EIA. False positive results in the doubtful category (6% of total samples) were associated with excessively dirty samples. EB-EIA also was used to compare varieties for reaction to RSD - intensity of reaction correlated well with field observations on varietal resistance to RSD infection.