3.3.19
DETECTION OF RATOON STUNTING DISEASE IN SUGARCANE USING PCR-ELISA AND REAL-TIME PCR (TAQMANTM) ASSAYS

J HENDERSON1, BJ CROFT2, RJ DIETZGEN3, GR SMITH4, PJ WHITTLE4 and DJ MACLEAN1

1Cooperative Research Centre for Tropical Plant Pathology, Department of Biochemistry, University of Queensland, Qld 4072, Australia; 2Tully Sugar Experiment Station, Bureau of Sugar Experiment Stations, PO Box 566, Tully, Qld 4854, Australia; 3Queensland Agricultural Biotechnology Centre, University of Queensland, Qld 4072, Australia; 4David North Plant Research Centre, Bureau of Sugar Experiment Stations, PO Box 86, Indooroopilly, Qld 4068, Australia

Background and objectives
Ratoon stunting disease (RSD), caused by the bacterium Clavibacter xyli subsp. xyli (Cxx), causes yield losses to the Australian sugar industry estimated in excess of AU$30 million annually. The disease is difficult to detect in the field as there are no external symptoms for reliable diagnosis. Stunting, a major sign of RSD infection, is not definitive, as poor growth can often be attributed to a variety of environmental factors including drought and poor farming techniques. In addition, these environmental stresses are capable of exacerbating the effects of RSD, causing increased crop losses. Currently, phase-contrast microscopy and a serological test, evaporative-binding (EB)-ELISA, are used by the sugar industry to monitor RSD outbreaks and ensure distribution of clean seed stocks; however, these methods lack sensitivity and specificity. PCR-based assays specific for Cxx would greatly assist the sugar industry in the monitoring and prevention of disease outbreaks in many situations, and two assay systems have been developed to this end; (i) PCR-ELISA and (ii) TaqManTM fluorescence PCR.

Materials and methods
Both the PCR-ELISA and TaqManTM assays have two levels of specificity based on PCR primers and an internal hybridization probe derived from the internal transcribed spacer (ITS) region between the 16S and 23S rRNA genes. The PCR-ELISA assay incorporates digoxigenin (DIG) antigen into PCR products using a Boehringer Mannheim kit and detects a 305-bp amplification product via a biotinylated hybridization probe. The TaqManTM assay, based on systems developed by Perkin Elmer [1], uses a Model 7700 Sequence Detector to detect a 68-bp amplification product via a dye-labelled probe that releases fluorescent product during PCR (real time fluorescence PCR).

Results and conclusions
The PCR-ELISA assay for Cxx is sensitive and semi-quantitative to a level of 50-100 cultured cells/PCR reaction. This figure is 102- to 103-fold more sensitive than EB-ELISA, the current industry standard. Field tests of PCR-ELISA have identified variability among cane varieties in detection limits of Cxx, and approaches to circumvent this problem are under investigation. A fluorescence PCR method based on the TaqManTM chemistry of Perkin Elmer has also been developed, is currently being assessed, and will be compared with PCR-ELISA.

References
1. Bassam BJ, Allen T, Flood S et al., 1996. Australasian Biotechnology, 6, 285-294.