3.3.12
SAMPLE COLLECTION AND STORAGE FOR EVAPORATIVE-BINDING ENZYME-LINKED IMMUNOASSAY DIAGNOSIS OF RATOON STUNTING DISEASE OF SUGARCANE

B CROFT

Bureau of Sugar Experiment Stations, Tully, Australia

Background and objectives
Ratoon stunting disease (RSD) caused by the bacterium Clavibacter xyli subsp. xyli (Cxx) is the most economically important disease of sugarcane. The disease has no specific external symptoms. Serological diagnostic techniques include immunofluorescence, dot-blot enzyme- linked immunoassay and evaporative-binding enzyme-linked immunoassay (EB-EIA) [1]. The EB-EIA is used extensively in Australia with 49,000 samples processed in 1997. The standard procedure for collection of samples for the EB-EIA is to collect xylem water by applying positive air-pressure to one end of a stalk piece. Collecting samples from the field is the most labour intensive component of the diagnostic procedure. This paper reports on a comparison of different methods of collecting samples for the EB-EIA and methods of preserving samples in the field.

Materials and methods
Four different extracts were collected from the same stalk of three Cxx inoculated and three uninoculated plants of 20 sugarcane cultivars. The extracts were collected by: 1. Positive air-pressure applied to one end of a stalk piece to collect xylem extract. 2. Stalk juice collected with a grooved metal probe. 3. Stalk juice collected with pliers. 4. Leaf mid-ribs cut into 2 x 2 mm cubes and placed in distilled water. The samples were processed with the EB-EIA [1].

Formaldehyde (1.4%) treated and untreated samples of Cxx-free and positive xylem were incubated for 0, 2, 4, 8, 16 and 24 days at room temperature. After incubation the samples were frozen until all incubation treatments were completed, the optical density of samples and the reaction with the EB-EIA were measured.

Results and conclusions
The EB-EIA rated 55 of the xylem extracts from 60 Cxx inoculated plants positive and all 60 xylem extracts from uninoculated plants negative. The mean EB-EIA absorbance for positive extracts was 1.205 (SD=0.413) and for negative extracts was -0.012 (SD=0.006). Juice extracted with the probe and pliers techniques gave EB-EIA positive readings for 46 and 49 respectively of the 55 plants rated positive with xylem extracts. The mean EB-EIA absorbance for positive juice samples was 0.250 (probe) and 0.278 (pliers). There were no false positive results for the juice extracts. The leaf extract was only able to detect 9 of the 55 samples rated positive with xylem extracts. These results clearly show that xylem extracts give the most sensitive diagnosis of RSD with the EB-EIA. Juice may be used when xylem extracts cannot be obtained from drought affected cane.

The optical density of extracts stored for up to 24 days at room temperature increased rapidly up to 8 days and then showed a gradual decline. Formaldehyde prevented any increase in optical density. The EB-EIA reaction of stored Cxx-positive extracts declined from 1.78 to 0.47 after eight days and then stabilised but the addition of formaldehyde prevented any reduction in EB-EIA reaction. There was no significant change in EB-EIA readings from Cxx-free extracts stored up to 24 days. The results of this experiment clearly show that formaldehyde is an excellent preservative of xylem extracts and will allow storage of samples collected in areas with no ready access to a freezer and during transport to diagnostic laboratories.

References
1. Croft BJ, Greet AD, Leaman TM, Teakle DS, 1994. Proceedings of the Australian Society of Sugar Cane Technologists 16,143-51.