INTRODUCTION  

DAS-ELISA (double antibody sandwich enzyme-linked inmunosorbent assay) is an immuno-enzymatic assay that uses a microtitration plate as a support for the samples and reagents.

The procedure for the detecting Ralstonia solanacearum in soil, consists of:

1. Coating a microtitration plate with Ralstonia solanacearum (Rs)-specific rabbit immunoglobulins (IgG)
2. Adding solutions prepared from soil samples to the wells of the microtitration plate
3. Adding enzyme-labeled Rs-specific rabbit immunoglobulins (conjugated IgG)
4. Adding the enzyme substrate; if Rs is present in the soil solution this produces a color reaction, the intensity of which is proportional to the bacterial concentration

The bacteria may be present in soil at only low concentrations, so before performing the DAS-ELISA an enrichment procedure must be carried out to allow the bacteria to multiply to detectable levels. This is done by incubating the soil solution in an enrichment broth at 30°C for 48 hours with constant agitation. By this procedure the method is capable of detecting original bacterial concentrations as low as 20 bacteria per gram of soil (instead of 10⁷ bacteria/g without enrichment).

All races, biovars and serotypes of Rs can be detected with the Rs-specific immunoglobulins provided in the kit. Some saprophytic bacteria may cross-react with the antibodies produced against Rs when in pure culture at concentrations equal to or higher than 10⁸  bacteria/ml. However, after enrichment of soil solutions containing these saprophytic bacteria, no cross-reactions have been detected in DAS-ELISA.

This kit can be used to monitor Rs in bare soil after harvest as well as in the rhizosphere, rhizoplane and roots of potato or other hosts, to study the epidemiology of the disease, to evaluate the effectiveness of a control method in reducing soil populations of the pathogen, and to determine the suitability of a field for seed production.

Protocol for sample collection

In fields planted with potato or other hosts, take samples from the plant rhizosphere. In fields already harvested (bare soil), samples should be taken at 20–30 cm depth to avoid areas of high moisture and temperature fluctuations that can influence bacterial populations. The soil moisture should be at its field capacity. Collect about 100 g of soil with a small hand hoe. After each sample, disinfect the hoe with 0.5% sodium hypochloride and rinse with water.

Collect the samples in polyethylene bags; keep the bags open until sampling is complete. Transport samples to the lab under conditions that avoid excess of heat. At the lab, open the bags immediately and store them in an aerated place to avoid the multiplication of antagonistic and anaerobic bacteria. If possible, process the samples as soon as they arrive. If not, samples can be kept for up to a week if they are stored in a cool place (temperature between 10 and 15°C). Maintain the moisture of soil samples at field capacity.

Sample size

Experimental field

To evaluate methods for controlling bacterial wilt in experimental units of 15–20 m² in a soil highly infested with Rs, take one sample of about 100 g from each 2 m². Mix all the samples well, then take two composite samples of 100 g of soil for each plot.

For a semi-quantification of the bacterial population in the soil make serial 10-fold dilutions of the supernatant of the soil solution in citrate buffer up to 10^–5 (10^–1, 10^–2, 10^–3, 10^–4 and 10^–5). These dilutions should be enriched by mixing 1 ml of each dilution with 9 ml of enrichment broth and incubating them at 30°C for 48 hours under constant agitation.

The kit is sufficient for two evaluations of an experimental field of 12 plots of about 15–20 m² each

Seed production

For a qualitative evaluation of the inoculum in the soil (presence or absence of Rs) analyze 20 composite samples per hectare. Each composite sample is a mixture of 5 sub-samples. The samples should be taken in a randomized design following a zig-zag or cross pattern. For each sample make two serial 10-fold dilutions of the supernatant of the soil solution in citrate buffer (10^–1 and 10^–2). These dilutions should be enriched by mixing 1 ml of each dilution with 9 ml of enrichment broth and incubating the mixture at 30°C for 48 hours under constant agitation.

The kit is sufficient to evaluate five fields of approximately 1 ha each.