Commission Directive 2006/63/CEShow full title

2. Methods for detection and identification of R. solanacearum in soil

Principles

The validated detection scheme, described in this section, is applicable for pathogen detection in soil samples but can also be used to test samples of solid potato processing waste or sewage sludge. However, it should be noted that these methods are insufficiently sensitive to guarantee detection of low and/or irregularly dispersed populations of Ralstonia solanacearum that may occur in naturally infested samples of these substrates.

The limitations in sensitivity of this test scheme should be considered when assessing the reliability of any negative results obtained and also when used in surveys to determine presence or absence of the pathogen in soils or sludges. The most reliable test for presence of the pathogen in a field soil is to plant a susceptible host and monitor it for infection, but even with this method low levels of contamination will escape detection.

2.1. Sample preparation

2.1.1. Sampling of field soil should follow standard principals used for nematode sampling. Collect 0,5 to 1 kg of soil per sample from 60 sites per 0,3 ha from a depth of 10 to 20 cm (or in a grid of 7 x 7 metres) If the pathogen is suspected to be present, increase the number of collection points to 120 per 0,3 ha. Maintain samples at 12 to 15 °C prior to testing. Sample potato processing and sewage sludges by collecting a total of 1 kg from sites representing the total volume of sludge to be tested. Mix each sample well before testing.

2.1.2. Disperse sub-samples of 10 to 25 g of soil or sludge by rotary shaking (250 rpm) in 60 to 150 ml extraction buffer (Appendix 4) for up to two hours. If required, addition of 0,02 % sterile Tween-20 and 10 to 20 g sterile gravel may assist dispersion.

2.1.3. Maintain the suspension at 4 °C during testing.

2.2. Testing

See flow chart and description of the tests in the relevant appendices.