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Textual Amendments
The standard sample size is 200 tubers per test. More intensive sampling requires more tests on samples of this size. Larger numbers of tubers in the sample will lead to inhibition or difficult interpretation of the results. However, the procedure can be conveniently applied for samples with less than 200 tubers where fewer tubers are available.
Validation of all detection methods described below is based on testing of samples of 200 tubers.
The potato extract described below can also be used for detection of the potato brown rot bacterium, Ralstonia solanacearum.
Optional pre-treatment in advance to sample preparation: U.K.
Wash the tubers. Use appropriate disinfectants (chlorine compounds when PCR-test is to be used in order to remove eventual pathogen DNA) and detergents between each sample. Air dry the tubers. This washing procedure is particularly useful (but not required) for samples with excess soil and if a PCR-test or direct isolation procedure is to be performed.
Set aside any tubers with suspected ring rot symptoms and test separately.
If during removal of the heel end core suspect symptoms of ring rot are observed, the tuber should be visually inspected after cutting near the heel end. Any cut tuber with suspected symptoms should be suberised at room temperature for two days and stored under quarantine (at 4 to 10 °C) until all tests have been completed. All tubers in the sample (including those with suspicious symptoms) should be kept according to Annex II.
cover the cores with sufficient volume (approximately 40 ml) of extraction buffer (Appendix 3) and agitate on a rotary shaker (50 to 100 rpm) for four hours below 24 °C or for 16 to 24 hours refrigerated,
or
homogenise the cores with sufficient volume (approximately 40 ml) of extraction buffer (Appendix 3), either in a blender (e.g. Waring or Ultra Thurax) or by crushing in a sealed disposable maceration bag (e.g. Stomacher or Bioreba strong guage polythene, 150 mm × 250 mm; radiation sterilised) using a rubber mallet or suitable grinding apparatus (e.g. Homex).
The risk of cross-contamination of samples is high when samples are homogenized using a blender. Take precautions to avoid aerosol generation or spillage during the extraction process. Ensure that freshly sterilised blender blades and vessels are used for each sample. If the PCR test is to be used, avoid carry-over of DNA on containers or grinding apparatus. Crushing in disposable bags and use of disposable tubes is recommended where PCR is to be used.
Repeated freezing and thawing is not advisable.
If transport of the extract is required, ensure delivery in a cool box within 24 to 48 hours.
For detection of latent C. m. subsp. sepedonicus populations it is advised to test composite samples. The procedure can be conveniently applied for composite samples of up to 200 stem parts. (Where surveys are performed they should be based on a statistically representative sample of the plant population under investigation.)
Disinfect stem segments briefly with ethanol 70 % and immediately blot dry on tissue paper.
Collect stem segments in a closed sterile container according to the following sampling procedures:
cover the segments with sufficient volume (approximately 40 ml) of extraction buffer (Appendix 3) and agitate on a rotary shaker (50 to 100 rpm) for four hours below 24 °C or for 16 to 24 hours refrigerated,
or
process immediately. By crushing the segments in a strong maceration bag (e.g. Stomacher or Bioreba) with an appropriate volume of extraction buffer (Appendix 3) using a rubber mallet or appropriate grinding apparatus (e.g. Homex). If this is not possible, store the stem segments refrigerated for not longer than 72 hours or for not longer than 24 hours at room temperature.