# Council Directive 98/57/EC of 20 July 1998 on the control of Ralstonia solanacearum (Smith) Yabuuchi et al., Appendix 3 Extent: E+W+S+N.I. A. Commercially available standardised control material (a) Bacterial isolates The following bacterial isolates are recommended for use as standard reference material either as positive controls (Table 1) or during optimisation of tests to avoid cross-reactions (Table 2). All strains are commercially available from: 1.. National Collection of Plant Pathogenic Bacteria (NCPPB), Central Science Laboratory, York, UK 2.. Culture Collection of the Plant Protection Service (PD), Wageningen, the Netherlands. 3.. Collection Française de Bactéries Phytopathogènes (CFBP), INRA Station Phytobactériologie, Angers, France. Table 1 SMT reference panel of isolates of R. solanacearum Use as standard reference strain of R. solanacearum biovar 2 (race 3). NCPPB code SMT # Other codes Country of origin Biovar NCPPB 4153 6 CFBP 4582, Pr 3020, EURS11 Egypt 2 NCPPB 4154 10 CFBP 4585, 550, EURS21 Turkey 2 NCPPB 3857 12 CFBP 4587, Pr 1140, EURS26 England 2 NCPPB 1584 23 CFBP 4598, EURS49 Cyprus 2 NCPPB 2505 24 CFBP 4599, EURS50 Sweden 2 NCPPB 4155 26 CFBP 4601, 502, EURS55 Belgium 2 NCPPB 4156 71 PD 2762, CFBP 3857 Netherlands 2 NCPPB 4157 66 LNPV 15.59 France 2 NCPPB 4158 39 CFBP 4608, Port 448, EURS80 Portugal 2 NCPPB 4160 69 IVIA-1632-2 Spain 2 NCPPB 4161 76 B3B Germany 2 NCPPB 325 41 CFBP 2047, KEL60-1, R842 USA 1 NCPPB 3967 42 CFBP 4610, R285, GONg7 Costa Rica 1 NCPPB 4028 43 CFBP 4611, R303/571, CIP310, SEQ205 Colombia 2 NCPPB 3985 44 CFBP 4612, R578, CIP312 Peru 2T NCPPB 3989 45 CFBP 4613, R568, CIP226 Brazil 2T NCPPB 3996 46 CFBP 3928, R276/355, CIP72, SEQ225 Peru 3 NCPPB 3997 47 CFBP 4614, R280/363, CIP49, HAY0131a Australia 3 NCPPB 4029 48 CFBP 4615, R297/349, CIP121, CMIb2861 Sri Lanka 4 NCPPB 4005 49 CFBP 4616, R470 Philippines 4 NCPPB 4011 50 CFBP 4617, R288, HEmps2 China 5 Note: Authenticity of the above strains can be guaranteed only if obtained from an authentic culture collection. Table 2: SMT reference panel of serologically- or genetically-related bacteria for use in optimisation of detection tests Potential cross-reacting strain in serological tests (IF and/or ELISA) with polyclonal antisera. Strain from which PCR product can be amplified in some laboratories of a similar size to that expected using specific primers OLI-1 and Y-2 (see Appendix 6). Likely to cross-react in most tests but known to occur only on banana in Indonesia. NCPPB code SMT # Other code Identification NCPPB 4162 51 CFBP 1954 Bacillus polymyxa NCPPB 4163 52 CFBP 1538 Pseudomonas marginalis pv. marginalis NCPPB 4164 — CFBP 2227 Burkholderia cepacia NCPPB 4165 — CFBP 2459 Ralstonia pickettii NCPPB 4166 58 CFBP 3567 CSL Pr1150 Ralstonia pickettii NCPPB 4167 60 CFBP 4618 PD 2778 Ralstonia sp. NCPPB 1127 53 CFBP 3575 Burkholderia andropogonis NCPPB 353 54 CFBP 3572 Burkholderia caryophylli NCPPB 945 55 CFBP 3569 Burkholderia cepacia NCPPB 3708 56 CFBP 3574 Burkholderia glumae NCPPB 3590 57 CFBP 3573 Burkholderia plantarii NCPPB 3726 59 CFBP 3568 Banana Blood Disease Bacterium NCPPB 4168 61 CFBP 4619 IPO S339 Enterobacter sp. NCPPB 4169 62 IPO 1695 Enterobacter sp. NCPPB 4170 63 CFBP 4621 IPO S306 Ochrobactrum anthropi NCPPB 4171 64 CFBP 4622 IPO 1693 Curtobacterium sp. NCPPB 4172 65 IPO 1696a Pseudomonas sp. NCPPB 4173 — PD 2318 Aureobacterium sp. NCPPB 4174 81 IVIA 1844.06 Flavobacterium sp. (b) Commercially available standardised control material The following standard control material is available from the NCPPB culture collection. Freeze dried pellet of potato extract from 200 healthy potato tubers as negative control for all tests. Freeze dried pellet of potato extract from 200 healthy potato tubers containing 10 3 to 10 4 and 10 4 to 10 6 cells R. solanacearum biovar 2 (strain NCPPB 4156 = PD 2762 = CFBP 3857) as positive controls for serological and PCR tests. Since cell viability is affected during freeze-drying, these are not suitable as standard controls for isolation or bioassay tests. Formalin-fixed suspensions of R. solanacearum biovar 2 (strain NCPPB 4156 = PD 2762 = CFBP 3857) at 10 6 cells per ml as positive controls for serological tests. B. Preparation of positive and negative controls for the core screening tests PCR/IF and FISH Produce a 48 hour culture of a virulent strain of R. solanacearum race3/biovar2 (e.g. strain NCPPB 4156 = PD 2762 = CFBP 3857) on basal SMSA medium and suspend in 10 mM phosphate buffer to obtain a cell density of approximately 2 × 10 8 cfu per ml. This is usually obtained by a faintly turbid suspension equivalent to an optical density of 0,15 at 600 nm. Remove the heel end cores of 200 tubers taken from a white skin variety production known to be free from R. solanacearum . Process the heel ends as usual and resuspend the pellet in 10 ml. Prepare 10 sterile 1,5 ml microvials with 900 µl of the resuspended pellet. Transfer 100 µl of the suspension of R. solanacearum to the first microvial. Vortex. Establish decimal levels of contamination by further diluting in the next five microvials. The six contaminated microvials will be used as positive controls. The four non-contaminated microvials will be used as negative controls. Label the microvials accordingly. Prepare aliquots of 100 µl in sterile 1,5 ml microvials thus obtaining nine replicas of each control sample. Store at -16 to -24 °C until use. The presence and quantification of R. solanacearum in the control samples should be first confirmed by IF. For the PCR test perform DNA extraction from positive and negative control samples with each series of test samples. For IF and FISH tests perform assays on positive and negative control samples with each series of test samples. For IF, FISH and PCR assays R. solanacearum must be detected in at least the 10 6 and 10 4 cells/ml of the positive controls and not in any of the negative controls. --- Source: https://www.legislation.gov.uk/eudr/1998/57/annex/II/appendix/3 (the official version) Page: https://openlegislation.uk/eu-directive-1998-57/annex-II/appendix-3 Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives.