[F1Appendix 3
Textual Amendments
A. Commercially available standardised control material U.K.
(a) Bacterial isolates U.K.
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:
National Collection of Plant Pathogenic Bacteria (NCPPB), Central Science Laboratory, York, UK
Culture Collection of the Plant Protection Service (PD), Wageningen, the Netherlands.
Collection Française de Bactéries Phytopathogènes (CFBP), INRA Station Phytobactériologie, Angers, France.
Table 1
SMT reference panel of isolates of R. solanacearum
| a 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 a | 71 a | 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. U.K.
Table 2:
SMT reference panel of serologically- or genetically-related bacteria for use in optimisation of detection tests
| a Potential cross-reacting strain in serological tests (IF and/or ELISA) with polyclonal antisera. | |||
| b 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). | |||
| c 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 a |
| NCPPB 4163 | 52 | CFBP 1538 | Pseudomonas marginalis pv. marginalis a |
| NCPPB 4164 | — | CFBP 2227 | Burkholderia cepacia b |
| NCPPB 4165 | — | CFBP 2459 | Ralstonia pickettii b |
| NCPPB 4166 | 58 | CFBP 3567 CSL Pr1150 | Ralstonia pickettii a |
| NCPPB 4167 | 60 | CFBP 4618 PD 2778 | Ralstonia sp. a |
| NCPPB 1127 | 53 | CFBP 3575 | Burkholderia andropogonis a |
| NCPPB 353 | 54 | CFBP 3572 | Burkholderia caryophylli a |
| NCPPB 945 | 55 | CFBP 3569 | Burkholderia cepacia a |
| NCPPB 3708 | 56 | CFBP 3574 | Burkholderia glumae a |
| NCPPB 3590 | 57 | CFBP 3573 | Burkholderia plantarii a |
| NCPPB 3726 | 59 | CFBP 3568 | Banana Blood Disease Bacterium a b c |
| NCPPB 4168 | 61 | CFBP 4619 IPO S339 | Enterobacter sp. a |
| NCPPB 4169 | 62 | IPO 1695 | Enterobacter sp. a |
| NCPPB 4170 | 63 | CFBP 4621 IPO S306 | Ochrobactrum anthropi a b |
| NCPPB 4171 | 64 | CFBP 4622 IPO 1693 | Curtobacterium sp. a b |
| NCPPB 4172 | 65 | IPO 1696a | Pseudomonas sp. a |
| NCPPB 4173 | — | PD 2318 | Aureobacterium sp. b |
| NCPPB 4174 | 81 | IVIA 1844.06 | Flavobacterium sp. a b |
(b) Commercially available standardised control material U.K.
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 U.K.
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.]