Elphinstone JG, Hennessy J, Wilson JK & Stead DE (1996) Sensitivity of detection of Ralstonia solanacearum in potato tuber extracts. Bulletin. OEPPIEPPO ...
Standardization of methods for detection of Ralstonia solanacearum in potato* J. G. Elphinstone’, D. E. Stead’, D. Caffier’, J. D. Janse’, M. M. Lopez2, U. Mazzucchi2, P. Muller ’, P. Persson2, E. Rauscher ’, E. Schiessendoppler 2, M. Sousa Santos 2, E. Stefani ’, J. van Vaerenbergh et a/.
’
‘Central Science Laboratory, Sand Hutton, York, YO41 ILZ (United Kingdom); e-mail: j.elphinstone@csl,gov.uk 2See Table I
In support of EU Council Directive 98/57/EC on the control of potato brown rot disease, caused by Ralstonia solanacearum, a ring test was conducted to evaluate and standardize currently recommended methods for official testing of potato tubers. Test tubers were either naturally infected (symptomatic) or vacuum-infiltrated with different R. solanacearum suspensions or water. Testing was performed independently in 19 EU plant health laboratories using standardized protocols based on those annexed to the Directive. With high pathogen concentrations, fully reliable results were obtained with dilution plating on a selective medium, immunofluorescent antibody staining (IFAS), enzyme-linked immunosorbent assay (ELISA), fluorescent in situ hybridization and a tomato bioassay. The reliability of diagnoses across all laboratories decreased with inoculum concentration in asymptomatic tubers, irrespective of the methods used. Comparable results were obtained with 3 commercial antisera in F A S or with 2 antisera in ELISA but the IFAS method was slightly more reliable. In several laboratories, diagnoses obtained using polymerase chain reaction (PCR) protocols were less reliable than those using the other methods. Independent optimization of PCR conditions in each laboratory and the introduction of internal positive PCR controls are recommended.
Introduction Ralstonia solanacearum, which causes potato brown rot, is a quarantine pest in Europe. Phytosanitary measures contained in EU Council Directive 98/57EC (EU 1998) aim to prevent entry of this bacterium into EU member states and, if found, to determine its distribution as well as contain and control it with a view to eradication. An interim test scheme, annexed to the Directive, details both compulsory and facultative methods for detection and identification of the pathogen. The following study was conducted as a ring-test in a number of official plant health laboratories in order to validate, standardize and improve the interim methods currently recommended for official testing of potato tubers. Specific objectives were: (1) to compare sensitivity and specificity of optimized procedures for detection of R. solanuceanun in potato tubers, and (2) to harmonize test procedures across EU plant health laboratories.
was co-ordinated at the Central Science Laboratory (CSL), York (GB). Replicated samples, distributed to each laboratory, consisted of 1 infected potato tuber (cv. Estima) with typical brown rot symptoms and 4 other tubers vacuuminfiltrated with aqueous suspensions of R. solanacearum of varying unknown population or sterile water controls. Each test tuber was mixed with 199 healthy tubers in each laboratory to represent a standard 200 tuber sample. Heel-end cores of vascular tissue were then removed (as directed in Annex I1 of EU Council Directive 98/57/EC) from each of the 200 tubers per sample and shaken in 50 mM phosphate buffer for 16 h at 4°C. The resulting suspensions were centrifuged at 7000g for 15 min at 4”C, the supernatant was discarded and the pellets were resuspended in 2mL of lOmh4 phosphate buffer.
Pathogen detection
Materials and methods Sample preparation
R. solanacearum was detected in aliquots of the resuspended pellets, as directed in Annex II of EU Council Directive 98/ 5 7 E C (Anon, 1998), using the following methods.
The ring-test involved a total of 19 laboratories (Table 1) and Dilution plating *Paper presented at the EPPO Conference on diagnostic techniques for plant pests, Wageningen (NL), 2000-02-01/04.
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Ten-fold dilution series of resuspended pellets were prepared in sterile water and lOOpL of each dilution was spread onto
39 1
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J. G. Elphinstone et al.
Table 1 European plant health laboratories participating in ring-tests
Laboratory
City
Country
Bundesamt fur Agrarbiologie' Bundesamt und Forschungszentrum fur Ldndwirtschaft' Departement Gewa~bescherming~ Plantedirektoratet" Central Science Laboratory' Laboratoire National de la Protection des VCgCtaux, Unit6 Bact6riologie6 Station de Quarantaine de la Pomme de Terre7 Biologische BundesanstaltX Ptlanzenschutzamt Hannover' State Laboratory'" lstituto di Patologia Vegetale' I Osservatorio Malattie Piantel' Nederlandse Algemene Keuringsdienst13 Plantenziektenkundige Dienst I" DirecGgo-Geral de ProtecGHo das Culturas" Scottish Agricultural Science Agency" Centro Diagnostic0 de Aldearrubia17 lnstituto Valenciano de Investigaciones Agrarias" Swedish University of Agricultural Sciences''
Linz Wien Merelbeke
AT AT BE DK GB (England)
LYWbY York Angers Le Rheu Kleinmachnow Hannover Dublin Bologna Verona Emmeloord Wageningen Lisbon Edinburgh Salamanca Valencia Uppsala
FR FR DE DE IE IT IT NL NL
PT GB (Scotland) ES ES SE
Principal scientists involved in ring-tests: IS. Bemkopf, 'E. Schiessendoppler, 'E. Rauscher, 'J. van Vaerenbergh, 'B. de Paepe, "I. Dinesen, 'H. Stanford, 'J. G. Elphinstone, 'D. E. Stead, "D. Caffier, 6M. Solano, 'J.-M. Expert, 6F. Noublanche, 7A. Herve, 7J. Martin, 'F. Ollivier, 8P. Muller, "K.-H. Pastrik. 'OD. Hayes. 'OM. Sutton, "U. Mazzucchi, "E. Stefani, "C. R. Messina, "A. Saccardi, "G. W van der Bovenkarnp, "E. G. de Haan, '"5. D. Janse, '"A. van Beuningen, I4J. Derks, ISM. Sousa Santos, "L. Henriques, "L. Cmz, "J. Wood, I6K. Breckenridge, I7J.-L. Palorno, I7P. Benavides, I'M. M. L6pez. '*B.Vicedo, "B. Lastra, "P. Persson.
modified SMSA semi-selective agar medium (Elphinstone et al., 1996). lmmunofluorescenceantibody staining (IFAS)
The IFAS procedure of Janse (1988) was performed on 20-pL aliquots of resuspended pellets before and after dilution (1: 100) in 10 mM phosphate buffer. Three commercially available polyclonal antisera were compared: IACR-278 (IACR-Rothamsted, GB), Adgen- 1091 (Adgen Diagnostics, GB) and IPO-9523 (Plant Research International, NL). Enzyme-linked immunosorbent assay (ELISA)
The indirect ELISA procedure of Robinson-Smith et al. (1995) was performed on 2 ~ 5 0 p Lof resuspended pellets after heating to 96°C for 4 min and dilution (1: 1) in doublestrength carbonate coating buffer. Three commercially available polyclonal antisera were compared: IACR-278 (IACR-Rothamsted, GB), Adgen-109 1 (Adgen Diagnostics, GB) and Loewe-7356 (Loewe Phytodiagnostica, DE).
Polymerase chain reaction (PCR)
Two pathogen-specific PCR procedures were performed using either primer pairs OLI-1 and Y-2 (Seal et al., 1993) or Psl and Ps2 (K.-H. Pastrik, pers. comm.) based on 16s rRNA gene sequences. All reagents were supplied from a central source except for Taq polymerase which was obtained locally in each case from either Perkin Elmer Applied Biosystems (AmpliTaq) or Gibco BRL. Four methods of DNA extraction prior to analysis by PCR were compared: 1 heating of 100 pL of resuspended pellet containing 50mM NaOH to > 96°C by placing in a boiling water bath or heating block for 4 min; 2 enrichment in semi-selective SMSA broth (Elphinstone et al., 1996) by incubation of 100pL of resuspended pellets in 10 mL broth for 72 h at 28"C, followed by heating of 100 pL enriched broth with 50 mM NaOH to < 96°C by placing in a boiling water bath or heating block for 4 min; 3 DNA purification using the 'Easy DNA' kit from Invitrogen BV (NL) according to the method of Pastrik & Rainey (1999); 4 DNA purification using an isopropanol extraction method of Llop et al. (1 999).
Fluorescent in situ hybridization (FISH)
The FISH technique of Wullings et al. (1998) was performed on 1OOpL of resuspended pellets using an R. solanacearumspecific oligoprobe to 16s rRNA gene sequence (Seal et al., 1993).
Tomato bioassay
The bioassay method of Janse (1988) was performed by injection of resuspended pellets (about 5 pL per site) into 2
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Ralstonia solanacearum in potato Table 2 Mean* Ralstonia solanacearum population (n = 17) in concentrated extracts from potato samples estimated after dilution plating on semi-selective SMSA agar
Table 4 Detection of Ralstonia solanacearum in concentrated extracts from potato samples by indirect ELISA with pathogen-specific polyclonal antibodies (pAb) from 3 commercial sources
LOG cfu per mL of resuspended pellet
Sample
7.7 (f 1.4) 4.1 ( 2 3.4) 6.6 ( 2 2.8) 8.7 (f2.0)
Mean* ELISA ODdo5nm ( n = 18) Sample
pAb IACR-278
pAb Adgen-1091
pAb Loewe-7356
1
1.4 (f0.9) 0.3 (% 0.4) 0.8 (% 0.8) 1.4 (f 0.9) 0.1 ( 2 0.1)
1.4 ( 2 0.9) 0.4 ( % 0.4) 0.9 (f0.8) 1.4 ( f 0.9) 0.2 ( 2 0.2)
0.9 ( 2 0.6) 0.2 ( 2 0.2) 0.4 (f0.4) 0.8 ( 2 0.5) 0.1 ( 2 0.2)
2 3 4 5
0.0
393
* Standard error of the mean shown in parentheses. sites per stem (one above and one below the cotyledons) of 10 susceptible tomato seedlings (cv. Moneymaker or cv. of equivalent susceptibility). Inoculated plants were maintained at 25-30°C for 28 days and observed for typical symptoms of wilting and epinasty. After 28 days, attempts were made to reisolate the pathogen from seedlings with and without symptoms.
Results and discussion The results of all tests are shown in Tables 2-7 and Fig. 1. As expected, R. solanacearum populations detected varied according to the original levels of inoculum used to infect the test tubers. Highest populations were consistently detected in sample 1 (which contained symptomatic tubers) and sample 4 (which contained asymptomatic tubers infiltrated with the highest inoculum level of 108cfu per mL). Lower mean population estimates in samples 2 and 3 also reflected the original levels of inoculum used to infiltrate the test tubers (lo4 and 106cfu per mL respectively). Mean population estimates for each sample were similar whether determined by dilution plating (Table 2) or IFAS (Table 3), whereas estimates determined by FISH (Table 4) were consistently lower. Mean ELISA readings (Table 5) and rates of infection of tomato seedlings (Table 6) also reflected initial inoculum concentrations. In general, the reliability of detection across all laboratories
Table 3 Mean* Ralstonia solanacearum population (n = 18) in concentrated extracts from potato samples estimated by immunofluorecence antibody staining (IFAS) with pathogen-specific polyclonal antibodies (pAb) from 3 commercial sources
*Standard error of the mean shown in parentheses. A positive result was indicated when the mean OD4o5nm for each sample exceeded 2 x the value for the non-inoculated control (sample 5).
varied with initial inoculum concentration irrespective of the methods used (Table 7). With samples containing the highest pathogen concentrations (samples 1 and 4), consistently reliable results were obtained using dilution plating (Table 2), IFAS (Table 3), ELISA (Table 4), FISH (Table 5) and the tomato bioassay (Table 6). Moreover, non-inoculated controls (sample 5) consistently tested negative using these tests. However, with samples containing low or medium levels of inoculum (samples 2 and 3 respectively), results obtained with each test were more variable (Tables 2-6) and fewer laboratories successfully detected the pathogen as inoculum levels decreased, irrespective of the test used (Table 7). At the lowest inoculum level, the unreliability of detection may have been due at least in part to an uneven distribution of inoculum among the test tubers. At all levels of inoculum, the most consistent results were obtained by dilution plating on SMSA medium. With the serological detection tests, comparable results were obtained with 3 different commercial antisera (IACR278, Adgen-1091 and PO-9523) in IFAS and with 2 antisera (IACR-278 and Adgen-1091) in ELISA. A third antiserum (Loewe 7356) resulted in a reduced ELISA signal. IFAS was slightly more reliable than ELISA, especially at low pathogen concentration, as low numbers of cells which were visible microscopically were insufficient to generate an ELISA signal.
Table 5 Mean* Ralstonia solanacearum population (n = 7) in concentrated extracts from potato samples estimated by fluorescent in situ hybridization (FISH)
LOG cells per mL of resuspended pellet Sample
pAb IACR-278
pAb Adgen-1091
pAb PO-9523
1 2 3 4 5
8.1 (t0.9) 3.3 ( 2 3.1) 5.9 ( f 3.0) 7.9 ( 2 0.7) 0.0
7.8 (f 1.1) 3.4 (t 3.2) 4.9 ( 2 3.4) 7.7 ( 2 0.8) 0.0
8.1 (t0.9) 3.4 (f 3.2) 5.3 ( 2 3.3) 7.8 (f0.8) 0.0
* Standard error of the mean shown in parentheses.
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Sample
LOG cells per mL resuspended pellet 6.7 ( 2 0.6) 3.1 ( 2 3.6) 4.4 ( 2 3.0) 6.3 ( 2 0.5) 0.0
*Standard error of the mean shown in parentheses.
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J. G. Elphinstone et al.
Table 6 Incidence of infection of tomato seedlings 28 days after inoculation with extracts from potato samples Sample
Mean* no. of tomato plants infected out of 10 ( n = 15)
1 2 3
9.5 ( 2 1.4) 3.8 ( 2 4.7) 7.5 ( 2 3.8) 9.4 ( 2 1.2) 0.0
4 5
*Standard error of the mean shown in parentheses
Although all infected samples were successfully detected using PCR protocols in some laboratories (Fig. l), the pathogen was generally less reliably detected across all laboratories when using PCR protocols than when using the other methods (Table 7). In addition, a low level of false positive results obtained when testing sample 5 indicated that cross-contamination had occurred in one laboratory during three of the DNA extraction methods (Table 7). This may in part reflect differences in levels of experience across laboratories in the use of the PCR technique. Variation in results may also have resulted from a lack of optimization of PCR reaction conditions in each laboratory to suit differences in thermal cyclers or sources of DNA polymerase used. Pathogen populations obtained in extracts from samples containing naturally infected symptomatic or latently infected
1
2
3
4
5
6
7
Fig. 1 PCR results obtained using Invitrogen DNA extraction and PCR protocol of K-H. Pastrik (unpublished). Lane 1 and 7, 100 bp DNA ladder; lane 2, sample 1 ; lane 3, sample 2; lane 4, sample 3; lane 5. sample 4; lane 6, sample 5.
tubers are usually high (Elphinstone et al., 1996; J. D. Janse, pers. COD.). With the exception of the PCR protocols used, the methods evaluated above should provide reliable levels of detection, particularly if more than one method is used for initial screening. Further investigation will, however, be required to determine the frequency of occurrence of samples containing low R. solanacearum populations and assess their
Table 7 Proportion of laboratories obtaining positive results with different detection methods following blind testing of samples of 200 potato tubers containing single symptomatic or asymptomatic tubers inoculated with different populations of Ra/sfoniasolanacearum No. of laboratories obtaining positive test results
No. of laboratories performing the test Sample I* Sample 21 Sample 3$ Sample 45 Sample 51
Detection method used Dilution plating on SMSA Tomato bioassay IFAS-1 (pAb IACR-278) IFAS-2 (pAb Adgen-1091) WAS-3 (pAb IPO-9523) ELISA-1 (pAb IACR-278) ELISA-2 (pAb Adgen-1091) ELISA-3 (pAb Loewe-7356) FISH PCR-I Seal ef al. (1993)maOH extraction PCR-2 K:H. Pastrik (unpublished)/NaOH extraction PCR-3 Seal ef a/. ( I 993)/enrichment NaOH extraction PCR-4 K.-H. Pastrik (unpublished)/enrichment NaOH extraction PCR-5 Seal ef al. (1993)Anvitrogen extraction PCR-6 K.-H. Pastrik (unpublished)/Invitrogenextraction PCR-7 Seal e f a /. (1993)llsopropanol extraction PCR-8 K.-H. Pastrik (unpublished)/Isopropanol extraction
+
*Containing tcontaining $Containing 5Containing TContaining
+
17 15 18 18 18
18 18 18
7 18
18 18 18 18 18 18 18
17 (100%) 12 (71%)
15 (100%) 18 (100%) 18(100%) 18 (100%) 18 (100%) 17 (94%) 17 (94%) 7(100%) 7(39%) 3(17%) I I (61%) 10 (56%) 9 (50%) 12 (67%) 10(56%) 9 (50%)
8 (53%) 12 (67%) 10(56%) 12 (67%) 7 (39%) 7 (39%) 4(23%) 5(71%) 1(6%)
I(6%) 4(22%) 5 (28%) 4 (22%) 8 (44%) 5 (28%) 5 (28%)
0
I6 (94%) 13 (87%) 16 (89%) 13(72%) I4 (78%) 12 (67%) 13 (72%) 12(67%) 6(86%) 5(28%) 4(22%)
17 (100%) 15 (100%) 18 (100%) 18(100%) 18 (100%) 18 (100%) 17 (94%) 17(94%) 7(100%)
10(56%)
I I (61%) 13 (72%) 1 1 (61%) 10 (56%)
I (6%) 1 (6%) 0 I (6%)
9 (50%) I1 (61%) I 1 (61%) 10(56%) 9 (50%)
0
0 0 0 0 1 (6%)
8(44%)
0 0 0
8(44%)
0
1 1 (61%) 10 (56%)
0 1 (6%)
1 naturally infected tuber with symptoms and 199 healthy tubers.
I asymptomatic tuber vacuum infiltrated with a suspension containing lo4 cfu R. solanacearum per mL and 199 healthy tubers. 1 asymtomatic tuber vacuum infiltrated with a suspension containing lo6 cfu R. solanacearum per mL and 199 healthy tubers. 1 asymptomatic tuber vacuum infiltrated with a suspension containing lo8 cfu R. solanacearum per mL and 199 healthy tubers. 1 asymptomatic tuber vacuum infiltrated with water only and 199 other healthy tubers.
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Ralstonia solanacearum in potato
395
epidemiological importance. For improved harmonization of PCR protocols, an additional ring-test is planned and will include the use of internal positive PCR controls, standardized positive and negative controls and independent optimization of reaction conditions in each laboratory.
Acknowledgements This ring-test was funded in full by the EC under project SMT-4-CT97-2 179 (DGXII-EGAA). The authors specifically thank all persons who have contributed to the ring-tests in laboratories mentioned in Table 1.
Normalisation des methodes de detection de Ralstonia solanacearum sur pomme de terre En appui B la Directive de I’m98/57/EC sur la lutte contre la poumture brune de la pomme de terre, causCe par Ralstonia solanacearum, un test cornparatif a 6th conduit pour Cvaluer et normaliser les mtthodes actuellement recommandkes pour les tests officiels des tubercules de pomme de terre. Les tubercules test& Ctaient soit naturellement infect& (presentant des sympt6mes) ou infiltrbs sous vide avec diffirentes suspensions de R. solanacearum ou d’eau. Les tests ont CtC effectu6s indkpendamment dans 19 laboratoires phytosanitaires de 1’UE h l’aide de protocoles normalisCs basts sur les protocoles annex& B la directive. Pour les concentrations Clevks du pathoghe, des rCsultats parfaitement fiables ont CtC obtenus avec la mise en culture de dilutions sur milieu sklectif (SMSA), la coloration des anticorps par immunofluorescence (IFAS), l’ELISA, l’hybridation fluorescente in situ (FISH) et un test biologique sur tomate. La fiabilitC du diagnostic pour tous les laboratoires diminuait avec la concentration d’inoculum dans les tubercules ne prksentant pas de sympt6mes, quelle que soit la mCthode utiliske. Des rksultats cornparables ont CtC obtenus avec 3 antisCrums commerciaux par IFAS ou avec 2 antiskrums par ELISA mais la mkthode IFAS ktait 1Cgkrement plus fiable. Dans plusieurs laboratoires, les diagnostics obtenus par les protocoles de PCR Ctaient moins fiables qu’avec les autres mCthodes. L’optimisation ind6pendante des conditions de la PCR dans chaque laboratoire et l’introduction de tCmoins positifs internes de PCR sont recommandbes.
References
Anonymous (1998) Council Directive 98157EC of 20 July 1998 on the control of Ralstonia solanacearum (Smith) Yabuuchi et al. Oflcial Journal of the European Communities No. L 235, 1-39. Elphinstone JG, Hennessy J, Wilson JK & Stead DE (1996) Sensitivity of detection of Ralstonia solanacearum in potato tuber extracts. Bulletin OEPPIEPPO Bulletin 26, 663-678. Janse JD (1988) A detection method for Pseudomonas solanacearum in symptomless potato tubers and some data on its sensitivity and specificity. Bulletin OEPP/EPPO Bulletin 18, 343-35 1. Llop P, Caruso P, Cubero J, Morente C & L6pez MM (1999) A simple extraction procedure for efficient routine detection of pathogenic bacteria in plant material by polymerase chain reaction. Journal of Microbiological Methods 31, 23-3 1. Pastrik K-H & Rainey RA (1999) Identification and differentiation of Clavibacter michigunensis subspecies by polymerase chain reactionbased techniques. Journal of Phytopathology 147, 687-693. Robinson-Smith A, Jones P, Elphinstone JG & Forde SMD (1995) Production of antibodies to Pseudomonas solanarearum, the causative agent of bacterial wilt. Food and Agricultural Immunology I,67-79. Seal SE, Jackson LA, Young JPW & Daniels MJ (1993) Differentiation of Pseudomonas solanacearum. Pseudomonas syzygii, Pseudomonas pickettii and the Blood Disease Bacterium by partial 16s rRNA sequencing: CTaHaaPTH3a4HFl MeTOa0B BblRBJleHHR constmction of oligonucleotide primers for sensitive detection by Ralstonia solanacearum Ha KapTo@ene polymerase chain reaction. Journal of General Microbiology 139, Onwpmcb Ha &ipeK’rUBY981571 CoeeTa EC no 6opb6e c 1587-1594. 6ypoii rHnnbto KapTo+ena, ebnb~sae~oiiRalstonia Wullings BA, Van Beuningen AR, Janse JD & Akkermans ADL (1998) solanacearum, npoBonuna cpaBHnTenbHbii4 a ~ m n 3 an^ Detection of Ralstonia solanacearum, which causes brown rot of oueHKn n c ~ a ~ n a p ~ w 3 a qpeKoMeHayeMHx nn B HacTonuee potato, by fluorescent in situ hybridization with 23s rRNA-targeted BpeMI MeTOAOB OI$nUUiWlbHbIX aHaJlU30B ~ n y 6 ~ e i i probes. Applied and Environmental Microbiology 64,4546-4554.
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