Evaluation of magnetic capture for the detection of Ralsfonia solanacearum in various substrates*

J. M.

F. Noublanche1s2,F. Poliakoff' and D. Caffier'

'Service Rigional de la Protection des Vigitaux des Pays de la Loire, Unite' Bactiriologie du Laboratoire National de la Protection des Vigitaux, 10 rue L . e NGtre, F-49044 Angers Cedex 01 (France); e - m i l : david.caffier@agriculture.gouv.j? 2Fidiration Rigionale des groupements de Dqense contre les Ennemis des Cultures, 10, rue L . e NGtre, F-49044 Angers Cedex 01 (France)

Ralstonia solanacearum, the causal agent of bacterial wilt, is an important quarantine pest for European countries. Available analytical tools give good results for the detection of the bacterium in potato tuber extracts and tomato macerates. The pathogen may be present in complex substrates such as environmental water, soils, sewage sludge and potato processing wastes, which therefore have to be tested. Immuno-magnetic separation and DNA magnetic capture combined with other tests have been investigated for this purpose and appear promising.

Introduction
The quarantine pest Ralstonia solanacearum (Yabuuchi et al., 1995) is the causal agent of potato brown rot. Infected seedpotato tubers are the main means for dissemination of the pathogen, but brown rot infections in Europe have also occurred after irrigation with surface water (Olsson, 1976; Elphinstone, 1996; De GuCnin, 1998; Elphinstone etal., 1998; Hayward et al., 1998; Janse et al., 1998). Moreover, R. solanacearum is able to survive in soil at low temperatures (van der Wolf et al., 1998) and possibly in industrial and household sewage sludge (Farag et al., 1999). Existing techniques require adaptation in order to detect low populations of this organism in these unusual substrates. Currently available techniques often give poor results with these substrates because of the high populations of competing non-target organisms and their high content of compounds inhibitory to serological and molecular detection methods. Magnetic capture offers a potential means for separating and concentrating target organisms or their DNA from complex substrates. It is based on synthetic paramagnetic microbeads coated either with: (1) antibodies which specifically recognize target cells (immunomagnetic separation or IMS); (2) a compound with high DNA affinity (DNA magnetic capture) (Fig. 1); or (3) probes which specifically recognize a target DNA sequence. When sensitized beads and sample are mixed, complexes are formed between beads and targets which can then be separated from the remainder of the sample using a strong magnetic field. This kind of technique has already been successfully applied in the detection of bacterial contaminants in foods, or in environmental water (Yu & Bruno, 1996), or in the detection of circulating tumour cells in blood (Hardingham et al., 1993). However, there are few
*Paper presented at the EPPO Conference on Diagnostic Techniques for Plant Pests, Wageningen (NL), 2000-02/01-04.

publications concerning plant-pathogenic bacteria (Pooler et al., 1997; Walcott & Gitaitis, 2000).

Materials and methods

Strains, media, antiserum and purified immunoglobulins

Details concerning the strains used in this study are given in Table 1. Bacteria were cultured on yeast peptone glucose agar medium (YPGA). SMSA broth (Engelbrecht, 1994; modified by Elphinstone et al., 1996) was used for liquid enrichment. Bacterial populations were estimated by plating of serial dilutions onto YPGA plates. Antiserum no. 421 was produced in rabbit against R. solanacearum. Its specificity is good in immunofluorescence, no cross reactions occumng with a range of bacterial strains including those listed in Table 1. Purification of IgG was performed by dialysis followed by chromatography through a DEAE cellulose column.
Beads and magnet

Synthetic paramagnetic sheep anti-rabbit Ig, beads (Dynabeads M280) were supplied by Dynal. Prior to use, the beads were coated, according to the manufacturer (Anonymous, 1996), with purified IgG from antiserum no. 421. Beads for DNA capture were obtained together with appropriate buffers as a kit (Dynabeads DNA Direct system I). Beads were recovered using a Dynal MPC magnet.
Samples

Samples consisted either of pure cultures of R. solanacearum or mixtures of pure cultures in potato or tomato macerates, sewage sludge or potato processing waste. Potato macerates

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J. M. Expert et al.

Fig. 1 Principle of magnetic capture of cells and of DNA. Beads are sold coated with goat anti-rabbit IgG.Trapping antibodies are directed against antigen specific to the targeted organism. The DNA trapping layer is either a chemical compound with high affinity for DNA or a probe specific to the targeted DNA sequence.

were prepared according to EU Directive 98/57/CE (EU, 1998) from naturally infected potato tubers (cv. Dbirie) stored at 80C. Pieces of healthy tomato stems were crushed in sterile distilled water and shaken at 180 rev m K ' on a rotary shaker for 2 h at room temperature. Uncontaminated sewage sludge collected from two different locations contained either high (sample A) or low (sample B) organic compounds. Similarly, potato processing waste (free from R. solanaceam) collected from two French potato processing companies, contained either high (sample C) or low (sample D) organic compounds. Pathogen-free samples were inoculated with a range of concentrations of R. solanacearum prior to use. When appropriate, samples were enriched in SMSA broth [10mL of concentrated (10-fold) SMSA broth mixed with 90 mL of sample] and shaken at 180 rev m i f for 48 h at room temperature.

washed and resuspended in sterile distilled water. Serial dilutions were then plated onto YPGA and incubated at 28C for 48-72 h.
Magnetic capture of DNA

'

lmmunomagneticseparation of cells

Samples of pure culture of R. solanacearum CFBP 3579 or other bacterial strains or inoculated potato macerates were mixed with approximately 200 pL of coated beads per mL of sample. After 30 min incubation at room temperature with gentle shaking, the beads were collected using the magnet,

Prior to use, the beads were resuspended by gentle shaking to obtain a homogeneous suspension. Volumes of 200 pL of pure culture of R. solanacearum (strain CFBP 3857) in distilled sterile water, other bacterial strains, potato or tomato macerates, sewage sludge (before and after enrichment in SMSA broth) or potato processing wastes (before and after enrichment in SMSA broth) were centrifuged at 13 000 g for 10min at 4"C, and the supernatant was discarded. Pellets were resuspended in approximately 200pL of beads and after 5 min were separated magnetically, collected with the magnet and washed with an appropriate washing buffer. Finally, pellets were resuspended in 40 pL of resuspension buffer and gently mixed by pipetting up and down 30-40 times. DNA was eluted by incubation at 65C over a 5-min period. Solutions containing DNA were then removed by pipette, while the beads were trapped magnetically. Samples were further analysed by PCR as described in EU

Table 1 Characteristics of bacterial atrains

Reference number of collection Species Ralstonia solanacearum Ralstonia solanacearum Enviniu herbicuh CFBP
3857 3579

Others PD 2762 PD 295 NCPPB 2971 ATCC 33243 PD 1596 ATCC 17430

Host plant Potato Tomato

Year of isolation
1995 1995 1981

Location Netherlands France Canada

Pseudomonns putidu Bacillus polvmyxa

3140 1954

1989

Potato

I979

France

CFBP, Collection Frangaise de Bactiries Phytopathogines, France; PD, Plantenziektenkundige Dienst, The Netherlands; ATCC, American Type Culture Collection, USA; NCPPB, National Collection of Plant Pathogenic Bacteria, United Kingdom.

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Magnetic capture of R. solanacearum

Table 2 Evaluation of immunocapture of cells of Ralstonia solanarearum (strain CFBP 3579)
Bacterial concentration (x lo6cfu) Before capture Pure culture in water
Pure culture in potato macerate

387

After capture 1.0 ( 2 0.2) 0.1 (? 0.2) 1.6 (t0.5) 1.6(2 0.1)

6.1 (2 1.8) 0.8 (? 2.0)

190.0 ( 2 30.0) l2.0(f 0.1)

According to Table 3, uncoated beads also non-specifically capture target bacteria. Specificity increased when beads were coated with specific Igc, but non-target bacteria were also captured at a low rate. The addition of a blocking step with 3% non-fat milk before the addition of the sample did not clearly reduce the numbers of unwanted cells captured.
Magnetic capture of DNA

Values are means (cfumL-') 2 standard deviation. Means were calculated from three replications.

Directive 98/57/CE (EU, 1998), with primers Olil and Y2 (Seal et al., 1993). Amplified DNA fragments were visualized in a 3% Nusieve 3:l (FMC Ltd) agarose gel in 0 . 5 ~ Tris Borate EDTA buffer (TBE).

Results and discussion

lmmunomagnetic separation of cells

The protocol used for immunomagnetic separation of cells differed slightly different from that recommended by the supplier but gave more reliable results. Using Dynabeads with the simplified protocol, approximately 15% of initial bacterial populations were successfully recovered from sterile water or potato macerates (Table 2). Potato extracts did not interfere with immunomagnetic separation. More than one bacterial cell stuck to each individual bead. As bacteria were not detached from beads prior to plating, each colony-forming unit probably originated from multiple target cells trapped on individual beads, thus resulting in an apparent poor rate of recovery of cells after capture. According to the supplier, four beads are required on average to trap a target cell successfully. Therefore, immunomagnetic separation can fail if target cells outnumber beads. Under these conditions, heavily contaminated samples could appear healthy (Table 2). This problem can be avoided by diluting the sample prior to analysis or by increasing the quantity of coated beads.

When performing DNA capture with the Dynal system, the elution step was necessary to maintain sensitivity. Because the protocol for DNA capture includes a concentration step (1 mL of sample is concentrated to 40pL),the sensitivity of the PCR test was similar with or without DNA magnetic capture (Table 4). The addition of non-target bacteria to the samples did not affect the sensitivity of target detection by PCR (Table 4). Potato macerates are known to inhibit PCR, but this did not occur following magnetic capture (Table 5) where inhibitory compounds were removed during the process. DNA capture may therefore be a useful technique for pretreatment of highly inhibitory samples such as soils, sewage sludge and effluents from potato processing industries. In addition to purification of DNA extracts the technique also permits concentration of target DNA prior to PCR.

Conclusion
A wide range of kits is now commercially available for magnetic capture of cells or of DNA. Useful results were obtained in this study using kits supplied by Dynal Ltd. The kits are not specifically designed for detection purposes but for purification of target, where yield of recovery is not the main concern. Promising results have also been obtained in our laboratory with immunomagnetic separation for Cluvibacter michigunensis subsp. sepedonicus using monoclonal antibodies. However, the high concentration of antibodies required to coat the beads does not permit a wide extension of immunomagnetic separation for routine analysis. Nevertheless, this technique could be helpful in some cases, e.g. when the bacterium needs to be isolated from complex substrates for legal reasons.

Table 3 Specificity of immunocapture of cells

of Ralstonia solanacearum (strain CFBP 3579) Treatment Ralstonia solanacearum (alone) Ralstonia solanacearum other bacteria (mixture) A B C A B C Number of cells (x lo3 cfu) R. solanacearum 410 ( 2 0.4) 31 (+ 0.3) 250 ( 2 2.5) 550 ( 2 5.0) 68 ( 2 0.6) 180 (f 1.9) Other bacteria
-

290 (? 3.1) 59 ( 2 0.5) 130(? 0.1)

Values are means (cfu mL-') + standard deviation. Means were calculated from three replications. A, no beads; B, beads without secondary antibody; C: beads with specific antibody. The bacterial mixture was made with Envinia herbicola PD 295, Pseudomonas putida CFBP 3 140 and Bacillus polymyxa CFBP 1954.

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J. M. Expert et al.

Table 4 DNA magnetic capture of Ralstoniu solanacearum from various substrates artificially contaminated with serial dilutions of strain CFBP 3857
Initial population (cfu mL-l) 2 x 10' Pure culture in water Bacterial mixture (2 x 10' cfu mL-') Tomato macerate Potato macerate 2~ 104 2 x lo3 2x
-I+ -I+
-I+ -/+

lo2

2 x 10'
-1-1-I+

+I+
+I+

+I+
+I+ +I+ -I+

+I+
+I+

+I+ +I+ -I+ -I+

-I-

+I+, Results obtained by PCR, respectively, before and after DNA capture. The bacterial mixture was made with Envinia herbicola PD 295, Pseudomonus putida CFBP 3140 and Bacillus polymyxa CFF3P 1954.

Table 5 Results obtained by PCR from various wastes artificially contaminated with serial dilutions of Ralstonia solanacearum strain CFBP 3857
PCR results Sewage A Initial population (cfu mL-') 7.7 7.7 7.7 4.4 4.4 4.4 8.5 8.5 8.5 3.7 3.7 3.7
t 2.4 x t 2.4 x 5 2.4 x 5 0.7 x 2 0.7 x t 0.7 x -C 1.7 x 2 1.7 x ? 1.7 x 5 1.3 x
5 1.3 x

Evaluation de la capture magnetique pour la detection de Ralstonia solanacearum dans differents substrats
Ralstonia solanacearum, I'agent responsable du flCtrissement bactirien, est un organisme de quarantaine important pour les pays europCens. Les outils analytiques disponibles donnent de bons rksultats pour dCtecter la bactCrie dans des extraits de tubercules de pomme de terre et des macCrats de tomates. Des milieux complexes, comme I'eau, le sol, les effluents de stations d'kpuration et les rejets d'usines de transformation de pomme de terre peuvent contenir la bactCrie et doivent donc Ctre analysks. La sCparation immunomagnCtique (IMS) et la capture magnCtique d' ADN, combinCes i d'autres tests, ont CtC CvaluCs et apparaissent prometteuses.

Without enrichment

With enrichment

10' loh

-I+ -I+
-I+ -I+ -I+ -I+ -I+ -I+ -If
+I+ -I+

+I+
-I+

lo4

-I+

inx
10' 104

+I+ -I+
-I+ -I+

lo8
10'

lo4

-I+ -I+
+I+ -I+

lo8
loh

2 1 . 3 104 ~

-I+

-I+

+I+, results from direct PCWPCR after DNA capture. A and B, domestic sewage sludges; C and D, potato processing waste from two factories.

Results obtained using magnetic capture of DNA were more adapted to routine analysis, because reagents are available in unlimited quantities. Nevertheless, this technique did not always give the expected results. For example, in a separate study, we did not succeed in detecting C. m. subsp. michigunensis in tomato seeds. Further study will be required to improve understanding of these techniques and adapt commercial kits for detection of plant bacteria.

Acknowledgements
This study was funded by the European Union through the Standards, Measurements and Testing programme SMT4CT97-2179 and by the French Ministry of Agriculture and Fisheries. We thank J. G. Elphinstone for reviewing the manuscript and for providing strain PD2762. Thanks are also due to M. Solano, C. Boisseau and A. Keters for excellent technical assistance. Antiserum no. 421 was produced in INRA Angers in collaboration with FNPPPT, and thanks are due to R. Samson and to A.-C. Leroux.

References
Anonymous ( 1996) Cell Separation and Protein Purification, 2nd edn. Dynal, Oslo (NO). De GuCnin MC (1998) Management and monitoring of Ralstoniu solunucearum in France. Bulletin OEPP/EPPO Bulletin 28, 109-1 12. Elphinstone JG (1996) Survival and possibilities of extinction of Pseudomonns solanacearum in cool climates. Potato Research 39.403-410. Elphinstone JG, Hennessy J, Wilson JK & Stead DE (1996) Sensitivity of different methods for the detection of Ralstonia .solanacearum in potato tuber extracts. Bulletin OEPP/EPPO Bulletin 26, 663-678. Elphinstone JG, Stanford HM & Stead DE (1998) Detection of Ralstnnia solanacearum in potato tubers, Solunum dulccinutra and associated

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irrigation water. In Bacterial Wilt Disease, Molecular and Ecological Aspects (eds Prior P, Allen C & Elphinstone JG), pp. 133-1 39. Springer Verlag, Berlin (DE). Engelbrecht MC (1994) Modification of a semi-selective medium for the isolation and quantification of Pseudomonas solanacearum. Bacterial Wilt Newsletter 10, 3-5. EU (1998) Directive 98/57/CE du Conseil du 20 juillet 1998 concernant la lutte contre Ralstonia solanacearum (Smith) Yabuuchi et al. Journal OfJ'iciel des Communautis Europiennes No. L 235, 1-39. Farag N, Stead DE & Janse JD (1999) Ralstonia (Pseudomonas)solanacearum race 3, biovar 2, detected in surface (irrigation) water in Egypt. Journal of Phytopathology 147, 485-487. Hardingham JE, Kotasek D, Farmer B, Butler RN, Mi J-X, Sage RE & Dobrovic A (1993) Immunobead-PCR: a technique for the detection of circulating tumour cells using immunomagnetic beads and the Polymerase Chain Reaction. Cancer Research 53,3455-3458. Hayward AC, Elphinstone JG, Caffier D, Janse J, Stefani E, French ER & Wright AJ (1998) Round table on bacterial wilt (brown rot) of potato. In Bacterial Wilt Disease, Molecular and Ecological Aspects (eds Prior P, Allen C & Elphinstone JG), pp. 420-430. Springer Verlag, Berlin (DE). Janse J, Araluppan FAX, Schans J, Wenneker M & Westerhuis W (1998) Experiences with bacterial brown rot Ralstonia solanacearum biovar 2, race 3 in the Netherlands. In Bacterial Wilt Disease, Molecular and Ecological Aspects (eds Prior P, Allen C & Elphinstone JG), pp. 146152. Springer Verlag, Berlin (DE). Olsson K (1976) Experience of brown rot caused by Pseudomonas solanacearum in Sweden. Bulletin OEPP/EPPO Bulletin 6, 199-207. Pooler MR, Myung IS, Bentz J, Sherald J & Hartung JS (1997) Detection

of Xylella fastidiosa in potential insect vectors by immunomagnetic separation and nested polymerase chain reaction. Letters of Applied Microbiology 25, 123- 126. Seal S, Jackson LA, Young JPW & Daniels MJ (1993) Differentiation of Pseudomonas solanacearum. Pseudomonas syzygii, Pseudomonus pickettii and the blood disease bacterium by partial 16s rRNA sequencing: construction of oligonucleotide primers for sensitive detection by polymerase chain reaction. Journal o f General Microbiology 139,1587-1594. Walcott RR & Gitaitis RD (2000) Detection of Acidovorax avenae subsp. citrulli in watermelon seed using immunomagnetic separation and the polymerase chain reaction. Plant Disease 84, 470-474. van der Wolf JM, van Bekkum PJ, van Elsas JD, Nijhuis EH, Vrien SGC & Ruissen M A (1998) Immunofluorescence colony staining and selective enrichment in liquid medium for studying the population dynamics of Ralstonia solanacearum (race 3) in soil. Bulletin OEPP/EPPO Bulletin 28, 7 1-79. Yabuuchi E, Kosako Y, Yano I. Hotta H & Nisbiuchi Y (1995) Transfer of two Burkholderia and an Alcaligenes species to Ralstonia Gen. Nov: proposal of Ralstonia pickettii (Ralston, Palleroni and Doudoroff 1973) Comb. Nov. Ralstonia solanacearum (Smith 1896) Comb. Nov. and Ralstonia eutropha (Davis 1969) Comb. Nov. Microbiological Immunology 39,897-904. Yu H & Bruno JG (1996) Immunomagnetic-electrochemiluminescent detection of Escherichia coli 0157 and Salmonella typhimurium in foods and environmental water samples. Applied and Environmental Microbiology 62,587-592.

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