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Sensitive and specific detection of Xanthomonas campestris pv. campestris by PCR using species-specific primers based on hrpF gene sequences
ARTICLE IN PRESS Microbiological Research 159 (2004) 419—423
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Sensitive and specific detection of Xanthomonas campestris pv. campestris by PCR using speciesspecific primers based on hrpF gene sequences Young Jin Park, Byoung Moo Lee, Jang Ho-Hahn, Gil Bok Lee, Dong Suk Park National Institute of Agricultural Biotechnology, Rural Development Administration, 441-707 Suwon, Republic of Korea Accepted 14 September 2004
KEYWORDS hrpF; PCR; Specific primer; Xanthomonas campestris pv. campestris
Summary A sensitive and specific assay was developed to detect bacterial black rot of crucifers caused by Xanthomonas campestris pv. campestris (X. c. pv. campestris), in cabbage seed and plant. Primers XCF and XCR from hrpF homologous to nolX, host recognition protein, were used to amplify a 525 bp DNA fragment. PCR technique was applied to detect the pathogen in naturally infected seed and plant of cabbage. The PCR product was only produced from X. c. pv. campestris among 40 isolates of Xanthomonas strains, Escherichia coli (O157:H7), Pectobacterium carotovorum subsp. carotovorum, and other reference bacteria. & 2004 Elsevier GmbH. All rights reserved.
Introduction Xanthomonas campestris pv. campestris (X. c. pv. campestris) is the causal agent of black rot, a serious disease that affects crucifers such as Brassica and Arabidopsis. X. c. pv. campestris is a vascular pathogen that invades the xylem and colonizes the mesophyll. The symptoms of black rot include marginal leaf chlorosis, necrosis, and darkening of leaf veins and vascular tissue within the stem. Full leaf yellowing, wilting, and necrosis
also occur as the disease advances (Hayward, 1993). To prevent black rot, the use of pathogenfree seeds is recommended. Testing of seed lots for the presence of pathogen is, therefore, essential. The general methods for detecting the pathogen in seed lots and plants are based on serological techniques or plating assays. These are considered to be reliable and efficient methods for routine detection. However, it takes 3 days to 1 week to detect the pathogen. Another disadvantage is
Corresponding author.
E-mail address: [email protected] (D.S. Park). 0944-5013/$ - see front matter & 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2004.09.002
ARTICLE IN PRESS 420 the possible presence of cells of other microorganisms, which may interfere by causing overgrowth or suppression of outgrowth of the pathogen. Furthermore, immunological techniques, such as the enzyme-linked immunosorbent assay (ELISA), and flow cytometry are relatively insensitive or time-consuming than PCR-based sensitive assay (Alvarez and Lou, 1985; Chitarra et al., 2002). In this study, specific primers from hrpF region of X. c. pv. campestris were designed. The primer set showed high sensitivity and specificity for detecting the pathogen in crude seed extracts and plants of cabbage.
Materials and methods Bacterial strains and culture conditions Bacterial and fungal strains were obtained from the Korean Agricultural Culture Collection (KACC) in Suwon, Korea and the Belgian Co-ordinated Collections of Micro-organisms (BCCMTM) in Belgium. All microorganisms used in this study are listed in Table 1. Xanthomonas strains were cultured on YGC medium (2.0% D- (+)-glucose, 2.0% CaCO3, 1.0% yeast extract, 1.5% agar) at 28 1C for two days, Escherichia coli on LB agar (Sambrook et al., 1989) at 37 1C for 18 h, and other bacteria on nutrient agar (NA, Difco) at 26–28 1C for 1–2 days.
Isolation of total DNA Xanthomonas strains were cultured on YGC medium and harvested with a scraper for total DNA extraction. Total DNA from Xanthomonas strains was prepared as described by Lazo and Gabriel (1987). Total DNA from other microorganisms was extracted using the genomic DNA extraction kit [Genomic-tips] supplied by Qiagen (Hilden, Germany).
Primer design and PCR amplification The primers XCF (50 -CGATTCGGCCATGAATGACT-30 ) and XCR (50 -CTGTTGATGGTGGTCTGC AA-30 ) were designed from hrpF of X. c. pv. campestris, with a predicted PCR product of 535 bp. PCR assays were performed with a PTC-200TM thermocycler (MJ Research, Watertown, MA, USA). All amplifications were carried out in a final volume of 50 ml containing 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, 0.2 mM of each dNTP, 10 pM of each primer,
Y.J. Park et al. and 2 units of Taq polymerase (Promega, Madison, Wisconsin, USA). The total amount of genomic DNA from various microorganisms added to the PCR mixture was approximately 50 ng. Reactions were run for 35 cycles, each consisting of 15 s at 94 1C, 15 s at 58 1C, and 30 s at 72 1C, with initial denaturation of 5 min at 94 1C and final extension of 5 min at 72 1C. An 8-ml aliquot of each amplified PCR product was electrophoresed on a 1.0% agarose gel, stained with ethidium bromide, and visualized on a UV transilluminator.
DNA dot-blot analysis DNA dot-blot analysis was carried out to confirm whether the hrpF gene was present in other microorganisms including xanthomonads used in this study. A 100-ng sample of genomic DNA isolated from Xanthomonas strains and other reference microorganisms were spotted onto Hybond-N+ nylon membrane (Amersham Pharmacia Biotech, UK). These were UV cross-linked to bind the labeled probe DNA. PCR product from X. c. pv. campestris (KACC10913) was labeled as probe with [32P]dCTP using the random primed method according to the manufacturer’s instructions (LaddermanTM Labeling kit, Takara, Japan). Prehybridization and hybridization were conducted in hybridization buffer (0.75 M NaCl, 75 mM sodium citrate, 0.5% SDS, 0.1% BSA, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, and 50 ml/ml denatured salmon sperm DNA) at 65 1C for 18 h. After hybridization, the filter was washed twice (10 min each) in 2 SSC containing 0.1% SDS at room temperature and twice (15 min each) in 0.1 SSC containing 0.1% SDS at 65 1C. Autoradiography was done at 70 1C with CURIX X-ray film (AGFA, Belgium).
Estimates of limits of detection To determine the limits of PCR detection in pure culture suspension, strain X. c. pv. campestris LMG 8091 was grown in NB, cells were pelleted and washed four times in distilled water. A suspension of 1.3 1010 CFU/ml was diluted seven times, in a ten-fold series. Aliquots of 10 ml of each dilution were directly used in PCR for amplification.
Detection of the pathogen by PCR in naturally infected cabbage plant To detect X. c. pv. campestris in naturally infected cabbages (Brassica oleracea var. capitata), symptomatic and symptomless cabbage leaves and seeds
ARTICLE IN PRESS Sensitive and specific detection of Xanthomonas campestris pv. campestris Table 1.
421
List of bacterial strains used in this study
No.
Bacterial isolate
Sourcea
Geographical origin
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
Xanthomonas campestris pv. campestris Xanthomonas campestris pv. campestris Xanthomonas campestris pv. campestris Xanthomonas campestris pv. campestris Xanthomonas campestris pv. campestris Xanthomonas campestris pv. campestris Xanthomonas campestris pv. campestris Xanthomonas campestris pv. campestris Xanthomonas campestris pv. campestris Xanthomonas campestris pv. campestris Xanthomonas campestris pv. carotae Xanthomonas campestris pv. glycines Xanthomonas campestris (Pammel 1895) Dowson Xanthomonas oryzae pv. oryzae (Xoo90) Xanthomonas oryzae pv. oryzae (Xoo85) Xanthomonas axonopodis pv. aurantifolii Xanthomonas oryzae pv. oryzae (PXO99) Xanthomonas axonopodis pv. citri Xanthomonas axonopodis pv. citri Xanthomonas axonopodis pv. axonopodis Xanthomonas fragariae Xanthomonas campestris pv. malvacearum Xanthomonas campestris pv. pelargonii Xanthomonas campestris pv. juglandii Xanthomonas arboricola pv. poinsettiicola Xanthomonas axonopodis pv. axonopodis Xanthomonas axonopodis pv. begoniae Xanthomonas axonopodis pv. dieffenbachiae Xanthomonas axonopodis pv. malvacearum Xanthomonas axonopodis pv. phaseoli Xanthomonas axonopodis pv. phyllanthi Xanthomonas axonopodis pv. vasculorum Xanthomonas axonopodis pv. vesicatoria Xanthomonas cassavae Xanthomonas cucurbitae Xanthomonas pisi Xanthomonas theicola Xanthomonas translucens pv. cerealis Xanthomonas translucens pv. hordei Xanthomonas translucens pv.phleipratensis Pseudomonas aeruginosa Pseudomonas fluorescens Pectobacterium carotovorum subsp. carotovorum Pectobacterium carotovorum subsp. carotovorum Escherichia coli (O157:H7) Mesorhizobium loti
KACC10913 (ATCC 33913)b LMG 568 LMG 582 LMG 584 LMG 8002 LMG 8052 LMG 8081 LMG 8091 LMG 8105 LMG 8110 KACC 10164 (ATCC 10547) KACC 10445 (LMG 7403) KACC 10490 (ATCC 33913) KACC 10312 KACC 10331 KACC 10161 (NCPPB 3655) KACC 10884 KACC 10443 KACC 10444 KACC 10935 (LMG 982) KACC 11115 (DSMZ 3587) KACC 11127 (DSMZ 1220) KACC 11128 (DSMZ50857) KACC 11116 (DSMZ 1049) LMG 5403 LMG 538 LMG 551 LMG 695 LMG 761 LMG 7455 LMG 844 LMG 901 LMG 905 LMG 673 LMG 8662 LMG 847 LMG 8684 LMG 679 LMG 882 LMG 843 KACC 10259 (ATCC 10145) KACC 10327 (LMG 1794) KACC 10441 (LMG 2412) KACC 10455 (LMG 2453) KACC 10765 (ATCC 35150) KACC 10644 (LMG 10644)
United Kingdom United Kingdom Belgium Belgium Malawi Netherland New Zealand USA Western Samoa Tonga USA Zambia United Kingdom Korea Korea Brazil Philippines Korea Korea Columbia USA — — United Kingdom New Zealand Columbia UK Brazil Sudan Vulgaris Sudan Mauritius — Malawi New Zealand Japan Japan USA Canada USA — United Kingdom United Kingdom Belgium — New Zealand
a
KACC, Korean Agricultural Culture Collection, Korea (http://kacc.rda.go.kr); ATCC, American Type Culture Collection, USA; LMG, The Belgian Co-ordinated Collections of Microorganisms (BCCMTM), Belgium; NCPPB, National Collection of Plant Pathogenic Bacteria, United Kingdom. ( ): Other Collection No. ‘‘—’’ not known. b Parenthesis: other collection No.
were obtained from fields in Icheon, Korea (Syngenta Korea Ltd.; http://www.syngenta.co.kr). One gram of leaves was sampled from each cabbage plants with sterile scissors and total DNA was
directly extracted from the sample by the C-TAB method (Murray and Thompson, 1980). Isolated DNA from plants was used in PCR assays as described above.
ARTICLE IN PRESS 422
Y.J. Park et al.
Results Specificity of primers The 20-mer oligonucleotide XCF and XCR were tested for X. c. pv. campestris. As expected, a 535 bp DNA fragment was amplified. To check the specificity of the primers, a large collection of other microorganisms, including Xanthomonas species and their pathovars, were tested in PCR assay with primers XCF and XCR. None of the other Xanthomonas strains and reference microorganisms reacted with the primers; only X. c. pv. campestris showed a single amplified DNA fragment (Fig. 1). The primer pair, XCF and XCR, amplified a 535 bp DNA fragment from X. c. pv. campestris, when 50 ng DNA was used as the template under optimized conditions (50 ml PCR reaction, containing 1.5 mM MgCl2, 0.2 mM of each dNTP, 10 pM of each primer, 2 units of Taq polymerase, and 5 ml of 10 PCR buffer). Similar conditions yielded reproducible results in the Perkin Elmer 9600 thermal cyclers (Perkin Elmer International, Rotkreuz). Taq polymerase enzymes supplied by different manufacturers (Promega, Takara, and Toyobo) also yielded similar PCR results (data not shown).
Figure 2. DNA dot-blot analysis of hrpF with PCRamplified fragment (535 bp) from X. campestris pv. campestris. Lanes 1, X. c. pv. campestris ATCC 33913; lane 2, X. c. pv. campestris LMG 582; lane 3, X. c. pv. campestris LMG 8002; lane 4, X. c. pv. campestris LMG 8081; lane 5, X. c. pv. campestris LMG 8105; lanes from 6 (corresponding to the number 11 in Table 1) to 41(corresponding to the number 46 in Table 1), other reference microbes.
DNA dot-blot analysis The presence (or homology) of the hrpF gene in Xanthomonas strains was confirmed by dot-blot analysis. The positive results in the dot-blot analysis suggested that the hrpF genes of Xanthomonas strains might be highly or slightly conserved, while the negative results indicated that the hrpF gene did not exist or share substantial homology with other bacteria and fungi (Fig. 2).
Figure 3. Limits of detection of X. c. pv. campestris cells LMG 8091. Lane M, size marker (1kb ladder, Gibco BRL); lane 1, 1.3 108 CFU/10 ml; lane 2, 1.3 107 CFU/10 ml; lane 3, 1.3 106 CFU/10 ml; lane 4, 1.3 105 CFU/10 ml; lane 5, 1.3 104 CFU/10 ml; lane 6, 1.3 103 CFU/10 ml; lane 7, 1.3 102 CFU/10 ml; lane 8, 1.3 10 CFU/10 ml.
Sensitivity of PCR It was possible to detect X. c. pv. campestris in pure culture suspensions tested by PCR. The minimum number of cells detected was about 1.3 104 CFU/ ml. A dilution series of cultured cells of strain LMG
Figure 1. Specific PCR amplification of partial hrpF gene from X. campestris pv. campestris with hrp-specific primers XCF and XCR. Lane M, Size marker (1Kb DNA ladder; Gibco BRL); lanes 1–46 listed in Table 1.
Figure 4. PCR detection of naturally infected cabbage leaves (Brassica oleracea var. capitata). M, size marker (1 Kb DNA ladder; Promega); Lane 1, X. c. pv. campestris KACC10913; Lane 2, X. c. pv. campestris LMG 568; Lane 3, X. c. pv. campestris LMG 582; Lane 4, X. c. pv. campestris LMG 584; Lane 5, X. c. pv. campestris LMG 8110; Lanes 6–8, symptomless leaves from cabbages; Lanes 9–12, symptomatic leaves from cabbages.
ARTICLE IN PRESS Sensitive and specific detection of Xanthomonas campestris pv. campestris 8091 yield a limit of detection of about 1.3 102 CFU per reaction after amplification (Fig. 3).
PCR detection of the pathogen in nature The pathogen was detected by PCR in naturally infected cabbage plant (Fig. 4). Fig. 4 shows a PCR assay of naturally infected cabbage plant. As expected, the pathogen was detected by PCR assay from infected cabbage leaves. On the other hand, a single DNA fragment was not showed from healthy cabbage leaves by PCR.
Discussion Development of specific primers and DNA probes for identification and detection has been reported for a number of plant pathogenic bacteria (Hartung et al., 1993; Leite et al., 1994; Rasmussen and Reeves, 1992) including xanthomonads. This study found hrpF to be a useful gene for detecting X. c. pv. campestris with PCR assay. Recently, it has been reported that the hrpF plays a crucial role in host recognition and after protein secretion across the bacterial envelope, i.e. translocation into the host cell (Rossier et al., 2000). This study revealed that the sequences of hrpF among xanthomonads were highly conserved or different from each other. Especially, Fig. 2 showed that a highly positive signal of Xanthomonas axonopodis pv. aurantifolii. It is thought that the nucleotide sequences of hrpF gene between X. c. pv. campestris and X. axonopodis pv. aurantifolii is very similar. In addition, hrpF amino acid sequences of X. campestris pv. campestris is 37.7% similar to nolX of the Mesorhizobium loti (data not shown). The role of nolX in nodulation and secretion in Rhizobium is not known clearly, but it may have a role in the release of bacteria from the infection threads like an hrpF of xanthomonads (Viprey et al., 1998). Successful detection of the pathogens using PCR techniques depends upon the specificity of primers. In this study, the specificity and sensitivity of PCR assays were evaluated through the detection of the pathogen in naturally infected plants. PCR conditions such as primers, template, concentration of Mg2+ (Bassam et al., 1992), thermocyclers, and thermostable polymerase origin (Schierwater and Ender, 1993) have been shown to affect amplification. In this study, all these parameters were optimized to avoid artifacts and to ensure reproducibility of amplification. Consistent results of amplification of a 535 bp fragment from X. c. pv. campestris by XCF and XCR were also
423
obtained by using various PCR machines and different Taq polymerase enzymes supplied by various manufacturers. In conclusion, the results presented here indicate that X. c. pv. campestris can be detected and identified by PCR assays with XCF and XCR primer set.
References Alvarez, A.M., Lou, K., 1985. Rapid identification of Xanthomonas campestris pv. campestris by ELISA. Plant Dis. 69, 1082–1086. Bassam, B.J., Caetano-Anolles, G., Gresshoff, P.M., 1992. DNA amplification fingerprinting of bacteria. Appl. Microbiol. Biotechnol. 38, 70–76. Chitarra, L.G., Langerak, C.J., Bergervoet, J.H.W., van den Bulk, R.W., 2002. Detection of the plant pathogenic bacterium Xanthomonas campestris pv. campestris in seed extracts of Brassica sp. applying fluorescent antibodies and flow cytometry. Cytometry 47, 118–126. Hartung, J.S., Daniel, J.F., Pruvost, O.P., 1993. Detection of Xanthomonas campestris pv. citri by the polymerase chain reaction method. Appl. Environ. Microbiol. 59 (4), 1143–1148. Hayward, A.C., 1993. The host of Xanthomonas. In: Swings, J.G., Civerolo, E.L. (Eds.), Xanthomonas. Chapman & Hall, London, pp. 51–54. Lazo, G.R., Gabriel, D.W., 1987a. Conservation of plasmid DNA sequences and pathovar identification of strains of Xanthomonas campestris. Phytopathology 77, 448–453. Leite Jr., R.P., Minsavage, G.V., Bonas, U., Stall, R.E., 1994. Detection and identification of phytopathogenic Xanthomonas strains by amplification of DNA sequences related to the hrp genes of Xanthomonas campestris pv. vesicatoria. Appl. Environ. Microbiol. 60 (4), 1068–1077. Murray, M.G., Thompson, W.F., 1980. Rapid isolation of high molecular-weight plant DNA. Nucl. Acids Res. 8, 4321. Rasmussen, O.F., Reeves, J.C., 1992. DNA probes for the detection of plant pathogenic bacteria. J. Biotechnol. 25, 203–220. Rossier, O., van den Ackerveken, G., Bonas, U., 2000. HrpB2 and HrpF from Xanthomonas are type IIIsecreted proteins and essential for pathogenicity and recognition by the host plant. Mol. Microbiol. 38, 828–838. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular cloning: A labolatory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Schierwater, B., Ender, A., 1993. Different thermostable DNA polymerases may amplify different RAPD products. Nucl. Acid Res. 21, 4647–4648. Viprey, V., Del Greco, A., Golinowski, W., Broughton, W.J., Perret, X., 1998. Symbiotic implications of type III protein secretion machinery in Rhizobium. Mol. Microbiol. 28, 1381–1389.
www.elsevier.de/micres
Sensitive and specific detection of Xanthomonas campestris pv. campestris by PCR using speciesspecific primers based on hrpF gene sequences Young Jin Park, Byoung Moo Lee, Jang Ho-Hahn, Gil Bok Lee, Dong Suk Park National Institute of Agricultural Biotechnology, Rural Development Administration, 441-707 Suwon, Republic of Korea Accepted 14 September 2004
KEYWORDS hrpF; PCR; Specific primer; Xanthomonas campestris pv. campestris
Summary A sensitive and specific assay was developed to detect bacterial black rot of crucifers caused by Xanthomonas campestris pv. campestris (X. c. pv. campestris), in cabbage seed and plant. Primers XCF and XCR from hrpF homologous to nolX, host recognition protein, were used to amplify a 525 bp DNA fragment. PCR technique was applied to detect the pathogen in naturally infected seed and plant of cabbage. The PCR product was only produced from X. c. pv. campestris among 40 isolates of Xanthomonas strains, Escherichia coli (O157:H7), Pectobacterium carotovorum subsp. carotovorum, and other reference bacteria. & 2004 Elsevier GmbH. All rights reserved.
Introduction Xanthomonas campestris pv. campestris (X. c. pv. campestris) is the causal agent of black rot, a serious disease that affects crucifers such as Brassica and Arabidopsis. X. c. pv. campestris is a vascular pathogen that invades the xylem and colonizes the mesophyll. The symptoms of black rot include marginal leaf chlorosis, necrosis, and darkening of leaf veins and vascular tissue within the stem. Full leaf yellowing, wilting, and necrosis
also occur as the disease advances (Hayward, 1993). To prevent black rot, the use of pathogenfree seeds is recommended. Testing of seed lots for the presence of pathogen is, therefore, essential. The general methods for detecting the pathogen in seed lots and plants are based on serological techniques or plating assays. These are considered to be reliable and efficient methods for routine detection. However, it takes 3 days to 1 week to detect the pathogen. Another disadvantage is
Corresponding author.
E-mail address: [email protected] (D.S. Park). 0944-5013/$ - see front matter & 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2004.09.002
ARTICLE IN PRESS 420 the possible presence of cells of other microorganisms, which may interfere by causing overgrowth or suppression of outgrowth of the pathogen. Furthermore, immunological techniques, such as the enzyme-linked immunosorbent assay (ELISA), and flow cytometry are relatively insensitive or time-consuming than PCR-based sensitive assay (Alvarez and Lou, 1985; Chitarra et al., 2002). In this study, specific primers from hrpF region of X. c. pv. campestris were designed. The primer set showed high sensitivity and specificity for detecting the pathogen in crude seed extracts and plants of cabbage.
Materials and methods Bacterial strains and culture conditions Bacterial and fungal strains were obtained from the Korean Agricultural Culture Collection (KACC) in Suwon, Korea and the Belgian Co-ordinated Collections of Micro-organisms (BCCMTM) in Belgium. All microorganisms used in this study are listed in Table 1. Xanthomonas strains were cultured on YGC medium (2.0% D- (+)-glucose, 2.0% CaCO3, 1.0% yeast extract, 1.5% agar) at 28 1C for two days, Escherichia coli on LB agar (Sambrook et al., 1989) at 37 1C for 18 h, and other bacteria on nutrient agar (NA, Difco) at 26–28 1C for 1–2 days.
Isolation of total DNA Xanthomonas strains were cultured on YGC medium and harvested with a scraper for total DNA extraction. Total DNA from Xanthomonas strains was prepared as described by Lazo and Gabriel (1987). Total DNA from other microorganisms was extracted using the genomic DNA extraction kit [Genomic-tips] supplied by Qiagen (Hilden, Germany).
Primer design and PCR amplification The primers XCF (50 -CGATTCGGCCATGAATGACT-30 ) and XCR (50 -CTGTTGATGGTGGTCTGC AA-30 ) were designed from hrpF of X. c. pv. campestris, with a predicted PCR product of 535 bp. PCR assays were performed with a PTC-200TM thermocycler (MJ Research, Watertown, MA, USA). All amplifications were carried out in a final volume of 50 ml containing 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, 0.2 mM of each dNTP, 10 pM of each primer,
Y.J. Park et al. and 2 units of Taq polymerase (Promega, Madison, Wisconsin, USA). The total amount of genomic DNA from various microorganisms added to the PCR mixture was approximately 50 ng. Reactions were run for 35 cycles, each consisting of 15 s at 94 1C, 15 s at 58 1C, and 30 s at 72 1C, with initial denaturation of 5 min at 94 1C and final extension of 5 min at 72 1C. An 8-ml aliquot of each amplified PCR product was electrophoresed on a 1.0% agarose gel, stained with ethidium bromide, and visualized on a UV transilluminator.
DNA dot-blot analysis DNA dot-blot analysis was carried out to confirm whether the hrpF gene was present in other microorganisms including xanthomonads used in this study. A 100-ng sample of genomic DNA isolated from Xanthomonas strains and other reference microorganisms were spotted onto Hybond-N+ nylon membrane (Amersham Pharmacia Biotech, UK). These were UV cross-linked to bind the labeled probe DNA. PCR product from X. c. pv. campestris (KACC10913) was labeled as probe with [32P]dCTP using the random primed method according to the manufacturer’s instructions (LaddermanTM Labeling kit, Takara, Japan). Prehybridization and hybridization were conducted in hybridization buffer (0.75 M NaCl, 75 mM sodium citrate, 0.5% SDS, 0.1% BSA, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, and 50 ml/ml denatured salmon sperm DNA) at 65 1C for 18 h. After hybridization, the filter was washed twice (10 min each) in 2 SSC containing 0.1% SDS at room temperature and twice (15 min each) in 0.1 SSC containing 0.1% SDS at 65 1C. Autoradiography was done at 70 1C with CURIX X-ray film (AGFA, Belgium).
Estimates of limits of detection To determine the limits of PCR detection in pure culture suspension, strain X. c. pv. campestris LMG 8091 was grown in NB, cells were pelleted and washed four times in distilled water. A suspension of 1.3 1010 CFU/ml was diluted seven times, in a ten-fold series. Aliquots of 10 ml of each dilution were directly used in PCR for amplification.
Detection of the pathogen by PCR in naturally infected cabbage plant To detect X. c. pv. campestris in naturally infected cabbages (Brassica oleracea var. capitata), symptomatic and symptomless cabbage leaves and seeds
ARTICLE IN PRESS Sensitive and specific detection of Xanthomonas campestris pv. campestris Table 1.
421
List of bacterial strains used in this study
No.
Bacterial isolate
Sourcea
Geographical origin
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
Xanthomonas campestris pv. campestris Xanthomonas campestris pv. campestris Xanthomonas campestris pv. campestris Xanthomonas campestris pv. campestris Xanthomonas campestris pv. campestris Xanthomonas campestris pv. campestris Xanthomonas campestris pv. campestris Xanthomonas campestris pv. campestris Xanthomonas campestris pv. campestris Xanthomonas campestris pv. campestris Xanthomonas campestris pv. carotae Xanthomonas campestris pv. glycines Xanthomonas campestris (Pammel 1895) Dowson Xanthomonas oryzae pv. oryzae (Xoo90) Xanthomonas oryzae pv. oryzae (Xoo85) Xanthomonas axonopodis pv. aurantifolii Xanthomonas oryzae pv. oryzae (PXO99) Xanthomonas axonopodis pv. citri Xanthomonas axonopodis pv. citri Xanthomonas axonopodis pv. axonopodis Xanthomonas fragariae Xanthomonas campestris pv. malvacearum Xanthomonas campestris pv. pelargonii Xanthomonas campestris pv. juglandii Xanthomonas arboricola pv. poinsettiicola Xanthomonas axonopodis pv. axonopodis Xanthomonas axonopodis pv. begoniae Xanthomonas axonopodis pv. dieffenbachiae Xanthomonas axonopodis pv. malvacearum Xanthomonas axonopodis pv. phaseoli Xanthomonas axonopodis pv. phyllanthi Xanthomonas axonopodis pv. vasculorum Xanthomonas axonopodis pv. vesicatoria Xanthomonas cassavae Xanthomonas cucurbitae Xanthomonas pisi Xanthomonas theicola Xanthomonas translucens pv. cerealis Xanthomonas translucens pv. hordei Xanthomonas translucens pv.phleipratensis Pseudomonas aeruginosa Pseudomonas fluorescens Pectobacterium carotovorum subsp. carotovorum Pectobacterium carotovorum subsp. carotovorum Escherichia coli (O157:H7) Mesorhizobium loti
KACC10913 (ATCC 33913)b LMG 568 LMG 582 LMG 584 LMG 8002 LMG 8052 LMG 8081 LMG 8091 LMG 8105 LMG 8110 KACC 10164 (ATCC 10547) KACC 10445 (LMG 7403) KACC 10490 (ATCC 33913) KACC 10312 KACC 10331 KACC 10161 (NCPPB 3655) KACC 10884 KACC 10443 KACC 10444 KACC 10935 (LMG 982) KACC 11115 (DSMZ 3587) KACC 11127 (DSMZ 1220) KACC 11128 (DSMZ50857) KACC 11116 (DSMZ 1049) LMG 5403 LMG 538 LMG 551 LMG 695 LMG 761 LMG 7455 LMG 844 LMG 901 LMG 905 LMG 673 LMG 8662 LMG 847 LMG 8684 LMG 679 LMG 882 LMG 843 KACC 10259 (ATCC 10145) KACC 10327 (LMG 1794) KACC 10441 (LMG 2412) KACC 10455 (LMG 2453) KACC 10765 (ATCC 35150) KACC 10644 (LMG 10644)
United Kingdom United Kingdom Belgium Belgium Malawi Netherland New Zealand USA Western Samoa Tonga USA Zambia United Kingdom Korea Korea Brazil Philippines Korea Korea Columbia USA — — United Kingdom New Zealand Columbia UK Brazil Sudan Vulgaris Sudan Mauritius — Malawi New Zealand Japan Japan USA Canada USA — United Kingdom United Kingdom Belgium — New Zealand
a
KACC, Korean Agricultural Culture Collection, Korea (http://kacc.rda.go.kr); ATCC, American Type Culture Collection, USA; LMG, The Belgian Co-ordinated Collections of Microorganisms (BCCMTM), Belgium; NCPPB, National Collection of Plant Pathogenic Bacteria, United Kingdom. ( ): Other Collection No. ‘‘—’’ not known. b Parenthesis: other collection No.
were obtained from fields in Icheon, Korea (Syngenta Korea Ltd.; http://www.syngenta.co.kr). One gram of leaves was sampled from each cabbage plants with sterile scissors and total DNA was
directly extracted from the sample by the C-TAB method (Murray and Thompson, 1980). Isolated DNA from plants was used in PCR assays as described above.
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Results Specificity of primers The 20-mer oligonucleotide XCF and XCR were tested for X. c. pv. campestris. As expected, a 535 bp DNA fragment was amplified. To check the specificity of the primers, a large collection of other microorganisms, including Xanthomonas species and their pathovars, were tested in PCR assay with primers XCF and XCR. None of the other Xanthomonas strains and reference microorganisms reacted with the primers; only X. c. pv. campestris showed a single amplified DNA fragment (Fig. 1). The primer pair, XCF and XCR, amplified a 535 bp DNA fragment from X. c. pv. campestris, when 50 ng DNA was used as the template under optimized conditions (50 ml PCR reaction, containing 1.5 mM MgCl2, 0.2 mM of each dNTP, 10 pM of each primer, 2 units of Taq polymerase, and 5 ml of 10 PCR buffer). Similar conditions yielded reproducible results in the Perkin Elmer 9600 thermal cyclers (Perkin Elmer International, Rotkreuz). Taq polymerase enzymes supplied by different manufacturers (Promega, Takara, and Toyobo) also yielded similar PCR results (data not shown).
Figure 2. DNA dot-blot analysis of hrpF with PCRamplified fragment (535 bp) from X. campestris pv. campestris. Lanes 1, X. c. pv. campestris ATCC 33913; lane 2, X. c. pv. campestris LMG 582; lane 3, X. c. pv. campestris LMG 8002; lane 4, X. c. pv. campestris LMG 8081; lane 5, X. c. pv. campestris LMG 8105; lanes from 6 (corresponding to the number 11 in Table 1) to 41(corresponding to the number 46 in Table 1), other reference microbes.
DNA dot-blot analysis The presence (or homology) of the hrpF gene in Xanthomonas strains was confirmed by dot-blot analysis. The positive results in the dot-blot analysis suggested that the hrpF genes of Xanthomonas strains might be highly or slightly conserved, while the negative results indicated that the hrpF gene did not exist or share substantial homology with other bacteria and fungi (Fig. 2).
Figure 3. Limits of detection of X. c. pv. campestris cells LMG 8091. Lane M, size marker (1kb ladder, Gibco BRL); lane 1, 1.3 108 CFU/10 ml; lane 2, 1.3 107 CFU/10 ml; lane 3, 1.3 106 CFU/10 ml; lane 4, 1.3 105 CFU/10 ml; lane 5, 1.3 104 CFU/10 ml; lane 6, 1.3 103 CFU/10 ml; lane 7, 1.3 102 CFU/10 ml; lane 8, 1.3 10 CFU/10 ml.
Sensitivity of PCR It was possible to detect X. c. pv. campestris in pure culture suspensions tested by PCR. The minimum number of cells detected was about 1.3 104 CFU/ ml. A dilution series of cultured cells of strain LMG
Figure 1. Specific PCR amplification of partial hrpF gene from X. campestris pv. campestris with hrp-specific primers XCF and XCR. Lane M, Size marker (1Kb DNA ladder; Gibco BRL); lanes 1–46 listed in Table 1.
Figure 4. PCR detection of naturally infected cabbage leaves (Brassica oleracea var. capitata). M, size marker (1 Kb DNA ladder; Promega); Lane 1, X. c. pv. campestris KACC10913; Lane 2, X. c. pv. campestris LMG 568; Lane 3, X. c. pv. campestris LMG 582; Lane 4, X. c. pv. campestris LMG 584; Lane 5, X. c. pv. campestris LMG 8110; Lanes 6–8, symptomless leaves from cabbages; Lanes 9–12, symptomatic leaves from cabbages.
ARTICLE IN PRESS Sensitive and specific detection of Xanthomonas campestris pv. campestris 8091 yield a limit of detection of about 1.3 102 CFU per reaction after amplification (Fig. 3).
PCR detection of the pathogen in nature The pathogen was detected by PCR in naturally infected cabbage plant (Fig. 4). Fig. 4 shows a PCR assay of naturally infected cabbage plant. As expected, the pathogen was detected by PCR assay from infected cabbage leaves. On the other hand, a single DNA fragment was not showed from healthy cabbage leaves by PCR.
Discussion Development of specific primers and DNA probes for identification and detection has been reported for a number of plant pathogenic bacteria (Hartung et al., 1993; Leite et al., 1994; Rasmussen and Reeves, 1992) including xanthomonads. This study found hrpF to be a useful gene for detecting X. c. pv. campestris with PCR assay. Recently, it has been reported that the hrpF plays a crucial role in host recognition and after protein secretion across the bacterial envelope, i.e. translocation into the host cell (Rossier et al., 2000). This study revealed that the sequences of hrpF among xanthomonads were highly conserved or different from each other. Especially, Fig. 2 showed that a highly positive signal of Xanthomonas axonopodis pv. aurantifolii. It is thought that the nucleotide sequences of hrpF gene between X. c. pv. campestris and X. axonopodis pv. aurantifolii is very similar. In addition, hrpF amino acid sequences of X. campestris pv. campestris is 37.7% similar to nolX of the Mesorhizobium loti (data not shown). The role of nolX in nodulation and secretion in Rhizobium is not known clearly, but it may have a role in the release of bacteria from the infection threads like an hrpF of xanthomonads (Viprey et al., 1998). Successful detection of the pathogens using PCR techniques depends upon the specificity of primers. In this study, the specificity and sensitivity of PCR assays were evaluated through the detection of the pathogen in naturally infected plants. PCR conditions such as primers, template, concentration of Mg2+ (Bassam et al., 1992), thermocyclers, and thermostable polymerase origin (Schierwater and Ender, 1993) have been shown to affect amplification. In this study, all these parameters were optimized to avoid artifacts and to ensure reproducibility of amplification. Consistent results of amplification of a 535 bp fragment from X. c. pv. campestris by XCF and XCR were also
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obtained by using various PCR machines and different Taq polymerase enzymes supplied by various manufacturers. In conclusion, the results presented here indicate that X. c. pv. campestris can be detected and identified by PCR assays with XCF and XCR primer set.
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