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The use of 16S and 16S–23S rDNA to easily detect and differentiate common Gram-negative orchard epiphytes
Journal of Microbiological Methods 44 Ž2001. 69–77 www.elsevier.comrlocaterjmicmeth
The use of 16S and 16S–23S rDNA to easily detect and differentiate common Gram-negative orchard epiphytes R.S. Jeng a , A.M. Svircev b,) , A.L. Myers b, L. Beliaeva a , D.M. Hunter b, M. Hubbes a b
a Faculty of Forestry, UniÕersity of Toronto, 33 Wilcox St., Toronto, ON, Canada N5S 3B3 Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 4902 Victoria AÕenue North, P.O. Box 6000, Vineland Station, ON, Canada L0R 2E0
Accepted 30 October 2000
Abstract The identification of Gram-negative pathogenic and non-pathogenic bacteria commonly isolated from an orchard phylloplane may result in a time consuming and tedious process for the plant pathologist. The paper provides a simple Aone-stepB protocol that uses the polymerase chain reaction ŽPCR. to amplify intergenic spacer regions between 16S and 23S genes and a portion of 16S gene in the prokaryotic rRNA genetic loci. Amplified 16S rDNA, and restriction fragment length polymorphisms ŽRFLP. following EcoRI digestion produced band patterns that readily distinguished between the plant pathogen Erwinia amyloÕora Žcausal agent of fire blight in pear and apple. and the orchard epiphyte Pantoea agglomerans Žformerly E. herbicola.. The amplified DNA patterns of 16S–23S spacer regions may be used to differentiate E. amyloÕora at the intraspecies level. Isolates of E. amyloÕora obtained from raspberries exhibited two major fragments while those obtained from apples showed three distinct amplified DNA bands. In addition, the size of the 16S–23S spacer region differs between Pseudomonas syringae and Pseudomonas fluorescens. The RFLP pattern generated by HaeIII digestion may be used to provide a rapid and accurate identification of these two common orchard epiphytes. q 2001 Published by Elsevier Science B.V. Keywords: Erwinia amyloÕora; Fire blight; Pseudomonas fluorescens; Pseudomonas syringae; Pantoea agglomerans
1. Introduction Bacteria commonly isolated from the canopy in pear and apple orchards belong to a variety of genera such as Arthrobacter, Bacillus, ClaÕibacter, Curtobacterium, Erwinia, Klebsiella, Micrococcus, Pantoea and Pseudomonas ŽJohnson and Stockwell, ) Corresponding author. Tel.: q1-905-562-4113 ext. 227; fax: q1-905-562-4335. E-mail address: [email protected] ŽA.M. Svircev..
1998.. The precise composition of the microbial population at any point in time may be influenced by a number of complex and changing environmental factors. The overall immigration, emigration, growth and death of viable propagules may have a direct effect on the microorganisms present on a leaf surface ŽKinkel, 1997.. The incidence of Pantoea agglomerans Ž E. herbicola ŽLonis ¨ . Dye. increases throughout the growing season. Pseudomonas spp. are the predominant bacterial species throughout the flowering period ŽKearns and Hale, 1995.. The popu-
0167-7012r01r$ - see front matter q 2001 Published by Elsevier Science B.V. PII: S 0 1 6 7 - 7 0 1 2 Ž 0 0 . 0 0 2 3 0 - X
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lation size of orchard epiphytes and pathogens may be greatly influenced by the plant host species. Fifteen different strains of Pseudomonas syringae Van Hall were isolated from a single host ŽO’Brien and Lindow, 1989.. Generally, there appears to be a succession in the phyllosphere over time and over a growing season: first bacteria, then yeasts and finally filamentous fungi were isolated ŽKinkel, 1997.. The classical methods used to differentiate the fire blight pathogen, Erwinia. amyloÕora ŽBurrill. Winslow et al. from leaf and flower epiphytes rely on the use of selective or semi-selective media for the initial isolation, differentiation and culture of the pathogen ŽKing et al., 1954; Kado and Heskett, 1970; Miller and Schroth, 1972; Bereswill et al., 1998.. Gram-staining of unknown bacterial cultures helps in the initial identification process ŽSchaad, 1994.. Pathogenicity tests used for the identification of E. amyloÕora may include the presence of bacterial ooze on inoculated immature pears, a hypersensitive reaction in tobacco ŽSteinberger and Beer, 1988; Brisset and Paulin, 1992. andror the presence of fire blight symptoms on apple or pear seedling shoots inoculated with pure cultures of the pathogen. It is relatively simple to isolate E. amyloÕora from plant tissue exhibiting fire blight symptoms. However, these techniques are labour intensive, requiring a constant source of either apple or pear seedlings, tobacco plants andror immature pear fruit. In our experience, occasionally, colonies of the fire blight pathogen E. amyloÕora and the epiphytic P. agglomerans appear identical on the selective media initially used by Miller and Schroth Ž1972.. Recent developments in the use of molecular markers for the identification of bacteria at the genus and species levels have carried over to research with plant pathogenic bacteria ŽClark et al., 1993; Bereswill et al., 1995; McManus and Jones, 1995a,b; Momol et al., 1997.. The major challenge in the identification of the fire blight pathogen has been to detect the pathogen when it is present in low numbers such as an epiphyte on leaves or in asymptomatic plant tissue ŽMomol et al., 1997; Llop et al., 2000.. The polymerase chain reaction ŽPCR. and specific primers based on sections of DNA fragments from the 29-kb plasmid were used to identify E. amyloÕora in asymptomatic plant tissue ŽBereswill et al., 1992.. A DNA hybridization method used to
detect low levels of E. amyloÕora in apple calyxes ŽClark et al., 1993. was highly sensitive but time and cost restrictive. rRNA is highly conserved and essential for the survival of living organisms ŽHirano and Upper, 1983.. Phylogenetic analysis based on the 16S rRNA gene became well established as a standard method for the identification of bacteria at the family, genera and species levels ŽWoese, 1987.. The rRNA is amplified from DNA using PCR techniques with primers specific to conserved regions ŽSaunders and Saunders, 1993.. Phylogenetic analysis of the Erwinia genus based on 16S rRNA Ž16S rDNA. has recently been published ŽKwon et al., 1997; Hauben et al., 1998.. The notation 16S rDNA includes the gene and the intergenic sequences while the notation rRNA excludes the spacer regions ŽGurtler and Stanisich, 1996.. Using a two primer PCR procedure, Bereswill et al. Ž1995. demonstrated that by the use of HaeIII restriction fragment length polymorphism ŽRFLP., E. amyloÕora was easily identified. Successful amplification of the 16S rDNA relied on the homogeneity of the bacterial populations and the quality of DNA preparations. The technique was therefore rated as unsuitable for analysis of field samples ŽBereswill et al., 1995.. Single round PCR, nested PCR and PCR dot-blot hybridization were successfully used to identify E. amyloÕora ŽMcManus and Jones, 1995a.. The sensitivity, difficulty level and time required to execute the technique will determine its use as a diagnostic tool or for the detection of E. amyloÕora in asymptomatic tissue ŽMcManus and Jones, 1995a.. In addition to 16S rRNA gene sequences for identification of bacteria, the heterogeneity present in bacterial 16S–23S rDNA spacer regions, referred to as the intergenic spacer region ŽITS., have been exploited for bacterial identification ŽTyler et al., 1995.. Oligonucleotide primers specific to the ITS regions of the genes coding for 16S–23S rRNA Ž16S–23S rDNA. allow specific identification and detection of many bacteria ŽJensen et al., 1993.. Using this technique, E. amyloÕora was identified at the species level ŽMcManus and Jones, 1995b. and many Pseudomonas spp. from human origin were easily differentiated ŽTyler et al., 1995.. Currently, many molecular techniques are available for the identification of the plant pathogen E.
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amyloÕora. In contrast, the identification of Pseudomonas spp. is always a cumbersome process. The objective of our study was to use an easy Aone-stepB PCR assay based on oligonucleotide sequences from 16S and 16S–23S rDNA ITS region to rapidly and efficiently identify E. amyloÕora, Pseudomonas spp. and other Gram-negative bacteria that may be commonly found in the orchard phylloplane.
Table 1 Sources and hosts of isolates used in this study Species
Isolate
Host
Source
E. amyloÕora
E2017P Ea G-5 Ea 6-4 Ea D-7 Ea P-1 Ea 17-1-1 Ea G-7 ATCC 15580 Ea 110 Ea 1-95 Ea 1-97 Ea 2-95 Ea 2-97 Ea 3-97 Ea 4-96 Ea 6-96b Ea 7-96b Ea 8-96 39VII ICMP 2697 ICMP 2696 ATCC 33243 At C58 ATCC 13525 A506 MA-4 MB-3b NSA-6 ATCC 19316
pear pear pear pear pear pear pear pear apple raspberry raspberry raspberry raspberry raspberry raspberry raspberry raspberry raspberry cotoneaster pear apricot
D. Cuppelsa D. Hunter b D. Hunter D. Hunter D. Hunter D. Hunter D. Hunter ATCC 15580 ATCC 29780 G. Braunc G. Braun G. Braun G. Braun G. Braun G. Braun G. Braun G. Braun G. Braun A. Svircev d D. Cuppels D. Cuppels ATCC 33243 L. Stobbse ATCC 13525 BlightBan e T. Zhouf T. Zhou T. Zhou deposited as X. pruni
2. Materials and methods 2.1. Bacterial strains Reference strains are summarised in Table 1 and include pear Ž Pyrus communis L.., apple Ž Malus X domestica Borkh.. and raspberry Ž Rubus spp.. isolates of E. amyloÕora, P. agglomerans, P. fluorescens, P. syringae, Xanthomonas arboricola Vauterin et al. pathovar pruni Ždeposited as X. pruni . and Agrobacterium Õitis Ophel and Kerr. Field isolates of E. amyloÕora from pear and apple orchards were initially isolated on Miller and Schroth medium ŽMS.ŽMiller and Schroth, 1972.. All bacterial isolates were cultured on a continual basis on nutrient agar ŽDifco, Detroit, MI. and incubated at 278C.
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P. agglomerans
A. Õitis P. fluorescens P. syringae
X. arboricola
2.2. DNA isolation and PCR amplification
grapes
apple apple apple peach
a
Freshly grown single colony from each strain was transferred to 1 ml of standard LB medium ŽDifco, Detroit, MI. and incubated overnight in shake culture at 218C. Genomic DNA was extracted using QIAamp Tissue Kit ŽCat. a 29304, QIAGEN, Canada, Mississauga, ON., as described by the supplier. Primer pairs were ITS16 Ž5X-TTGTACACACCGCCCGTC. and ITS23 Ž5X-GGTACCTTAGATGTTTCAG. for intergenic spacer ŽITS. ŽMcManus and Jones 1995b.; 16SF Ž5X-GAGTTTGATCATGGCTCAG. and 16SR Ž5X-ACGGTTACCTTGTTACGAC. for 16S rDNA ŽBereswill et al., 1995.. Each PCR reaction contained 50 pmol of each primer, 50–100 ng of genomic DNA, 10 mM Tris– HCl, 1.5 mM MgCl 2 , 0.01% gelatin, 0.1% Triton-X, I unit of Taq DNA polymerase, 50 mM KCl and 200 mM each of dATP, dCTP, dGTP and dTTP in a final volume of 50 ml. Amplification was performed un-
Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, London, ON, Canada N5V 4T3. b Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, 4902 Victoria Ave. N., Vineland Station, ON, Canada L0R 2E0. c Agriculture and Agri-Food Canada, Kentville Agricultural Centre, Kentville, NS, Canada B4N J15. d Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, 4902 Victoria Ave. N., Vineland Station, ON, Canada L0R 2E0. e Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, 4902 Victoria Ave. N., Vineland Station, ON, Canada L0R 2E0. f Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, 4902 Victoria Ave. N., Vineland Station, ON, Canada L0R 2E0.
der mineral oil in a thermal cycler ŽHybaid, Ashford, UK. with the following cycle program: Ž1. denaturation at 958C for 3 min, Ž2. cycle to 958C, 2 min; 508C, 50 s; 728C, 1 min for 35 times and Ž3.
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extension at 728C for 10 min. This PCR protocol was repeated 10 times and it demonstrated consistent reproducibility. In addition, the protocol was used on a routine basis for the identification of E. amyloÕora, from apple and pear orchards where fire blight was suspected. For molecular size analysis of 16S and 16Sr23S spacer regions, PCR products were directly separated on a 1.8% agarose gel ŽBioShop Canada , Burlington, ON, L7N 1E8.. For restriction analysis, PCR products were digested with EcoRI or HaeIII endonucleases and the resulting fragments were also separated on a 1.8% agarose gel. 2.3. Cloning and DNA sequence analysis PCR products were cloned into a pCR 2.1-TOPO plasmid vector using the TOPO TA cloning kit
ŽInvitrogen, San Diego, CA. following the manufacturer’s instructions. Plasmid DNA containing the PCR-amplified fragment was verified by EcoRI restriction digestion. The nucleotide sequence of the inserted DNA was determined by the chain termination method of Sanger et al. Ž1977. with w35 Sx-radiolabeled dATP by the T7 Sequencing kit ŽAmersham Pharmacia Biotech., Baie d’Urfe, QC.. The DNA fragments were then separated in a model S2 sequencing apparatus ŽGibco BRL, Life Technologies, Rockville, MD.. Complete DNA sequence was obtained using five different primers. The ends of 16S rDNAs were sequenced by using a forward sequencing primer pUCrM13 Ž5X-GTAAAACGACGGCCAGT. and a reverse primer Ž5X -CAGGAAACAGCTATGAC.. The internal regions were sequenced using a set of
Fig. 1. Agarose-gel electrophoresis of PCR-amplified 16S rRNA gene from isolates of P. fluorescens ATCC 13525 Žlane 1., P. fluorescens A506 Žlane 2., P. syringae MA-4 Žlane 3., P. syringae MA-3b Žlane 4., P. syringae NSA-6 Žlane 5., X. arboricola ATCC 19316 Žlane 6., A. Õitis At C58 Žlane 7., E. amyloÕora 39VII Žlane 8., E. amyloÕora Ea 110 Žlane 9., E. amyloÕora EA 6-4 Žlane 10., E. amyloÕora Ea 4-96 Žlane 11., P. agglomerans ICMP 2697 Žlane 12., P. agglomerans ICMP 2696 Žlane 13. and P. agglomerans ATCC 33243 Žlane 14.. M, 100-bp DNA ladder molecular weight marker. Note the molecular size of 16S rRNA gene in A. Õitis At C58 is smaller than other isolates.
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internal primers which we designed. The primer sequences were as follows: 16SF1 Ž5X-TACGGGAGGCAGCAGTGG., 16SR1 Ž5X-GCCATGATGACTTGAGCG. and 16SR2 Ž5X-CAATTCATTTGAGTTTTACC..
exhibiting three fragments Žapproximately 700, 500, and 350 bp. while those of P. agglomerans and X. arboricola each showed only two bands Žapproximately 850 and 700 bp.. PCR products of P. fluorescens, P. syringae and A. Õitis were not digested by EcoRI.
3. Results
3.2. DNA sequence analysis of 16S rDNA
3.1. PCR amplification of 16S rDNA
The 16S rDNA for P. fluorescens, P. syringae and A. Õitis did not have EcoRI restriction sites. Sequence search of NCBI GenBank database confirmed the restriction analysis for these three species. Nucleotide sequences of 16S rDNA were determined in order to verify the PCR products and the EcoRI restriction sites ŽGAATTC. for E. amyloÕora ŽGenBank Accession No. AF289542 . , P. agglomerans ŽGenBank Accession No. AF290417. and X.
A single DNA fragment of 16S rDNA was amplified for each of the bacterial isolates used in this study. The PCR product of all isolates except A. Õitis was about 1550 bp while isolates of A. Õitis produced a smaller DNA fragment. Characteristic RFLP patterns were obtained when 16S rDNA was digested with EcoRI ŽFig. 1. with E. amyloÕora
Fig. 2. EcoRI restriction pattern of the PCR-amplified 16S rRNA gene from isolates of P. fluorescens ATCC 13525 Žlane 1., P. fluorescens A506 Žlane 2., P. syringae MA-4 Žlane 3., P. syringae MA-3b Žlane 4., P. syringae NSA-6 Žlane 5., X. arboricola ATCC 19316 Žlane 6., A. Õitis At C58 Žlane 7., E. amyloÕora 39VII Žlane 8., E. amyloÕora Ea 110 Žlane 9., E. amyloÕora Ea 6-4 Žlane 10., E. amyloÕora Ea 4-96 Žlane 11., P. agglomerans ICMP 2697 Žlane 12., P. agglomerans ICMP 2696 Žlane 13. and P. agglomerans ATCC 33243 Žlane 14.. M, 100 bp DNA ladder molecular weight marker. Note that X. arboricola and P. agglomerans showed the same RFLP profile.
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Fig. 3. Agarose-gel electrophoresis of PCR-amplified 16Sr23S intergenic spacer of rRNA gene from isolates of P. fluorescens ATCC 13525 Žlane 1., P. fluorescens A506 Žlane 2., P. syringae MA-4 Žlane 3., P. syringae MA-3b Žlane 4., P. syringae NSA-6 Žlane 5., X. arboricola ATCC 19316 Žlane 6., A. Õitis At C58 Žlane 7., E. amyloÕora 39VII Žlane 8., E. amyloÕora Ea 110 Žlane 9., E. amyloÕora Ea 6-4 Žlane 10., E. amyloÕora Ea 4-96 Žlane 11.. In lane 11, the band at 700 bp is variable in intensity and can be ignored. M, 100-bp DNA ladder molecular weight marker.
the species as well as the genus level ŽFig. 3.. Isolates of P. fluorescens Žlanes 1–2. had a single band ; 850 bp, slightly smaller than the ; 900 bp band of P. syringae isolates Žlanes 3–5.. The single band of X. arboricola Žlane 6. was ; 800 bp while that of A. Õitis Žlane 7. was ; 1500 bp. Isolates of E. amyloÕora Žlanes 8–11. exhibited multiple bands. The profiles of E. amyloÕora isolates from apple Žlane 9. and pear Žlane 10. were similar with four bands amplified, but were different from E. amyloÕora isolated from cotoneaster Žtwo bands, lane 8. or raspberry Žthree bands, lane 11.. Following restriction digest of ITS fragments with HaeIII endonuclease, P. fluorescens isolates produced 520- and 350-bp fragments, while those of P. syringae isolates were 520 and 380 bp ŽFig. 4.. All isolates of E. amyloÕora obtained from infected apple or pear had three distinct amplified ITS bands, as compared to two major DNA fragments for isolates of E. amyloÕora from raspberry ŽFig. 5.. Sequencing two of the bands for the E. amyloÕora apple isolate Ea G-5, showed that the smallest ITS region contained the tRNA glu gene ŽGenBank Accession No. AF290419., while the larger fragment
arboricola ŽGenBank Accession No. AF290420.. The length of the PCR-amplified 16S rDNA including primers was 1501 bp for E. amyloÕora and P. agglomerans isolates while that of X. arboricola was 1506 bp. These three sequences were compared using computer software PCrGENE ŽIntelliGenetics, Mountain View, CA. which automatically compares and truncates the nucleotide sequence into 1200 bp for similarity analysis. The 16S rDNA of E. amyloÕora contained two EcoRI restriction sites Žq661 and q993. while only one site was noted for P. agglomerans Žq661. and X. arboricola Žq668.. Other differences such as nucleotide deletion, transversion and transition were also evident. 3.3. PCR amplification of 16S r 23S rDNA intergenic spacer (ITS) The 16Sr23S intergenic spacer PCR product can be used to differentiate among 11 bacterial isolates at
Fig. 4. HaeIII restriction profile of PCR-amplified ITS region of P. fluorescens ATCC 13525 Žlane 1., P. fluorescens A506 Žlane 2., P. syringae MA-4 Žlane 3., P. syringae MB-3b Žlane 4. and P. syringae NSA-6 Žlane 5.. M, 100-bp DNA ladder molecular weight marker.
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Ž; 1200 bp. contained the tRNAala gene ŽGenBank Accession No. 290418..
4. Discussion The combined use of PCR amplification of the 16S and 16S–23S rDNA ITS regions allowed the rapid and accurate means of differentiating among Gram-negative bacteria that are commonly found in the orchard phylloplane. Restriction fragment length polymorphisms of PCR-amplified 16S rRNA gene ŽFig. 1. produced by EcoRI digestion proved suitable for differentiating between E. amyloÕora and P. agglomerans ŽFig. 2.. This is in agreement with results previously reported by Bereswill et al. Ž1995. who used HaeIII restriction digest. Bereswill et al.
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Ž1995. felt that this method depended on homogenous bacterial populations, thus the method was not deemed suitable for analysis of field samples. The use of semi-selective media in our initial isolation procedures circumvents the concerns expressed by Bereswill et al. Ž1995.. The rapid differentiation of E. amyloÕora from P. agglomerans was particularly important since the colonies of the two organisms grown on the semi-selective MS media may on occasion appear identical. PCR amplification of the ITS region operons allowed detection of considerable length and sequence variations ŽFigs. 3–5.. ITS polymorphisms may form the PCR-based identification of many bacterial species ŽJensen et al., 1993., as well as differentiating between certain bacteria at subspecies level. In apple and pear orchards infected with E. amyloÕora, rapid and accurate identification of this
Fig. 5. Agarose-gel electrophoresis of PCR amplified ITS region of E. amyloÕora isolates obtained from apple and pear Žlanes 1–7. and E. amyloÕora isolates from raspberry Žlane 8–16.. M, 1-kb DNA ladder molecular weight marker.
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pathogen at the subspecies level is imperative. This study confirmed the findings of McManus and Jones Ž1995b. that strains of E. amyloÕora isolated from pear Ž Pyrus . and apple Ž Malus ., Rosaceae subfamily Pomoideae, may be easily differentiated from strains isolated from raspberry, Rubus, using ITS rDNA region ŽFig. 5.. The size of the ITS region and the RFLP pattern generated by HaeIII digestion makes the differentiation between P. fluorescens and P. syringae, rapid and accurate ŽFig. 4.. Tyler et al. Ž1995. used 16S– 23S rRNA ITS regions to differentiate between eight species of pathogenic pseudomonads from human infections and 24 Pseudomonas spp. from the American Type Culture Collection. The present study also demonstrated that small and large 16S–23S rRNA ITS of E. amyloÕora contained genes for tRNA glu and tRN ala , respectively as described by Graham et al. Ž1996.. Similarly, in Pseudomonas spp. the ITS region was found to contain tRNA sequences coding for isoleucine andror alanine ŽTyler et al., 1995. In addition to ITS pattern, the RFLP profile of the amplified 16S rDNA fragments generated by EcoRI restriction provided another means to screen Erwinia isolates from other bacteria at the species level. Kearns and Hale Ž1995. relied on the use of differential media and the oxidase reaction to differentiate between non-fluorescent and fluorescent pseudomonads, P. syringae and P. agglomerans, isolated from the orchard and DNA hybridization to differentiate between AErwinia-likeB and E. amyloÕora. This PCR technique together with the use of selective or semi-selective media allows for the rapid and accurate Aone stepB identification of Gram-negative bacteria commonly isolated from an orchard phylloplane.
Acknowledgements The authors wish to thank Agriculture and AgriFood Canada for the financial support of this work through an Ontario Research Enhancement Program grant. Our thanks to Drs. J. Gong and D. Errampalli of Agriculture and Agri-Food Canada, for their help and suggestions in the preparation of this manuscript.
References Bereswill, S., Pahl, A., Bellemann, P., Zeller, W., Geider, K., 1992. Sensitive and species-specific detection of Erwinia amyloÕora by PCR-analysis. Appl. Environ. Microbiol. 58, 3522–3526. Bereswill, S., Bugert, P., Bruchmuller, I., Geider, K., 1995. Identification of the fire blight pathogen, Erwinia amyloÕora, by PCR assays with chromosomal DNA. Appl. Environ. Microbiol. 61, 2636–2642. Bereswill, S., Jock, S., Bellemann, P., Geiger, K., 1998. Identification of Erwinia amyloÕora by growth morphology on agar containing copper sufate and by capsule staining with lectin. Plant Dis. 82, 158–164. Brisset, M.N., Paulin, J.-P., 1992. A reliable strategy for the study of disease and hypersensitive reactions induced by Erwinia amyloÕora. Plant Sci. 85, 171–177. Clark, R.G., Hale, C.N., Harte, D., 1993. A DNA approach to Erwinia amyloÕora detection in large scale apple testing and in epidemiological studies. Acta Hortic. 338, 59–66. Graham, T., Golsteyn-Thomas, E., Gannon, V.P.J., Thomas, J.E., 1996. Genus- and species-specific detection of Listeria monocytogenes using polymerase chain reaction assays targeting the 16Sr23S intergenic spacer region of the rRNA operon. Can. J. Microbiol. 42, 1155–1162. Gurtler, V., Stanisich, V.A., 1996. New approaches to typing and identification of bacteria using the 16s–23s rDNA spacer region. Microbiology 142, 3–16. Hauben, L., Moore, E.R.B., Vauterin, L., Steenackers, M., Mergaert, J., Verdonck, L., Swings, J., 1998. Phylogenetic position of phytopathogens within the Enterobacteriaceae. Syst. Appl. Microbiol. 21, 384–397. Hirano, S.S., Upper, C.D., 1983. Ecology and epidemiology of foliar bacterial plant pathogens. Ann. Rev. Phytopathol. 21, 243–269. Jensen, M.A., Webster, J.A., Straus, N., 1993. Rapid identification of bacteria on the basis of the polymerase chain reactionamplified ribosomal DNA spacer polymorphisms. Appl. Environ. Microbiol. 59, 945–952. Johnson, K.B., Stockwell, V.O., 1998. Management of fire blight: a case study in microbial ecology. Ann. Rev. Phytopathol. 36, 227–248. Kado, E.I., Heskett, M.G., 1970. Selective media for the isolation of Agrobacterium, Erwinia, Pseudomonas and Xanthomonas. Phytopathology 60, 969–976. Kearns, L.P., Hale, C.N., 1995. Incidence of bacteria inhibitory to Erwinia amyloÕora from blossoms in New Zealand apple orchards. Plant Pathol. 44, 918–924. Kinkel, L.L., 1997. Microbial population dynamics on leaves. Ann. Rev. Phytopathol. 35, 327–347. King, E.O., Ward, M.K., Raney, D.E., 1954. Two simple media for the demonstration of phycocyanin and fluorescein. J. Lab. Clin. Med. 4, 301–307. Kwon, S.W., Go, S.J., Kang, H.W., Ryu, J.C., Jo, J.K., 1997. Phylogenetic analysis of Erwinia species based on 16S rRNA gene sequences. Int. J. Syst. Bacteriol. 47, 1061–1067.
R.S. Jeng et al.r Journal of Microbiological Methods 44 (2001) 69–77 Llop, P., Bonaterra, A., Penalver, J., Lopez, M., 2000. Development of highly sensitive nested-PCR procedure using a single closed tube for detection of Erwinia amyloÕora in asymptomatic plant material. Appl. Environ. Microbiol. 66, 2071– 2078. McManus, P.S., Jones, A.L., 1995a. Detection of Erwinia amyloÕora by nested PCR and PCR-dot-blot and reverse-blot hybridizations. Phytopathology 85, 618–623. McManus, P.S., Jones, A.L., 1995b. Genetic fingerprinting of Erwinia amyloÕora strains isolated from tree-fruit crops and Rubus spp. Phytopathology 85, 1547–1553. Miller, T.D., Schroth, M.N., 1972. Monitoring the epiphytic population of Erwinia amyloÕora on pear with a selective medium. Phytopathology 62, 1175–1182. Momol, M.T., Momol, E.A., Lamboy, W.F., Norelli, J.L., Beer, S.V., Aldwinckle, H.S., 1997. Characterization of Erwinia amyloÕora strains using random amplified polymorphic DNA fragments ŽRAPDs.. J. Appl. Microbiol. 82, 389–398. O’Brien, R.D., Lindow, S.E., 1989. Effect of plant species and environmental conditions on epiphytic population sizes of Pseudomonas syringae and other bacteria. Phytopathology 79, 619–627.
77
Sanger, F., Nicklen, S., Coulson, A.R., 1977. DNA sequencing with chain-terminating inhibitors. Proc. Nat. Acad. Sci. U. S. A. 74, 5463–5467. Saunders, J.R., Saunders, V.A., 1993. Genotypic and phenotypic methods for the detection of specific released microorganisms. In: Edwards, C. ŽEd.., Monitoring Genetically Manipulated Microorganisms in the Environment. Wiley & Son Ltd., pp. 27–59. Schaad, N.W. ŽEd.. 1994. Laboratory Guide for the Identification of Plant Pathogenic Bacteria. Am. Phytopath. Society Press, St. Paul, Minnesota, pp. 157. Steinberger, E.M., Beer, S.V., 1988. Creation and complementation of pathogenicity mutants of Erwinia amyloÕora. Mol. Plant-Microbe Interact. 1, 135–144. Tyler, S.D., Strathdee, C.A., Rozee, K.R., Johnson, W.M., 1995. Oligonucleotide primers designed to differentiate pathogenic Pseudomonads on the basis of the sequencing of genes coding for 16S–23S rRNA internal transcribed spacers. Clin. Diag. Lab. Immunol. 2, 448–453. Woese, C.R., 1987. Bacterial evolution. Microbiol. Rev. 51, 221– 271.
The use of 16S and 16S–23S rDNA to easily detect and differentiate common Gram-negative orchard epiphytes R.S. Jeng a , A.M. Svircev b,) , A.L. Myers b, L. Beliaeva a , D.M. Hunter b, M. Hubbes a b
a Faculty of Forestry, UniÕersity of Toronto, 33 Wilcox St., Toronto, ON, Canada N5S 3B3 Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 4902 Victoria AÕenue North, P.O. Box 6000, Vineland Station, ON, Canada L0R 2E0
Accepted 30 October 2000
Abstract The identification of Gram-negative pathogenic and non-pathogenic bacteria commonly isolated from an orchard phylloplane may result in a time consuming and tedious process for the plant pathologist. The paper provides a simple Aone-stepB protocol that uses the polymerase chain reaction ŽPCR. to amplify intergenic spacer regions between 16S and 23S genes and a portion of 16S gene in the prokaryotic rRNA genetic loci. Amplified 16S rDNA, and restriction fragment length polymorphisms ŽRFLP. following EcoRI digestion produced band patterns that readily distinguished between the plant pathogen Erwinia amyloÕora Žcausal agent of fire blight in pear and apple. and the orchard epiphyte Pantoea agglomerans Žformerly E. herbicola.. The amplified DNA patterns of 16S–23S spacer regions may be used to differentiate E. amyloÕora at the intraspecies level. Isolates of E. amyloÕora obtained from raspberries exhibited two major fragments while those obtained from apples showed three distinct amplified DNA bands. In addition, the size of the 16S–23S spacer region differs between Pseudomonas syringae and Pseudomonas fluorescens. The RFLP pattern generated by HaeIII digestion may be used to provide a rapid and accurate identification of these two common orchard epiphytes. q 2001 Published by Elsevier Science B.V. Keywords: Erwinia amyloÕora; Fire blight; Pseudomonas fluorescens; Pseudomonas syringae; Pantoea agglomerans
1. Introduction Bacteria commonly isolated from the canopy in pear and apple orchards belong to a variety of genera such as Arthrobacter, Bacillus, ClaÕibacter, Curtobacterium, Erwinia, Klebsiella, Micrococcus, Pantoea and Pseudomonas ŽJohnson and Stockwell, ) Corresponding author. Tel.: q1-905-562-4113 ext. 227; fax: q1-905-562-4335. E-mail address: [email protected] ŽA.M. Svircev..
1998.. The precise composition of the microbial population at any point in time may be influenced by a number of complex and changing environmental factors. The overall immigration, emigration, growth and death of viable propagules may have a direct effect on the microorganisms present on a leaf surface ŽKinkel, 1997.. The incidence of Pantoea agglomerans Ž E. herbicola ŽLonis ¨ . Dye. increases throughout the growing season. Pseudomonas spp. are the predominant bacterial species throughout the flowering period ŽKearns and Hale, 1995.. The popu-
0167-7012r01r$ - see front matter q 2001 Published by Elsevier Science B.V. PII: S 0 1 6 7 - 7 0 1 2 Ž 0 0 . 0 0 2 3 0 - X
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lation size of orchard epiphytes and pathogens may be greatly influenced by the plant host species. Fifteen different strains of Pseudomonas syringae Van Hall were isolated from a single host ŽO’Brien and Lindow, 1989.. Generally, there appears to be a succession in the phyllosphere over time and over a growing season: first bacteria, then yeasts and finally filamentous fungi were isolated ŽKinkel, 1997.. The classical methods used to differentiate the fire blight pathogen, Erwinia. amyloÕora ŽBurrill. Winslow et al. from leaf and flower epiphytes rely on the use of selective or semi-selective media for the initial isolation, differentiation and culture of the pathogen ŽKing et al., 1954; Kado and Heskett, 1970; Miller and Schroth, 1972; Bereswill et al., 1998.. Gram-staining of unknown bacterial cultures helps in the initial identification process ŽSchaad, 1994.. Pathogenicity tests used for the identification of E. amyloÕora may include the presence of bacterial ooze on inoculated immature pears, a hypersensitive reaction in tobacco ŽSteinberger and Beer, 1988; Brisset and Paulin, 1992. andror the presence of fire blight symptoms on apple or pear seedling shoots inoculated with pure cultures of the pathogen. It is relatively simple to isolate E. amyloÕora from plant tissue exhibiting fire blight symptoms. However, these techniques are labour intensive, requiring a constant source of either apple or pear seedlings, tobacco plants andror immature pear fruit. In our experience, occasionally, colonies of the fire blight pathogen E. amyloÕora and the epiphytic P. agglomerans appear identical on the selective media initially used by Miller and Schroth Ž1972.. Recent developments in the use of molecular markers for the identification of bacteria at the genus and species levels have carried over to research with plant pathogenic bacteria ŽClark et al., 1993; Bereswill et al., 1995; McManus and Jones, 1995a,b; Momol et al., 1997.. The major challenge in the identification of the fire blight pathogen has been to detect the pathogen when it is present in low numbers such as an epiphyte on leaves or in asymptomatic plant tissue ŽMomol et al., 1997; Llop et al., 2000.. The polymerase chain reaction ŽPCR. and specific primers based on sections of DNA fragments from the 29-kb plasmid were used to identify E. amyloÕora in asymptomatic plant tissue ŽBereswill et al., 1992.. A DNA hybridization method used to
detect low levels of E. amyloÕora in apple calyxes ŽClark et al., 1993. was highly sensitive but time and cost restrictive. rRNA is highly conserved and essential for the survival of living organisms ŽHirano and Upper, 1983.. Phylogenetic analysis based on the 16S rRNA gene became well established as a standard method for the identification of bacteria at the family, genera and species levels ŽWoese, 1987.. The rRNA is amplified from DNA using PCR techniques with primers specific to conserved regions ŽSaunders and Saunders, 1993.. Phylogenetic analysis of the Erwinia genus based on 16S rRNA Ž16S rDNA. has recently been published ŽKwon et al., 1997; Hauben et al., 1998.. The notation 16S rDNA includes the gene and the intergenic sequences while the notation rRNA excludes the spacer regions ŽGurtler and Stanisich, 1996.. Using a two primer PCR procedure, Bereswill et al. Ž1995. demonstrated that by the use of HaeIII restriction fragment length polymorphism ŽRFLP., E. amyloÕora was easily identified. Successful amplification of the 16S rDNA relied on the homogeneity of the bacterial populations and the quality of DNA preparations. The technique was therefore rated as unsuitable for analysis of field samples ŽBereswill et al., 1995.. Single round PCR, nested PCR and PCR dot-blot hybridization were successfully used to identify E. amyloÕora ŽMcManus and Jones, 1995a.. The sensitivity, difficulty level and time required to execute the technique will determine its use as a diagnostic tool or for the detection of E. amyloÕora in asymptomatic tissue ŽMcManus and Jones, 1995a.. In addition to 16S rRNA gene sequences for identification of bacteria, the heterogeneity present in bacterial 16S–23S rDNA spacer regions, referred to as the intergenic spacer region ŽITS., have been exploited for bacterial identification ŽTyler et al., 1995.. Oligonucleotide primers specific to the ITS regions of the genes coding for 16S–23S rRNA Ž16S–23S rDNA. allow specific identification and detection of many bacteria ŽJensen et al., 1993.. Using this technique, E. amyloÕora was identified at the species level ŽMcManus and Jones, 1995b. and many Pseudomonas spp. from human origin were easily differentiated ŽTyler et al., 1995.. Currently, many molecular techniques are available for the identification of the plant pathogen E.
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amyloÕora. In contrast, the identification of Pseudomonas spp. is always a cumbersome process. The objective of our study was to use an easy Aone-stepB PCR assay based on oligonucleotide sequences from 16S and 16S–23S rDNA ITS region to rapidly and efficiently identify E. amyloÕora, Pseudomonas spp. and other Gram-negative bacteria that may be commonly found in the orchard phylloplane.
Table 1 Sources and hosts of isolates used in this study Species
Isolate
Host
Source
E. amyloÕora
E2017P Ea G-5 Ea 6-4 Ea D-7 Ea P-1 Ea 17-1-1 Ea G-7 ATCC 15580 Ea 110 Ea 1-95 Ea 1-97 Ea 2-95 Ea 2-97 Ea 3-97 Ea 4-96 Ea 6-96b Ea 7-96b Ea 8-96 39VII ICMP 2697 ICMP 2696 ATCC 33243 At C58 ATCC 13525 A506 MA-4 MB-3b NSA-6 ATCC 19316
pear pear pear pear pear pear pear pear apple raspberry raspberry raspberry raspberry raspberry raspberry raspberry raspberry raspberry cotoneaster pear apricot
D. Cuppelsa D. Hunter b D. Hunter D. Hunter D. Hunter D. Hunter D. Hunter ATCC 15580 ATCC 29780 G. Braunc G. Braun G. Braun G. Braun G. Braun G. Braun G. Braun G. Braun G. Braun A. Svircev d D. Cuppels D. Cuppels ATCC 33243 L. Stobbse ATCC 13525 BlightBan e T. Zhouf T. Zhou T. Zhou deposited as X. pruni
2. Materials and methods 2.1. Bacterial strains Reference strains are summarised in Table 1 and include pear Ž Pyrus communis L.., apple Ž Malus X domestica Borkh.. and raspberry Ž Rubus spp.. isolates of E. amyloÕora, P. agglomerans, P. fluorescens, P. syringae, Xanthomonas arboricola Vauterin et al. pathovar pruni Ždeposited as X. pruni . and Agrobacterium Õitis Ophel and Kerr. Field isolates of E. amyloÕora from pear and apple orchards were initially isolated on Miller and Schroth medium ŽMS.ŽMiller and Schroth, 1972.. All bacterial isolates were cultured on a continual basis on nutrient agar ŽDifco, Detroit, MI. and incubated at 278C.
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P. agglomerans
A. Õitis P. fluorescens P. syringae
X. arboricola
2.2. DNA isolation and PCR amplification
grapes
apple apple apple peach
a
Freshly grown single colony from each strain was transferred to 1 ml of standard LB medium ŽDifco, Detroit, MI. and incubated overnight in shake culture at 218C. Genomic DNA was extracted using QIAamp Tissue Kit ŽCat. a 29304, QIAGEN, Canada, Mississauga, ON., as described by the supplier. Primer pairs were ITS16 Ž5X-TTGTACACACCGCCCGTC. and ITS23 Ž5X-GGTACCTTAGATGTTTCAG. for intergenic spacer ŽITS. ŽMcManus and Jones 1995b.; 16SF Ž5X-GAGTTTGATCATGGCTCAG. and 16SR Ž5X-ACGGTTACCTTGTTACGAC. for 16S rDNA ŽBereswill et al., 1995.. Each PCR reaction contained 50 pmol of each primer, 50–100 ng of genomic DNA, 10 mM Tris– HCl, 1.5 mM MgCl 2 , 0.01% gelatin, 0.1% Triton-X, I unit of Taq DNA polymerase, 50 mM KCl and 200 mM each of dATP, dCTP, dGTP and dTTP in a final volume of 50 ml. Amplification was performed un-
Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, London, ON, Canada N5V 4T3. b Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, 4902 Victoria Ave. N., Vineland Station, ON, Canada L0R 2E0. c Agriculture and Agri-Food Canada, Kentville Agricultural Centre, Kentville, NS, Canada B4N J15. d Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, 4902 Victoria Ave. N., Vineland Station, ON, Canada L0R 2E0. e Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, 4902 Victoria Ave. N., Vineland Station, ON, Canada L0R 2E0. f Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, 4902 Victoria Ave. N., Vineland Station, ON, Canada L0R 2E0.
der mineral oil in a thermal cycler ŽHybaid, Ashford, UK. with the following cycle program: Ž1. denaturation at 958C for 3 min, Ž2. cycle to 958C, 2 min; 508C, 50 s; 728C, 1 min for 35 times and Ž3.
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extension at 728C for 10 min. This PCR protocol was repeated 10 times and it demonstrated consistent reproducibility. In addition, the protocol was used on a routine basis for the identification of E. amyloÕora, from apple and pear orchards where fire blight was suspected. For molecular size analysis of 16S and 16Sr23S spacer regions, PCR products were directly separated on a 1.8% agarose gel ŽBioShop Canada , Burlington, ON, L7N 1E8.. For restriction analysis, PCR products were digested with EcoRI or HaeIII endonucleases and the resulting fragments were also separated on a 1.8% agarose gel. 2.3. Cloning and DNA sequence analysis PCR products were cloned into a pCR 2.1-TOPO plasmid vector using the TOPO TA cloning kit
ŽInvitrogen, San Diego, CA. following the manufacturer’s instructions. Plasmid DNA containing the PCR-amplified fragment was verified by EcoRI restriction digestion. The nucleotide sequence of the inserted DNA was determined by the chain termination method of Sanger et al. Ž1977. with w35 Sx-radiolabeled dATP by the T7 Sequencing kit ŽAmersham Pharmacia Biotech., Baie d’Urfe, QC.. The DNA fragments were then separated in a model S2 sequencing apparatus ŽGibco BRL, Life Technologies, Rockville, MD.. Complete DNA sequence was obtained using five different primers. The ends of 16S rDNAs were sequenced by using a forward sequencing primer pUCrM13 Ž5X-GTAAAACGACGGCCAGT. and a reverse primer Ž5X -CAGGAAACAGCTATGAC.. The internal regions were sequenced using a set of
Fig. 1. Agarose-gel electrophoresis of PCR-amplified 16S rRNA gene from isolates of P. fluorescens ATCC 13525 Žlane 1., P. fluorescens A506 Žlane 2., P. syringae MA-4 Žlane 3., P. syringae MA-3b Žlane 4., P. syringae NSA-6 Žlane 5., X. arboricola ATCC 19316 Žlane 6., A. Õitis At C58 Žlane 7., E. amyloÕora 39VII Žlane 8., E. amyloÕora Ea 110 Žlane 9., E. amyloÕora EA 6-4 Žlane 10., E. amyloÕora Ea 4-96 Žlane 11., P. agglomerans ICMP 2697 Žlane 12., P. agglomerans ICMP 2696 Žlane 13. and P. agglomerans ATCC 33243 Žlane 14.. M, 100-bp DNA ladder molecular weight marker. Note the molecular size of 16S rRNA gene in A. Õitis At C58 is smaller than other isolates.
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internal primers which we designed. The primer sequences were as follows: 16SF1 Ž5X-TACGGGAGGCAGCAGTGG., 16SR1 Ž5X-GCCATGATGACTTGAGCG. and 16SR2 Ž5X-CAATTCATTTGAGTTTTACC..
exhibiting three fragments Žapproximately 700, 500, and 350 bp. while those of P. agglomerans and X. arboricola each showed only two bands Žapproximately 850 and 700 bp.. PCR products of P. fluorescens, P. syringae and A. Õitis were not digested by EcoRI.
3. Results
3.2. DNA sequence analysis of 16S rDNA
3.1. PCR amplification of 16S rDNA
The 16S rDNA for P. fluorescens, P. syringae and A. Õitis did not have EcoRI restriction sites. Sequence search of NCBI GenBank database confirmed the restriction analysis for these three species. Nucleotide sequences of 16S rDNA were determined in order to verify the PCR products and the EcoRI restriction sites ŽGAATTC. for E. amyloÕora ŽGenBank Accession No. AF289542 . , P. agglomerans ŽGenBank Accession No. AF290417. and X.
A single DNA fragment of 16S rDNA was amplified for each of the bacterial isolates used in this study. The PCR product of all isolates except A. Õitis was about 1550 bp while isolates of A. Õitis produced a smaller DNA fragment. Characteristic RFLP patterns were obtained when 16S rDNA was digested with EcoRI ŽFig. 1. with E. amyloÕora
Fig. 2. EcoRI restriction pattern of the PCR-amplified 16S rRNA gene from isolates of P. fluorescens ATCC 13525 Žlane 1., P. fluorescens A506 Žlane 2., P. syringae MA-4 Žlane 3., P. syringae MA-3b Žlane 4., P. syringae NSA-6 Žlane 5., X. arboricola ATCC 19316 Žlane 6., A. Õitis At C58 Žlane 7., E. amyloÕora 39VII Žlane 8., E. amyloÕora Ea 110 Žlane 9., E. amyloÕora Ea 6-4 Žlane 10., E. amyloÕora Ea 4-96 Žlane 11., P. agglomerans ICMP 2697 Žlane 12., P. agglomerans ICMP 2696 Žlane 13. and P. agglomerans ATCC 33243 Žlane 14.. M, 100 bp DNA ladder molecular weight marker. Note that X. arboricola and P. agglomerans showed the same RFLP profile.
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Fig. 3. Agarose-gel electrophoresis of PCR-amplified 16Sr23S intergenic spacer of rRNA gene from isolates of P. fluorescens ATCC 13525 Žlane 1., P. fluorescens A506 Žlane 2., P. syringae MA-4 Žlane 3., P. syringae MA-3b Žlane 4., P. syringae NSA-6 Žlane 5., X. arboricola ATCC 19316 Žlane 6., A. Õitis At C58 Žlane 7., E. amyloÕora 39VII Žlane 8., E. amyloÕora Ea 110 Žlane 9., E. amyloÕora Ea 6-4 Žlane 10., E. amyloÕora Ea 4-96 Žlane 11.. In lane 11, the band at 700 bp is variable in intensity and can be ignored. M, 100-bp DNA ladder molecular weight marker.
the species as well as the genus level ŽFig. 3.. Isolates of P. fluorescens Žlanes 1–2. had a single band ; 850 bp, slightly smaller than the ; 900 bp band of P. syringae isolates Žlanes 3–5.. The single band of X. arboricola Žlane 6. was ; 800 bp while that of A. Õitis Žlane 7. was ; 1500 bp. Isolates of E. amyloÕora Žlanes 8–11. exhibited multiple bands. The profiles of E. amyloÕora isolates from apple Žlane 9. and pear Žlane 10. were similar with four bands amplified, but were different from E. amyloÕora isolated from cotoneaster Žtwo bands, lane 8. or raspberry Žthree bands, lane 11.. Following restriction digest of ITS fragments with HaeIII endonuclease, P. fluorescens isolates produced 520- and 350-bp fragments, while those of P. syringae isolates were 520 and 380 bp ŽFig. 4.. All isolates of E. amyloÕora obtained from infected apple or pear had three distinct amplified ITS bands, as compared to two major DNA fragments for isolates of E. amyloÕora from raspberry ŽFig. 5.. Sequencing two of the bands for the E. amyloÕora apple isolate Ea G-5, showed that the smallest ITS region contained the tRNA glu gene ŽGenBank Accession No. AF290419., while the larger fragment
arboricola ŽGenBank Accession No. AF290420.. The length of the PCR-amplified 16S rDNA including primers was 1501 bp for E. amyloÕora and P. agglomerans isolates while that of X. arboricola was 1506 bp. These three sequences were compared using computer software PCrGENE ŽIntelliGenetics, Mountain View, CA. which automatically compares and truncates the nucleotide sequence into 1200 bp for similarity analysis. The 16S rDNA of E. amyloÕora contained two EcoRI restriction sites Žq661 and q993. while only one site was noted for P. agglomerans Žq661. and X. arboricola Žq668.. Other differences such as nucleotide deletion, transversion and transition were also evident. 3.3. PCR amplification of 16S r 23S rDNA intergenic spacer (ITS) The 16Sr23S intergenic spacer PCR product can be used to differentiate among 11 bacterial isolates at
Fig. 4. HaeIII restriction profile of PCR-amplified ITS region of P. fluorescens ATCC 13525 Žlane 1., P. fluorescens A506 Žlane 2., P. syringae MA-4 Žlane 3., P. syringae MB-3b Žlane 4. and P. syringae NSA-6 Žlane 5.. M, 100-bp DNA ladder molecular weight marker.
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Ž; 1200 bp. contained the tRNAala gene ŽGenBank Accession No. 290418..
4. Discussion The combined use of PCR amplification of the 16S and 16S–23S rDNA ITS regions allowed the rapid and accurate means of differentiating among Gram-negative bacteria that are commonly found in the orchard phylloplane. Restriction fragment length polymorphisms of PCR-amplified 16S rRNA gene ŽFig. 1. produced by EcoRI digestion proved suitable for differentiating between E. amyloÕora and P. agglomerans ŽFig. 2.. This is in agreement with results previously reported by Bereswill et al. Ž1995. who used HaeIII restriction digest. Bereswill et al.
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Ž1995. felt that this method depended on homogenous bacterial populations, thus the method was not deemed suitable for analysis of field samples. The use of semi-selective media in our initial isolation procedures circumvents the concerns expressed by Bereswill et al. Ž1995.. The rapid differentiation of E. amyloÕora from P. agglomerans was particularly important since the colonies of the two organisms grown on the semi-selective MS media may on occasion appear identical. PCR amplification of the ITS region operons allowed detection of considerable length and sequence variations ŽFigs. 3–5.. ITS polymorphisms may form the PCR-based identification of many bacterial species ŽJensen et al., 1993., as well as differentiating between certain bacteria at subspecies level. In apple and pear orchards infected with E. amyloÕora, rapid and accurate identification of this
Fig. 5. Agarose-gel electrophoresis of PCR amplified ITS region of E. amyloÕora isolates obtained from apple and pear Žlanes 1–7. and E. amyloÕora isolates from raspberry Žlane 8–16.. M, 1-kb DNA ladder molecular weight marker.
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pathogen at the subspecies level is imperative. This study confirmed the findings of McManus and Jones Ž1995b. that strains of E. amyloÕora isolated from pear Ž Pyrus . and apple Ž Malus ., Rosaceae subfamily Pomoideae, may be easily differentiated from strains isolated from raspberry, Rubus, using ITS rDNA region ŽFig. 5.. The size of the ITS region and the RFLP pattern generated by HaeIII digestion makes the differentiation between P. fluorescens and P. syringae, rapid and accurate ŽFig. 4.. Tyler et al. Ž1995. used 16S– 23S rRNA ITS regions to differentiate between eight species of pathogenic pseudomonads from human infections and 24 Pseudomonas spp. from the American Type Culture Collection. The present study also demonstrated that small and large 16S–23S rRNA ITS of E. amyloÕora contained genes for tRNA glu and tRN ala , respectively as described by Graham et al. Ž1996.. Similarly, in Pseudomonas spp. the ITS region was found to contain tRNA sequences coding for isoleucine andror alanine ŽTyler et al., 1995. In addition to ITS pattern, the RFLP profile of the amplified 16S rDNA fragments generated by EcoRI restriction provided another means to screen Erwinia isolates from other bacteria at the species level. Kearns and Hale Ž1995. relied on the use of differential media and the oxidase reaction to differentiate between non-fluorescent and fluorescent pseudomonads, P. syringae and P. agglomerans, isolated from the orchard and DNA hybridization to differentiate between AErwinia-likeB and E. amyloÕora. This PCR technique together with the use of selective or semi-selective media allows for the rapid and accurate Aone stepB identification of Gram-negative bacteria commonly isolated from an orchard phylloplane.
Acknowledgements The authors wish to thank Agriculture and AgriFood Canada for the financial support of this work through an Ontario Research Enhancement Program grant. Our thanks to Drs. J. Gong and D. Errampalli of Agriculture and Agri-Food Canada, for their help and suggestions in the preparation of this manuscript.
References Bereswill, S., Pahl, A., Bellemann, P., Zeller, W., Geider, K., 1992. Sensitive and species-specific detection of Erwinia amyloÕora by PCR-analysis. Appl. Environ. Microbiol. 58, 3522–3526. Bereswill, S., Bugert, P., Bruchmuller, I., Geider, K., 1995. Identification of the fire blight pathogen, Erwinia amyloÕora, by PCR assays with chromosomal DNA. Appl. Environ. Microbiol. 61, 2636–2642. Bereswill, S., Jock, S., Bellemann, P., Geiger, K., 1998. Identification of Erwinia amyloÕora by growth morphology on agar containing copper sufate and by capsule staining with lectin. Plant Dis. 82, 158–164. Brisset, M.N., Paulin, J.-P., 1992. A reliable strategy for the study of disease and hypersensitive reactions induced by Erwinia amyloÕora. Plant Sci. 85, 171–177. Clark, R.G., Hale, C.N., Harte, D., 1993. A DNA approach to Erwinia amyloÕora detection in large scale apple testing and in epidemiological studies. Acta Hortic. 338, 59–66. Graham, T., Golsteyn-Thomas, E., Gannon, V.P.J., Thomas, J.E., 1996. Genus- and species-specific detection of Listeria monocytogenes using polymerase chain reaction assays targeting the 16Sr23S intergenic spacer region of the rRNA operon. Can. J. Microbiol. 42, 1155–1162. Gurtler, V., Stanisich, V.A., 1996. New approaches to typing and identification of bacteria using the 16s–23s rDNA spacer region. Microbiology 142, 3–16. Hauben, L., Moore, E.R.B., Vauterin, L., Steenackers, M., Mergaert, J., Verdonck, L., Swings, J., 1998. Phylogenetic position of phytopathogens within the Enterobacteriaceae. Syst. Appl. Microbiol. 21, 384–397. Hirano, S.S., Upper, C.D., 1983. Ecology and epidemiology of foliar bacterial plant pathogens. Ann. Rev. Phytopathol. 21, 243–269. Jensen, M.A., Webster, J.A., Straus, N., 1993. Rapid identification of bacteria on the basis of the polymerase chain reactionamplified ribosomal DNA spacer polymorphisms. Appl. Environ. Microbiol. 59, 945–952. Johnson, K.B., Stockwell, V.O., 1998. Management of fire blight: a case study in microbial ecology. Ann. Rev. Phytopathol. 36, 227–248. Kado, E.I., Heskett, M.G., 1970. Selective media for the isolation of Agrobacterium, Erwinia, Pseudomonas and Xanthomonas. Phytopathology 60, 969–976. Kearns, L.P., Hale, C.N., 1995. Incidence of bacteria inhibitory to Erwinia amyloÕora from blossoms in New Zealand apple orchards. Plant Pathol. 44, 918–924. Kinkel, L.L., 1997. Microbial population dynamics on leaves. Ann. Rev. Phytopathol. 35, 327–347. King, E.O., Ward, M.K., Raney, D.E., 1954. Two simple media for the demonstration of phycocyanin and fluorescein. J. Lab. Clin. Med. 4, 301–307. Kwon, S.W., Go, S.J., Kang, H.W., Ryu, J.C., Jo, J.K., 1997. Phylogenetic analysis of Erwinia species based on 16S rRNA gene sequences. Int. J. Syst. Bacteriol. 47, 1061–1067.
R.S. Jeng et al.r Journal of Microbiological Methods 44 (2001) 69–77 Llop, P., Bonaterra, A., Penalver, J., Lopez, M., 2000. Development of highly sensitive nested-PCR procedure using a single closed tube for detection of Erwinia amyloÕora in asymptomatic plant material. Appl. Environ. Microbiol. 66, 2071– 2078. McManus, P.S., Jones, A.L., 1995a. Detection of Erwinia amyloÕora by nested PCR and PCR-dot-blot and reverse-blot hybridizations. Phytopathology 85, 618–623. McManus, P.S., Jones, A.L., 1995b. Genetic fingerprinting of Erwinia amyloÕora strains isolated from tree-fruit crops and Rubus spp. Phytopathology 85, 1547–1553. Miller, T.D., Schroth, M.N., 1972. Monitoring the epiphytic population of Erwinia amyloÕora on pear with a selective medium. Phytopathology 62, 1175–1182. Momol, M.T., Momol, E.A., Lamboy, W.F., Norelli, J.L., Beer, S.V., Aldwinckle, H.S., 1997. Characterization of Erwinia amyloÕora strains using random amplified polymorphic DNA fragments ŽRAPDs.. J. Appl. Microbiol. 82, 389–398. O’Brien, R.D., Lindow, S.E., 1989. Effect of plant species and environmental conditions on epiphytic population sizes of Pseudomonas syringae and other bacteria. Phytopathology 79, 619–627.
77
Sanger, F., Nicklen, S., Coulson, A.R., 1977. DNA sequencing with chain-terminating inhibitors. Proc. Nat. Acad. Sci. U. S. A. 74, 5463–5467. Saunders, J.R., Saunders, V.A., 1993. Genotypic and phenotypic methods for the detection of specific released microorganisms. In: Edwards, C. ŽEd.., Monitoring Genetically Manipulated Microorganisms in the Environment. Wiley & Son Ltd., pp. 27–59. Schaad, N.W. ŽEd.. 1994. Laboratory Guide for the Identification of Plant Pathogenic Bacteria. Am. Phytopath. Society Press, St. Paul, Minnesota, pp. 157. Steinberger, E.M., Beer, S.V., 1988. Creation and complementation of pathogenicity mutants of Erwinia amyloÕora. Mol. Plant-Microbe Interact. 1, 135–144. Tyler, S.D., Strathdee, C.A., Rozee, K.R., Johnson, W.M., 1995. Oligonucleotide primers designed to differentiate pathogenic Pseudomonads on the basis of the sequencing of genes coding for 16S–23S rRNA internal transcribed spacers. Clin. Diag. Lab. Immunol. 2, 448–453. Woese, C.R., 1987. Bacterial evolution. Microbiol. Rev. 51, 221– 271.