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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1996, p. 3489–3493 0099-2240/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 62, No. 9

Use of Repetitive Intergenic DNA Sequences To Classify Pathogenic and Disease-Suppressive Streptomyces Strains† MICHAEL J. SADOWSKY,1* LINDA L. KINKEL,2 JOHN H. BOWERS,2

AND

JANET L. SCHOTTEL3

1

Department of Soil, Water, and Climate and Department of Microbiology, Department of Plant Pathology,2 and Department of Biochemistry and Plant Molecular Genetics Institute,3 University of Minnesota, St. Paul, Minnesota 55108 Received 18 March 1996/Accepted 28 June 1996

PCR DNA fingerprinting using repetitive intergenic DNA sequences (rep-PCR) was investigated as a means of differentiating between closely related strains of Streptomyces which were, in some cases, indistinguishable by other classification methods. Our results demonstrated that the majority of strains had unique rep-PCR DNA fingerprints and established that the technique could be a very useful tool in rapidly determining strain identity.

could be used to differentiate and group pathogenic and disease-suppressive Streptomyces strains. rep-PCR DNA fingerprints of Streptomyces sp. strains. The Streptomyces strains used in this study are listed in Table 1. Total genomic DNA from 17 scab pathogens, 2 sweet potato pathogens, and 10 nonpathogenic Streptomyces sp. strains was isolated essentially as described previously (1) and treated with 20 mg of RNAse (Boehringer Mannheim) per ml. The DNA was used as a template for PCR with the BOXA1R primer (59-CTACGGCAAGGCGACGCTGACG-39) (30), which was synthesized with an Applied Biosystems DNA synthesizer by the Macromolecular Structure, Sequence, and Synthesis Facility at Michigan State University. The REP1R-I and REP2 primers were also used in PCR mixtures (30), but the DNA fingerprints were less discriminating than those from the BOXA1R primer and were not used further (data not shown). The PCRs were performed as previously described (6), except that only a single primer was used at a final concentration of 50 pmol/ml with 50 ng of Streptomyces template DNA. Products from the PCR were separated on 1.5% agarose gels, stained with ethidium bromide (6), and analyzed as described previously (14). The primers yielded multiple DNA products ranging in size from approximately 0.25 kb to 2.3 kb (Fig. 1). A visual examination of banding patterns indicated that the Streptomyces strains were genetically diverse. However, some of the isolates were closely related to each other. For example, Streptomyces sp. strains PonSSII and PonSSI (lanes 17 and 18) shared a large number of DNA products in common and were virtually indistinguishable from each other on the basis of rep-PCR. Similarly, S. ipomoea B16450 and B16453 (lanes 26 and 27, respectively) had nearly identical DNA fingerprint patterns. Other strains had various degrees of relatedness to each other on the basis of the number of shared PCR products. As has been demonstrated for Streptococcus pneumoniae, Escherichia coli, and Salmonella typhimurium (30), BOX-like sequences can provide specific DNA fingerprint patterns for a variety of streptomycete isolates. Stability and reproducibility of rep-PCR DNA fingerprints. Previous studies have reported that the genomes of Streptomyces strains are relatively unstable (2, 5). To determine the stability of DNA fingerprint patterns over successive generations, DNA was extracted from 22 single-colony isolates of Streptomyces sp. strain 93 and from the original culture, which

Streptomyces strains are gram-positive, filamentous soil bacteria that are well known for their abilities to produce antibiotics and other secondary metabolites. These organisms have been implicated in the antagonism of a wide variety of plant pathogenic fungi, bacteria, and nematodes and are currently under investigation for their potential use as biological disease control agents (4, 7, 11, 21). Some Streptomyces sp. strains are plant pathogens. S. scabies (Thax.) is the causal agent of scab on a variety of underground vegetables, including beets, radishes, and potatoes. Other streptomycetes, including S. acidiscabies and S. albidoflavus, have also been reported to cause potato scab (8–10, 19), whereas S. ipomoea causes soil rot of sweet potatoes (18). The lack of a rapid and consistent means for distinguishing pathogenic, nonpathogenic, or pathogensuppressive Streptomyces strains represents a significant impediment to investigations of their dynamics, ecology, and interactions in soil. Numerous schemes have been developed to classify Streptomyces strains. Most of these, however, have relied on subjectively chosen morphological and biochemical traits (15, 16, 26, 29, 32). Analyses of DNA and cellular fatty acids have indicated that pathogenic Streptomyces strains show a high degree of strain diversity and do not comprise a distinct phylogenetic or taxonomic group (8, 10, 25). However, recent studies have demonstrated that pathogenic S. scabies and pathogen-suppressive Streptomyces strains from Minnesota soils were consistently distinguished from other strains on the basis of their cellular fatty acid profiles (3, 24). Another approach to classification has used DNA primers corresponding to repetitive extragenic palindromic (28), enterobacterial repetitive intergenic consensus (13), and repetitive BOX (23) sequences, coupled with the PCR technique (rep-PCR), to fingerprint the genomes of a variety of gram-negative and -positive soil bacteria (6, 14, 30, 33). The relative positions of the repeated sequences in the genome of a particular bacterial isolate appear to be conserved in closely related strains and are distinct in diverse species and genera (14, 22, 30). The goal of the present study was to determine if rep-PCR DNA fingerprinting

* Corresponding author. Mailing address: Department of Soil, Water, and Climate, University of Minnesota, 1991 Upper Buford Circle, 439 Borlaug Hall, St. Paul, MN 55108. Phone: (612) 624-2706. † Manuscript number 22,504 in the University of Minnesota Agricultural Experiment Station Series. 3489

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APPL. ENVIRON. MICROBIOL. TABLE 1. Streptomyces isolates used in this study

Species

S. scabies

Strain(s)

Pathogenicity Pathogen (potato)a inhibitorb

Sourcec

RB5, Beet, PonC, BC, NC, RB3, RB4, PonP, RB2 Roy FLII FLI

Yes Yes Yes No

No No No No

87, 88, 89

Yes

No

S. lividans

TK24

No

No

D. Hopwood, John Innes Institute, Norwich, United Kingdom

S. ipomoea

B12321, B16450d, B16453d

No

No

D. Labeda, U.S. Department of Agriculture, Peoria, Ill.

S. acidiscabies

RL104, RL182

Yes

No

R. Loria, Cornell University, Ithaca, N.Y.

S. diastatochromogenes PonSSI, PonSSII

No

Yes

Grand Rapids, Minn.

Not determined

No Yes

Yes No

Grand Rapids, Minn. Grand Forks, N.Dak.

15, PonSSR, 32, 93, 91 Crystal

Becker, Minn. Royalton, Minn. N. Anderson, University of Minnesota, St. Paul B. Goth, U.S. Department of Agriculture, Beltsville, Md. Grand Rapids, Minn.

a

Pathogenicity of all isolates was determined with a leaf-bud tuber assay (20). Yes, inhibition zone .3mm in an antibiotic inhibition assay (31) against strains 87 and RB4. c All strains isolated in Minnesota were obtained from D. Liu and N. Anderson, University of Minnesota, St. Paul. d Pathogenic on sweet potatoes. b

FIG. 1. rep-PCR fingerprint patterns of genomic DNA from Streptomyces sp. strains with the BOXA1R primer. The PCRs were repeated at least three times, and the patterns of PCR products were consistently reproducible. The PCR conditions were as follows: denaturation for 7 min at 958C, 35 cycles of 908C for 30 s, 538C for 1 min, and 568C for 8 min; final extension at 658C for 16 min; and a 48C soak. Lanes: 1, S. scabies 88; 2, S. scabies 89; 3, S. scabies 87; 4, S. scabies PonC; 5, S. scabies PonP; 6, S. scabies NC; 7, S. scabies Roy; 8, S. scabies RB2; 9, S. scabies RB3; 10, S. scabies RB4; 11, S. scabies RB5; 12, S. scabies BC; 13, S. scabies Beet; 14, S. scabies FL2; 15, Streptomyces sp. strain Crystal; 16, S. scabies FL1; 17, S. diastatochromagenes PonSSII; 18, S. diastatochromagenes PonSSI; 19, Streptomyces sp. strain 15; 20, Streptomyces sp. strain 32; 21, Streptomyces sp. strain 93; 22, Streptomyces sp. strain PonSSR; 23, Streptomyces sp. strain 91; 24, S. lividans TK24; 25, S. ipomoea B12321; 26, S. ipomoea B16450; 27, S. ipomoea B16453; 28, S. acidiscabies RL182; and 29, S. acidiscabies RL104. Values to the right are in kilobase pairs and represent molecular size markers (M).

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FIG. 2. rep-PCR fingerprint patterns of genomic DNA from Streptomyces sp. strain 93. The pattern of PCR products was generated with the BOXA1R primer. Lane 93 is the rep-PCR fingerprint pattern of parent strain 93, and lanes 1 to 22 are rep-PCR DNA fingerprint patterns of 22 independent single-colony isolates of strain 93. Values to the right are in kilobase pairs and represent molecular size markers (M).

was transferred en masse. The DNA samples were subjected to PCR with the BOXA1R primers, and the resulting patterns of DNA fragments were compared with that of the original strain 93 culture (Fig. 2). Only two of the DNA samples from isolated single colonies showed a change in the banding pattern that affected a major PCR product (lanes 15 and 20). In addition, three of the first-generation single colonies were used to isolate a second and subsequently a third generation of single colonies. When DNA was analyzed by rep-PCR from these single colonies, no significant difference in the DNA fingerprints was found (data not shown). Taken together, these results indicate that DNA fingerprints of Streptomyces strains obtained by PCR with BOXA1R primers are reproducible over successive generations, and if genomic instability occurs in these isolates, it does not preclude the use of rep-PCR for strain identification purposes. Cluster analysis on the basis of DNA fingerprints. The dendrogram in Fig. 3 shows that the Streptomyces strains could be divided into two major groups on the basis of cluster analysis of the rep-PCR DNA fingerprints. In general, the pathogenic and suppressive Streptomyces strains did not form distinct groups. While there were some suppressive isolates that were very similar to one another on the basis of DNA fingerprints (e.g., strains PonSSI and PonSSII clustered together at a euclidean distance of under 1.5, and strains 32 and 93 were distinguished at a distance of just over 2.5), the suppressive isolates and the pathogenic S. scabies isolates were intermingled throughout the two major groups. The S. acidiscabies (RL104 and RL182) and S. ipomoea (B16450, 16454, and B12321) isolates formed discrete subgroups separated from one another and from all other isolates at a euclidean distance of 3.5 or greater. It should also be noted that all non-scabies pathogens were contained in group 1, with the exception of Streptomyces sp. strain

Crystal. Our rep-PCR results are in agreement with the DNA analyses of Doering-Saad and coworkers (8), who reported that restriction fragment length polymorphism groupings of scab-forming Streptomyces strains did not correlate with numerical taxonomic classification and that these strains represent a genetically heterogeneous group of organisms. However, in our study, the average distance between clusters was relatively small among all strains tested, indicating that strains were quite closely related to one another. Cluster analyses of the DNA fingerprint patterns provided a very different picture of the relationships among the pathogenic and the suppressive strains than has been revealed by analysis of cellular fatty acids (3, 24). Cluster and principal component analyses of the total cellular fatty acid profiles have indicated that scab-forming pathogenic Streptomyces strains could be separated from scab-suppressive and other nonpathogenic strains at a euclidian distance of about 20 (24). However, there was no distinction between these two groups on the basis of DNA analyses. This lack of correspondence between classification methods was also noted in groupings of Bradyrhizobium japonicum on the basis of their rep-PCR DNA fingerprints, which were correlated with their ability to nodulate specific soybean genotypes (14) but which did not always correlate with previous classifications (12, 17). The lack of correlation in the clustering obtained with the DNA and the fatty acid analyses is intriguing, particularly in light of the apparently greater correspondence between the fatty acid clustering and the functional roles of the strains in soil (pathogenic versus pathogen suppressive). These data suggest that pathogenicity has not represented a major genetic bottleneck or constraint to the evolution of diversity within this group. Instead, factors not related to the pathogenicity of a strain have conferred greater selection pressure on the ge-

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APPL. ENVIRON. MICROBIOL. REFERENCES

FIG. 3. Dendrogram of relatedness of Streptomyces sp. strains derived from rep-PCR DNA fingerprints generated from the BOXA1R primer. Relationships between strains were determined as described by Judd et al. (14) by cluster analysis and the unweighted pair-group method with arithmetic averages (27). Analyses consisted of the dendrogram routine in the MIS software and the CLUSTER procedure in SAS (release 6.08; SAS Institute, Inc., Cary, N.C.) on an IBM 9121 mainframe computer. Cluster analyses in SAS were conducted with METHOD 5 AVERAGE and the NOSQUARE and NONORM options. The scale represents the average euclidian distance between clusters.

nome. The genetic variability observed among the pathogens may be the result of transfer of pathogenicity genes between somewhat diverse Streptomyces strains (8, 25). Further studies of the relationships among genome structure, fatty acid profiles, and taxonomic and functional groupings of Streptomyces strains are needed. An important outcome of this study was our finding that rep-PCR DNA fingerprints of Streptomyces strains with BOXA1R primers were relatively unique, stable, and reproducible. Consequently, the patterns of DNA fragments produced by rep-PCR could be a very useful tool in rapidly determining strain identity and tracking specific strains for epidemiological and ecological studies. This study was supported in part by grants from the University of Minnesota Agriculture Experiment Station (to M.J.S., J.L.S., and L.L.K.), by a grant from the USDA North Central Region Integrated Pest Management Program (to L.L.K.), by a grant from the Biological Process Technology Institute (to M.J.S., L.L.K., and J.L.S.), and by a USDA-APHIS National Biological Control Institute Postdoctoral Fellowship (to J.H.B.). We thank N. Anderson and D. Liu for providing the Minnesota isolates and B. Goth, D. Hopwood, D. Labeda, and R. Loria for providing all other Streptomyces strains.

1. Babcock, M. J., and K. E. Kendrick. 1988. Cloning of DNA involved in sporulation of Streptomyces griseus. J. Bacteriol. 170:2802–2808. 2. Birch, A., A. Ha ¨usler, and R. Hu ¨tter. 1990. Genome rearrangement and genetic instability in Streptomyces spp. J. Bacteriol. 172:4138–4142. 3. Bowers, J. H., L. L. Kinkel, and R. K. Jones. 1996. Influence of diseasesuppressive strains of Streptomyces on the native Streptomyces community in soil as determined by the analysis of cellular fatty acids. Can. J. Microbiol. 42:27–37. 4. Crawford, D. L., J. M. Lynch, J. M. Whipps, and M. A. Ousley. 1993. Isolation and characterization of actinomycete antagonists of a fungal root pathogen. Appl. Environ. Microbiol. 59:3899–3905. 5. Cullum, J., F. Flett, and W. Piendl. 1989. Genetic instability, deletions, and DNA amplification in Streptomyces species, p. 127–132. In C. L. Hershberger, S. W. Queener, and G. Hegeman (ed.), Genetics and molecular biology of industrial microorganisms. American Society for Microbiology, Washington, D.C. 6. de Bruijn, F. J. 1992. Use of repetitive (repetitive extragenic palindromic and enterobacterial repetitive intergenic consensus) sequences and the polymerase chain reaction to fingerprint the genomes of Rhizobium meliloti isolates and other soil bacteria. Appl. Environ. Microbiol. 58:2180–2187. 7. Dicklow, M. B., N. Acosta, and B. M. Zuckerman. 1993. A novel Streptomyces species for controlling plant-parasitic nematodes. J. Chem. Ecol. 19:159–173. 8. Doering-Saad, C., P. Ka ¨mpfer, S. Manulis, G. Kritzman, J. Schneider, J. Zakrzewska-Czerwinska, H. Schrempf, and I. Barash. 1992. Diversity among Streptomyces strains causing potato scab. Appl. Environ. Microbiol. 58:3932– 3940. 9. Faucher, E., T. Savard, and C. Beaulieu. 1992. Characterization of actinomycetes isolated from common scab lesions on potato tubers. Can. J. Plant Pathol. 14:197–202. 10. Healy, F. G., and D. H. Lambert. 1991. Relationships among Streptomyces spp. causing potato scab. Int. J. Syst. Bacteriol. 41:479–482. 11. Hodges, C. F., D. A. Campbell, and N. Christians. 1993. Evaluation of Streptomyces for biocontrol of Bipolaris sorokiniana and Sclerotinia homoeocarpa on the phylloplane of Poa pratensis. J. Phytopathol. 139:103–109. 12. Hollis, A. B., W. E. Kloos, and G. H. Elkan. 1981. DNA:DNA hybridization studies of Rhizobium japonicum and related Rhizobiaceae. J. Gen. Microbiol. 123:215–222. 13. Hulton, C. S. J., C. F. Higgins, and P. M. Sharp. 1991. ERIC sequences: a novel family of repetitive elements in the genomes of Escherichia coli, Salmonella typhimurium and other enteric bacteria. Mol. Microbiol. 5:825–834. 14. Judd, A. K., M. Schneider, M. J. Sadowsky, and F. J. de Bruijn. 1993. Use of repetitive sequences and the polymerase chain reaction technique to classify genetically related Bradyrhizobium japonicum serocluster 123 strains. Appl. Environ. Microbiol. 59:1702–1708. 15. Kampfer, P., R. M. Kroppenstedt, and W. Dott. 1991. A numerical classification of the genera Streptomyces and Streptoverticillium using miniaturized physiological tests. J. Gen. Microbiol. 137:1831–1891. 16. Ku ¨ster, E. 1972. Simple working key for the classification and identification of named taxa included in the International Streptomyces Project. Int. J. Syst. Bacteriol. 22:139–148. 17. Kuykendall, L. D., M. A. Roy, J. J. O’Neill, and T. E. Devine. 1988. Fatty acids, antibiotic resistance, and deoxyribonucleic acid homology groups of Bradyrhizobium japonicum. Int. J. Syst. Bacteriol. 38:358–361. 18. Labeda, D. P., and A. J. Lyons. 1992. DNA relatedness among strains of the sweet potato pathogen Streptomyces ipomoea (Person and Martin 1940) Waksman and Henrici 1948. Appl. Environ. Microbiol. 58:532–535. 19. Lambert, D. H., and R. Loria. 1989. Streptomyces acidiscabies sp. nov. Int. J. Syst. Bacteriol. 39:393–396. 20. Lauer, F. I. 1977. Tubers from leaf-bud cuttings: a tool for potato seed certification and breeding programs. Am. Potato J. 54:457–464. 21. Liu, D., N. A. Anderson, and L. L. Kinkel. 1995. Biological control of potato scab in the field with antagonistic Streptomyces scabies. Phytopathology 85: 827–831. 22. Lupski, J. R., and G. M. Weinstock. 1992. Short, interspersed repetitive DNA sequences in prokaryotic genomes. J. Bacteriol. 174:4525–4529. 23. Martin, B., O. Humbert, M. Camara, E. Guenzi, J. Walker, T. Mitchell, P. Andrew, M. Prudhome, G. Alloing, R. Hakenbeck, D. A. Morrison, G. J. Boulnois, and J.-P. Claverys. 1992. A highly conserved repeated DNA element located in the chromosome of Streptococcus pneumoniae. Nucleic Acids Res. 20:3479–3483. 24. Ndowora, T. C. R., L. L. Kinkel, R. K. Jones, and N. A. Anderson. 1996. Fatty acid analysis of pathogenic and suppressive strains of Streptomyces species isolated in Minnesota. Phytopathology 86:138–143. 25. Paradis, E., C. Goyer, N. C. Hodge, R. Hogue, R. E. Stall, and C. Beaulieu. 1994. Fatty acid and protein profiles of Streptomyces scabies strains isolated in eastern Canada. Int. J. Syst. Bacteriol. 44:561–564. 26. Shirling, E. B., and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16:313–340. 27. Sneath, P. H. A., and R. R. Sokal. 1973. Numerical taxonomy: the principles and practice of numerical classification. W. H. Freeman, San Francisco.

VOL. 62, 1996 28. Stern, M. J., G. F.-L. Ames, N. H. Smith, E. C. Robinson, and C. F. Higgins. 1984. Repetitive extragenic palindromic sequences: a major component of the bacterial genome. Cell 37:1015–1026. 29. Szabo, I. M., M. Marton, I. Buti, and C. Fernandez. 1975. A diagnostic key for the identification of ‘species’ of Streptomyces and Streptoverticillium included in the International Streptomyces Project. Acta Bot. Acad. Sci. Hung. 21:387–418. 30. Versalovic, J., M. Schneider, F. J. de Bruijn, and J. R. Lupski. 1994. Genomic fingerprinting of bacteria using repetitive sequence-based polymer-

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ase chain reaction. Methods Mol. Cell. Biol. 5:25–40. 31. Vidaver, A. K., M. L. Mathys, M. E. Thomas, and M. L. Schuster. 1972. Bacteriocins of the phytopathogens Pseudomonas syringae, P. glycinea, and P. phaseolicola. Can. J. Microbiol. 18:705–713. 32. Williams, S. T., M. Goodfellow, G. Alderson, E. M. H. Wellington, P. H. A. Sneath, and M. J. Sackin. 1983. Numerical classification of Streptomyces and related genera. J. Gen. Microbiol. 129:1743–1813. 33. Woods, S. A., and S. T. Cole. 1990. A family of dispersed repeats in Mycobacterium leprae. Mol. Microbiol. 4:1745–1751.