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Applied and Environmental Microbiology, February 2002, p. 738-744, Vol. 68, No. 2
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.2.738-744.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Horizontal Transfer of the Plant Virulence Gene, nec1, and Flanking Sequences among Genetically Distinct Streptomyces Strains in the Diastatochromogenes Cluster

R. A. Bukhalid,1 T. Takeuchi,2 D. Labeda,3 and R. Loria1*

Department of Plant Pathology, Cornell University, Ithaca, New York 14853,1 Department of Plant Pathology, Hokkaido Central Agricultural Experiment Station, Naganuma, Hokkaido 069-1395, Japan,2 Microbial Genomics and Biocatalysis Research, National Agricultural Utilization Research, Agricultural Research Service, US Department of Agriculture, Peoria, Illinois 616043

Received 25 June 2001/ Accepted 27 November 2001


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Evidence for the horizontal transfer of a pathogenicity island (PAI) carrying the virulence gene nec1 and flanking sequences among Streptomyces strains in the Diastatochromogenes cluster is presented. Plant-pathogenic, thaxtomin-producing Streptomyces strains, previously classified as S. scabiei based on the conventionally used phenotypic characteristics, were found to be genetically distinct from the type strain of S. scabiei based on DNA relatedness and 16S rDNA sequence analysis. Pairwise DNA-DNA hybridizations between some of these strains and the S. scabiei type strain were as low as 36%, a value much below what is conventionally accepted for species identity (70%). The sequence of the nec1 gene, however, was identical in all the S. scabiei and S. scabiei-like strains tested, irrespective of their DNA relatedness to the type strain of S. scabiei, their geographic origin, or the isolation host. Furthermore, a 26-kb DNA fragment including and flanking nec1 was also conserved among these strains based on restriction and Southern analyses. These data indicate that the etiology of potato scab is more complex than previously recognized; this result has important implications for potato scab management strategies. Previous research has suggested that horizontal transfer of a PAI was the mechanism for evolution of pathogenicity in S. acidiscabies and S. turgidiscabies, species that lie outside of the Diastatochromogenes cluster. Data presented here support this model and indicate that PAI transfer also has occurred frequently in species closely related to S. scabiei.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Hundreds of soil-dwelling, saprophytic streptomycetes have been described, while relatively few species are known pathogens of plants (see reference 20 for a review); hence, pathogenicity is considered a rare phenotype in the genus Streptomyces. Streptomycetes are filamentous, gram-positive prokaryotes that produce an array of agriculturally and clinically important secondary metabolites. Members of this genus have a complex morphology: spores germinate to produce substrate mycelium from which aerial hyphae arise, which, in turn, fragment into spore chains.

Three well-characterized, genetically diverse species, S. scabiei, S. acidiscabies, and S. turgidiscabies, cause scab symptoms on potato and tap root crops (20). S. scabiei (18) was the first species to be described and occurs worldwide. S. acidiscabies (17) and S. turgidiscabies (22) were first reported as a cause of potato scab in 1977 and 1996, respectively. In contrast to S. scabiei, S. acidiscabies and S. turgidiscabies have very limited geographic distributions, the northeastern United States and the island of Hokkaido in Japan, respectively. The similarity in symptoms and host range of the three pathogenic species suggests a common mechanism of pathogenicity. Members of S. scabiei, S. acidiscabies, and S. turgidiscabies are known to produce a family of dipeptide phytotoxins, thaxtomins (3, 14, 19), which have recently been shown to be required for disease development (10). Although these three species seem to have evolved similar mechanisms for pathogenicity, they are morphologically and genetically distinct, based on DNA-DNA relatedness and ribosomal sequence analysis (9, 22, 33).

There is evidence that S. scabiei is genetically complex relative to S. acidiscabies (9) and S. turgidiscabies (22), in spite of phenotypic uniformity among S. scabiei strains. DNA relatedness studies (9), fatty acid profiles (23, 24), and inhibitory reactions (23) between paired strains all support a model for genetic diversity among strains currently classified in this species. A comprehensive study of biochemical characteristics, DNA-DNA relatedness, and 16S ribosomal DNA (rDNA) sequence analysis placed S. scabiei strains into three genomic species (1, 2). The species names S. europaeiscabiei and S. stelliscabiei were assigned to two of the genomic species, separating the S. scabiei phenetic cluster into three genomic species.

Our long-term research goal is to understand the evolution and molecular mechanisms of plant pathogenicity in the genus Streptomyces. We have previously demonstrated that the virulence gene, nec1, as well as flanking open reading frames have been horizontally transferred between strains of S. scabiei, S. acidiscabies, and S. turgidiscabies (3). Moreover, diversity in the restriction fragment length polymorphism profiles of an IS element, IS 1629, suggests that the direction of horizontal transfer has been from S. scabiei to S. acidiscabies and S. turgidiscabies (8). These data are consistent with the paradigm of horizontal transfer of pathogencity islands (PAIs) among gram-negative plant and animal pathogens; PAIs are clusters of pathogenicity genes that are mobilizable and have evolved through multiple recombination events (6, 7).

This study addresses the role of horizontal gene transfer in the evolution of plant pathogenicity within the Diastatochromogenes cluster. Elesawy and Szabó (4) proposed the assignment of S. scabiei to the Diastatochromogenes cluster, which encompasses S. neyagawaensis, S. bottropensis, S. diastatochromogenes, S. eurythermus, and S. griseosporeus. Lambert and Loria (18) and Healy and Lambert (9) confirmed the placement of S. scabiei in this cluster using morphochemical characteristics and genomic characteristics, respectively, on a larger collection of strains. We evaluated the diversity of strains isolated from the United States, Egypt, South Africa, and Japan that were classified as S. scabiei based on morphology and pathogenicity on the potato. We analyzed 16S rDNA sequence data for all strains and performed DNA-DNA hybridization analysis on a subset of these strains. We also compared the nec1 sequence and evaluated the conservation of 26 kb of flanking sequences among the strains. Our data support the subdivision of S. scabiei into multiple species and document horizontal transfer of nec1 and at least 26 kb of flanking DNA into those species. These data indicate that the etiology of potato scab is more complex than currently recognized (20). Previous research (3, 8) suggested horizontal transfer of a PAI as the mechanism for the recent emergence of pathogenicity in S. acidiscabies and S. turgidiscabies, species that lie outside of the Diastatochromogenes cluster. Data presented here support this model and indicate that PAI transfer also has occurred frequently in species closely related to S. scabiei.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Media and growth conditions.
The International Streptomyces Project (ISP) Medium 2 (ISP2; Difco) and Medium 4 (ISP4; Difco) were used for the routine cultivation of Streptomyces strains. Strains were grown in CRM medium (26) for DNA extractions. Oatmeal broth and oatmeal agar were used to assess thaxtomin A production and to produce inoculum for pathogenicity tests (19). Spore suspensions of all the Streptomyces strains were prepared as previously described (12).

Streptomyces strains and their characterization.
Type strains of S. bottropensis ATCC 25435, S. diastatochromogenes ATCC 12309, and S. neyagawaensis ATCC 27449 were obtained from the American Type Culture Collection. These nonpathogenic species were selected for this study because they belong to the Diastatochromogenes group, which includes S. scabiei, based on morphological characteristics, DNA relatedness, and 16S rDNA sequence homology (9, 18, 33). Pathogenic strains (Table 1) were from a culture collection maintained by the authors and were originally isolated from scab lesions on potato or peanut in South Africa, Japan, Egypt, and the United States. S. acidiscabies strains ATCC 49003T, 84.104, 85.06, and 90.25 and S. turgidiscabies strains ATCC 700249, Hi-C-13, Ko-G-6, Hi-R-15, and Car-8, which have been previously characterized (3), were used in the Southern analysis only. Strains were maintained as spore suspensions in 20% glycerol at -80°C. Morphological and physiological characterization was based on methods of the ISP as described by Shirling and Gottlieb (31).


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  		TABLE 1. Source, phenotypic and genetic characteristics of pathogenic Streptomyces strains and selected nonpathogens

 
Pathogenicity and thaxtomin A production.
Strains isolated from scab lesions were evaluated for pathogenicity on immature potato tubers, as previously described (3). Methods for evaluation of thaxtomin A production by Streptomyces strains in culture media were similar to those used previously (19). Thaxtomins were extracted from the supernatant of oatmeal broth culture and fractionated by thin-layer chromatography. The chloroform-soluble fraction of the culture supernatants was also tested in the potato tuber disk assay for necrogenic activity typical of thaxtomins (19).

Recombinant DNA techniques.
Streptomyces cultures were grown in CRM for 24 to 48 h at 30°C. Total genomic DNA was extracted using a miniprep modification of the procedure of Rao et al. (27) as previously described (3). Purified total genomic DNA was used for Southern analysis and PCR amplification (3).

Southern blot analysis.
Total genomic DNA was digested with BamHI, separated by agarose gel electrophoresis, UV nicked, transferred to Hybond-N+ nylon membranes (Amersham, Little Chalfont, England) using the manufacturer's alkaline transfer method, and hybridized to random-primed [{alpha}-32P]dCTP-labeled DNA probes made using the Prime-It II labeling kit from Stratagene. Prehybridization and hybridization were done at 65°C overnight in HYB-9 buffer (PURGENE; Gentra Systems, Minneapolis, Minn.). The most stringent wash was 0.1x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 0.5% sodium dodecyl sulfate for 15 min at 65°C. Autoradiography was performed with X-OMAT AR X-ray film (Eastman Kodak) at -80°C.

PCR amplification.
The primers used for nec1 and 16S rDNA amplification are listed in Table 2. Amplification for nec1 was carried out as previously described (3). Universal primers 16S-1F and 16S-1R (33) were used for the amplification and sequencing of the 16S rDNA of Streptomyces strains. Primers 16S-2R and 16S-3F were used to sequence the internal 500 bp of the 16S rDNA. Amplification of both the 16S rDNA and nec1 was carried out using Taq polymerase (0.5 U; PE Applied Biosystems) under the following conditions: DNA denaturing at 95°C for 40 s, annealing at 55°C for the 16S rDNA and 60°C for nec1 for 40 s, and elongation at 72°C for 2 min for 30 cycles. The amplified DNA was separated on 1% agarose gels to verify the presence and homogeneity of the 1,500- and 700-bp 16S rDNA and nec1 fragments, respectively. The PCR amplification experiments were repeated twice from two independent cultures for each strain.


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  		TABLE 2. Primers used for the amplification and sequencing of the 16S rDNA and nec1 genes

 
Sequencing and sequence analysis of the 16S rDNA and nec1.
Following amplification, DNA from the PCR reactions was purified using the QIAquick PCR purification columns (QIAGEN). The sequences of both DNA strands of nec1 and the 16S rDNA of the test strains were determined by direct sequencing of the PCR products using the primers used for amplifications in addition to primers 16S-2R and 16S-3F for 16S rDNA (Table 2). DNA was sequenced by the dideoxy-chain termination method with dye terminator labeling using the ABI 377 Automated DNA Sequencer from PE Applied Biosystems (DNA Services, Biotechnology; Cornell University, Ithaca, N.Y.).

Phylogenetic analysis.
MEGALIGN and SEQMAN (DNASTAR Inc., Madison, Wis.) were used for the multiple sequence alignment with the CLUSTAL V method (11) and for sequence assembly and contig management, respectively. 16S rDNA sequences were either generated in this study or obtained from the public nucleotide database at NCBI. Sequences were also compared to the public nucleotide database using the BLAST algorithm to identify 16S rDNA sequences with high ratios of similarity to our sequences. The PAUP 4.0 software, beta version b8 (Sinauer Associates, Inc., D. L. Swofford), was used for computing the matrix-based genetic distances using Jukes and Cantor assumptions (13) and to deduce distance trees using the neighbor-joining method of Saitou and Nei (28). The topology of the resulting unrooted tree was evaluated by carrying out bootstrap analysis of the neighbor-joining tree based on 1,000 resamplings. Phylogenetic trees were computed using 1,433 nucleotides based on the 16S rRNA sequence of S. ambofaciens (from nt 41 to 1475) (25).

DNA-DNA hybridizations.
For hybridization studies, DNA was extracted from cells of strains DNK-G10, DNK-G01, 84.174, and S. scabiei ATCC 49173 by the method of Marmur (21) after breakage by passage through a French pressure cell (SLM Instruments, Inc., Urbana, Ill.) at 10,000 lb/in2. DNA was further purified by cesium chloride density gradient centrifugation. The percentage of DNA relatedness between strains was calculated from C0t0.5 determinations in 5x SSC supplemented with 20% dimethyl sulfoxide at 65°C (Tm - 23°) by the method of Seidler et al. (29) and Seidler and Mandel (30) as described in Kurtzman et al. (15).

Nucleotide sequence accession numbers.
The nucleotide sequences were deposited in GenBank under the accession numbers listed in Table 1 for the 16S rDNA sequences and under the following accession numbers for nec1: AF385166 for 84.102, AF385167 for 84.174, AF385168 for 87.70, AF385169 for 87.76, AF385170 for 87.79, AF385171 for B-4, AF385172 for CEK-037A, AF385173 for DNK-043, AF385174 for DNK-G01, AF385175 for DNK-G02, AF385176 for DNK-G10, AF385177 for E-1, AF385178 for K(D)75173, AF385179 for SBH-3, and AF385180 for SNS-26.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Strain phenotypes.
All strains that were labeled as pathogens were confirmed to be pathogenic on potato tubers and to produce thaxtomin A (Table 1). Nonpathogenic strains included in this study did not produce thaxtomins or cause disease symptoms on potato tubers. Strains were characterized based on the morphological, pigmentation, and carbon source utilization properties specified by the International Streptomyces Project. Six of the pathogenic strains did not produce spiral spore chains typical of S. scabiei (Table 1). Five of these strains (DNK-G10, 84.102, 84.174, DNK-G01,and DNK-G02) were indistinguishable from the type strain S. scabiei ATCC 49173 in all other phenotypic properties, including mycelium color, spore mass color, pigment production, and carbon source utilization (18). These strains produced spore chains with a mixture of terminal hooks, primitive open spirals with only one or two turns, and some flexuous (F) chains, a spore chain type known as retinaculiaperti (RA), which is qualitatively similar to the spiral spore chain type. One of these strains, DNK-G10, produced only F spore chains, distinctly different from the spiral and RA spore chain types. Strain E-1 from Egypt produced spore chains of the RA type in addition to the production of a diffusable orange-brown pigment on YME that was not produced by any other strains.

Three strains (SBH-3, SNS-26, and K(D)75173) from Japan (34) lacked the ability to produce melanoid pigments on either yeast-malt agar or peptone-yeast extract-iron agar but were indistinguishable from the S. scabiei type strain in all other ISP characteristics (Table 1).

Phylogenetic analysis.
Pairwise DNA-DNA hybridization of S. scabiei ATCC 49173T and strains 84.174, DNK-G10,and DNK-G01 were 37, 36, and 50%, respectively (Table 1). These hybridization values are well below the 70% threshold conventionally used to determine whether strains belong to a single genomic species (5, 35).

The 16S rDNA gene from all of the pathogenic strains was PCR amplified, using primers 16S-1F and 16S-1R (Table 2), and sequenced. An almost complete sequence (1,460 bp) was obtained for all the strains. The 16S rDNA sequence alignment (Fig. 1) identified sequence heterogeneity in the {gamma} (around position 190), {alpha} (around position 1000), and around position 1435 but not in the ß (around position 1100) variable regions of the streptomycetes 16S rRNA, as defined by Stackebrandt et al. (32). Diversity in the 16S rDNA sequence among the pathogenic strains was restricted to the variable regions of the gene. The 16S rDNA sequences from seven of the pathogenic strains (group 1 in Table 1) were identical to that of the S. scabiei type strain, whereas the sequences of the strains in group 2 differed in only one nucleotide from that of the type strain (Fig. 1). Interestingly, the 16S rDNA sequences of the four pathogenic strains in group 3 (87.70, 87.76, DNK-G01, and DNK-G02), which originated from South Africa and the United States, were identical to each other and differed from any other 16S rDNA sequence in the public databases. Furthermore, the consensus 16S rDNA sequence from these four strains was more similar to the sequences of the type strains of S. bottropensis (99.0%) and S. diastatochromogenes (98.7%) than to the type strain of S. scabiei (98.1%). Strain E-1 (group 4), originally isolated from potatoes in Egypt, had a 16S rDNA sequence that was unique among the pathogenic strains and identical to that of S. bottropensis (100%).



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  		FIG. 1. Multiple sequence alignment of the 16S rDNA sequences from pathogenic and nonpathogenic Streptomyces strains around the {gamma} and {alpha} variable regions and around position 1435. The nucleotide position on the consensus bar is based on the S. ambofaciens 16S rRNA sequence nomenclature (25). The strains in bold represent the four different groups of pathogenic streptomycetes used in this study.

 
Unrooted phylogenetic trees were computed using 1,433 nucleotides based on the 16S rRNA sequence of S. ambofaciens (from nt 41 to 1475) (25). Potato scab-causing and nonpathogenic species for which 16S rDNA data were available were also included in the analysis. It is clear from the neighbor-joining tree (Fig. 2) that the pathogenic strains from groups 1 and 2 (Table 1) formed a tight cluster with the type strains of S. scabiei and S. europeiascabiei CFBP4497T (1). The five pathogenic strains from groups 3 and 4, on the other hand, did not cluster with S. scabiei. The nearest neighbor to the strains from the 87.70 group was S. stelliscabiei CFBP4521T (1), a recently circumscribed species that causes netted scab in Europe. Strain E-1 and S. bottropensis were monophyletic and clustered with strain 87.70 and S. stelliscabiei (Fig. 2). The potato scab-causing species S. turgidiscabies and S. acidiscabies did not cluster with any of the pathogenic strains included in this study.



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  		FIG. 2. Unrooted neighbor-joining tree based on 1,433 bp of the 16S rDNA sequences of 41 Streptomyces strains. Streptomyces strains that are known to cause plant diseases are indicated by asterisks. Strains that cause the common scab disease of potato and harbor the horizontally transferred PAI are indicated in bold. Each number in parentheses indicates the number of strains represented in that group. The numbers at the nodes indicate the level of bootstrap support (1,000 resamplings). Only values of >40% are given. The length of the bar at the bottom represents 5 nucleotide substitutions.

 
nec1 and flanking DNA sequence homology.
Total genomic DNA from 16 Streptomyces strains included in this study was subjected to Southern analysis using three probes from the nec1 region: an internal fragment of nec1 and two restriction fragments from a 26-kb DNA region upstream of nec1 (Fig. 3). Four S. acidiscabies strains and five S. turgidiscabies strains previously shown to produce thaxtomin A and to be pathogenic on potato tubers (3) were also included in this analysis. DNA from all of the pathogenic strains hybridized to all three probes (Fig. 3A), while DNA from the nonpathogenic strains did not hybridize to any of the probes (data not shown). These data demonstrate that a 26-kb DNA fragment that includes nec1 is present in pathogenic strains but absent from nonpathogenic strains in the Diastatochromogenes cluster (Fig. 2), including nonpathogenic strains that are closely related to S. scabiei. Based on the data in Fig. 3A, it appears that the sequences of the 26-kb DNA region are almost identical in pathogenic strains. Restriction fragment B, which hybridizes to probe 3 (Fig. 3B), is identical in size (13 kb) in all the pathogenic strains. Differences in the size of fragment A (Fig. 3A) among pathogenic strains are the result of the insertion of one copy of IS1629 in some S. scabiei strains and in all the S. turgidiscabies strains (Fig. 3B) or the presence of two copies of IS1629 in the S. acidiscabies strains (8).



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  		FIG. 3. (A) Southern analysis of BamHI-digested total genomic DNA from representative Streptomyces strains hybridized with 32P-labeled nec1, probe 2, and probe 3. (B) Map of the 26-kb DNA fragment depicting the location of the three probes within the BamHI restriction fragments. Plant-pathogenic strains included S. scabiei and related strains (ATCC49173T, CEK-037A, DNK-043, DNK-G10, SNS-26, K(D)75173, 84.102, 87.79, 87.70, 87.76, DNK-G01,and E-1) (lanes a to l), S. acidiscabies strains (ATCC 49003, 84.104, 85.06, and 90.25) (lanes m to p), and S. turgidiscabies strains (ATCC700249, Hi-C-13, Ko-G-6, Hi-R-15, and Car-8) (lanes q to u). Size markers are shown on the left in panel A. Location and size of probes on the 26-kb DNA fragment are represented by brackets. B, BamHI.

 
The level of nucleotide similarity of the nec1 gene among the pathogenic strains was evaluated by sequence analysis. The sequence of nec1 was identical or almost identical in all pathogenic strains, irrespective of their geographic origin, isolation host, or level of DNA relatedness to the S. scabiei type strain (Table 1). The nec1 sequences of two South African strains, DNK-043 and DNK-G10, and one Japanese strain, B-4, each differed in 1 bp from that of the type strain, while the sequence of nec1 in the rest of the pathogenic strains was identical to that of the type strain (Table 1). Interestingly, the single nucleotide substitutions in DNK-G10 and DNK-043 at Arg136 (C_T) and in B-4 at Asn191 (C_T) did not cause a change in the amino acid sequence of nec1 because they both occurred at the third or wobble position of their respective codons.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our previous work with S. scabiei, S. acidiscabies, and S. turgidiscabies demonstrated that the virulence gene, nec1, and a flanking open reading frame (2.4 kb) were horizontally transferred between strains of these Streptomyces species. The phenotype, the low G+C content, and the presence of the nec1 gene only in pathogenic strains suggested that it had been mobilized within a transmissible PAI (3). In this paper, we provide further support for the PAI model as a mechanism for evolution of pathogenicity in genetically diverse Streptomyces species. The heterogeneity of strains classified as S. scabiei is well documented (8, 9, 23, 24) and is in contrast to S. acidiscabies (9), S. turgidiscabies (8, 22), and S. ipomoeae (16), which consist of clonal populations. Our previous research demonstrated that the nec1 sequence is conserved in 22 S. scabiei strains examined (3). Data provided here demonstrate that a DNA fragment, of >26 kb, has been transferred into strains that are in the Diastatochromogenes cluster but are genetically distinguishable from S. scabiei. Specifically, transfer has occurred into groups represented by strains E-1, 87.70, 84.102, and DNK-G10.

Most of the strains investigated here were very similar to S. scabiei based on ISP criteria. However, spore chain type was atypical of S. scabiei in some of them. These strains also were genetically distinct from S. scabiei based on DNA reassociation values or 16S rDNA sequences. The DNA relatedness between ATCC 49173T and strain 84.174 was previously found to be 37% (9), which is consistent with the value that was obtained in this study and excludes this strain from the S. scabiei genomic species. Bacterial genomic species are defined as taxa that encompass strains that share 70% or more DNA-DNA relatedness with a difference of 5% or less in thermal stability ({Delta}Tm, 112), irrespective of their growth requirements or their morphological heterogeneity (5, 35). Two other strains used in this study, 84.102 and 87.79, were previously reported to have DNA reassociation values of 49 and 28%, respectively (9), which also excludes them from the same genomic species as the type strain of S. scabiei. Furthermore, the DNA reassociation values for the S. scabiei type strain and some of these strains are as low as 28% (strain 87.79), which is lower than the values obtained for S. bottropensis (37%) and S. diastatochromogenes (42%) when paired with the S. scabiei type strain (Table 1). Conversely, the DNA homology data previously obtained for the strains originating from Japan (22) show that these strains belong to a single genomic species, along with the type strain of S. scabiei. The results obtained in this study support the separation of S. scabiei strains into at least three distinct species based on DNA relatedness and 16S rDNA sequence as proposed by Boucheck-Mechiche et al. (1).

It is clear from the 16S rDNA phylogenetic analysis that the strains in the 87.70 group (Table 1) form a distinct clade and that they are more closely related to the nonpathogenic species S. bottropensis and S. diastatochromogenes than they are to S. scabiei. Strain E-1 appears to belong to the S. bottropensis phenon, based on 16S rDNA analysis. It has been previously shown that some of the pathogenic strains classified as S. scabiei exhibit species-level DNA relatedness to S. bottropensis (9). The 16S rDNA sequence of the remaining 11 S. scabiei-like strains was identical to that of the type strain, irrespective of their origin, isolation host, or DNA relatedness to the type strain. These strains formed a single clade with the type strain of S. scabiei.

The identity of the nec1 and flanking sequences among closely related but genetically distinct species provides strong evidence for the horizontal rather than vertical transmission of the 26-kb DNA fragment among these strains. These data support a model for relatively recent horizontal transfer of nec1 into multiple strains within the Diastatochromogenes cluster. These data are also consistent with previously published results that demonstrate identity in the nec1 sequence as well as functional conservation of this gene among pathogenic strains in S. acidiscabies, S. turgidiscabies, and S. scabiei (3). In addition, the conserved substitutions in the amino acid sequence of nec1 in strains B-4, DNK-G10, and DNK-043 strongly suggest that nec1 plays a role in the potato-Streptomyces disease interaction. Previous studies indicate that nec1 is not required for pathogenicity or thaxtomin A production, based on evaluation of a naturally occurring S. scabiei {Delta}nec1 strain in laboratory assays (3). The role of nec1 in host-pathogen interactions is under investigation. We have recently cloned a peptide synthase, txtAB, from S. acidiscabies. This synthase is required for thaxtomin A biosynthesis and for pathogenicity (10). Further, txtAB is conserved among strains of S. scabiei and S. turgidiscabies based on Southern analysis. We hypothesize that nec1 and txtAB are located on a transmissible PAI and that transfer of the island has conferred plant pathogenicity on saprophytic strains, including those in the Diastatochromogenes cluster and strains of S. acidiscabies and S. turgidiscabies. It is also evident from the phylogenetic analysis that the frequency of transfer of the PAI among closely related species is higher than the frequency of transfer into distantly related species, such as S. acidiscabies and S. turgidiscabies. It is well known that genetic exchange among closely related taxa is common and is mostly due to the lack of physical barriers such as host restriction and modification systems. If our model is correct, this will be the first description of a PAI in gram-positive plant pathogens and the first evidence of emergence of multiple plant-pathogenic species through the mobilization of a PAI.


   ACKNOWLEDGMENTS
 
We thank R. King for providing strain E-1, R. Gouws for DNK-G02 and DNK-G10, and A. De Klerk for CEK-037A, DNK-043, and DNK-G01.

This work was supported by a United States Department of Agriculture National Research Initiative grant, no. 99-35303-8084.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Plant Pathology, 344 Plant Science Building, Cornell University, Ithaca, NY 14853-4203. Rosemary Loria. Phone: (607) 255-7831. Fax: (607) 255-4471. E-mail: RL21{at}cornell.edu. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bouchek-Mechiche, K., L. Gardan, P. Normand, and B. Jouan. 2000. DNA relatedness among strains of Streptomyces pathogenic to potato in France: description of three new species, S. europaeiscabiei sp. nov. and S. stelliscabiei sp. nov. associated with common scab, and S. reticuliscabiei sp. nov. associated with netted scab. Int. J. Syst. Evol. Microbiol. 50:91-99.[Abstract]
  2. Bouchek-Mechiche, K., C. Guerin, B. Jouan, and L. Gardan. 1998. Streptomyces species isolated from potato scabs in France: numerical analysis of "Biotype-100" carbon source assimilation data. Res. Microbiol. 149:653-663.[Medline]
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Applied and Environmental Microbiology, February 2002, p. 738-744, Vol. 68, No. 2
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.2.738-744.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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