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1 INRA, UMR BiO3P, Domaine de la Motte, BP 35327, F-35653 Le Rheu, France
2 UMR INRA-INH-Université d'Angers PaVé, BP 57, 42 rue G. Morel, F-49071 Beaucouzé, France
3 Ecologie Microbienne, UMR CNRS 5557 Université Claude Bernard Lyon I, F-69622 Villeurbanne Cedex, France
Correspondence
K. Bouchek-Mechiche
karima.bouchek{at}rennes.inra.fr
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ABSTRACT |
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A phenogram showing the relationships between S. turgidiscabies, S. reticuliscabiei and other Streptomyces species and images of potato and radish cultivars infected with S. turgidiscabies and S. reticuliscabiei are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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, b, 1969) as micro-organisms dwelling mostly in the soil, but also in other environments (seawater, sediments, plant surfaces, etc). Almost all are saprophytic and only a few are known to be pathogens. The most important and widespread plant diseases caused by Streptomyces species are potato scabs which appear as superficial or deep lesions of the tuber skin. Two main types of scabs, common scab and netted scab, have been identified and characterized from diseased potato tubers in Europe. Recent data showed that common and netted scab are two separate diseases, differing in the type of symptoms produced, the causal Streptomyces species (Bouchek-Mechiche et al., 2000a), the range of host species affected, the response of potato cultivars to infection and the optimum soil temperature required for disease expression (Bouchek-Mechiche et al., 2000b).
Streptomyces scabiei is the main species isolated from common scab lesions (Lambert & Loria, 1989a). It is prevalent in most potato-growing countries around the world. However, other Streptomyces species, including Streptomyces acidiscabies (Bonde & McIntyre, 1968; Lambert & Loria, 1989b), Streptomyces turgidiscabies (Miyajima et al., 1998), Streptomyces europaeiscabiei, Streptomyces stelliscabiei (Bouchek-Mechiche et al., 2000a), Streptomyces luridiscabiei, Streptomyces puniciscabiei and Streptomyces niveiscabiei (Park et al., 2003) have also been recently described as able to cause common scab. The similarity in symptoms and host range of these species suggests a common mechanism of pathogenicity, even though they are morphologically and genetically distinct. S. scabiei, S. acidiscabies and S. turgidiscabies are known to produce a dipeptide phytotoxin, thaxtomin (Bukhalid et al., 1998; King et al., 1991; Loria et al., 1995) required for disease development (Healy et al., 2000). Recently, Kers et al. (2005) showed that the pathogenicity determinant in pathogenic Streptomyces species involves genes encoding thaxtomin (txtAtxtB, txtC and nos), tomatinase (tomA) and a necrosis protein gene (nec1). These pathogenicity genes are located in a large cluster which corresponds to a pathogenicity island (PAI), which could be transferred horizontally from pathogenic species to saprophytic ones and lead to the emergence of new pathogenic forms. This is the case for S. acidiscabies and S. turgidiscabies, which have emerged independently over recent years in the USA and Japan.
S. reticuliscabiei (Bouchek-Mechiche et al., 2000a) was identified as the main species producing netted scab in France. Thaxtomin A is not associated with the development of netted scab lesions (Loria et al., 1997), suggesting that S. reticuliscabiei might possess other pathogenicity determinants. Previously published data (Bukhalid et al., 2002) showed that S. reticuliscabiei identified in France and S. turgidiscabies identified in Japan have similar 16S rRNA gene sequences. Nevertheless, the pathogenicity characteristics of the two species (Miyajima et al., 1998; Bouchek-Mechiche et al., 2000b) showed clearly that S. turgidiscabies and S. reticuliscabiei cause different diseases (common scab and netted scab, respectively). In order to clarify the phylogenetic relationships between S. reticuliscabiei and S. turgidiscabies, we investigated (i) the phenotypic relatedness of representative strains of the two species, using the 99 carbon sources of Biotype-100 strips (bioMérieux) and the 14 conventional International Streptomyces Project (ISP) tests (Bouchek-Mechiche et al., 1998); (ii) the genomic relatedness of the two species by DNA–DNA hybridization using labelled DNA of S. turgidiscabies SY9113T; (iii) the similarity of complete 16S rRNA gene sequences, based on previously and recently published datasets; (iv) the presence of a nec1 gene homologue as a marker of pathogenicity in S. reticuliscabiei, S. turgidiscabies and in other pathogenic species described in France and (v) a comparison of disease symptoms caused by each species on potato and radish.
This study included three strains of S. reticuliscabiei, CFBP 9530, CFBP 9531T and CFBP 9532, isolated from netted scab lesions in France and two strains of S. turgidiscabies, SY9103 and SY9113T (=ATCC 700248T), isolated from common scab lesions in Japan. An additional S. turgidiscabies strain (Car 8) isolated from carrot in Japan was also included in the pathogenicity tests. Pathogenic strains of S. europaeiscabiei (CFBP 4497T), S. scabiei (ATCC 49173T), S. stelliscabiei (CFBP 4521T) and S. acidiscabies (ATCC 49003T) were used as reference strains. The strains were routinely cultured on yeast extract–malt extract agar (Pridham et al., 1956) and were stored at –20 °C in tryptone–yeast extract broth (Pridham & Gottlieb, 1948) containing 20 % v/v glycerol.
Phenotypic tests were conducted as described by Bouchek-Mechiche et al. (1998). The distance matrix was calculated using the Jaccard coefficient and cluster analysis was performed using the unweighted pair group method with arithmetic means (UPGMA) (Sneath & Sokal, 1973). DNA extraction and DNA–DNA hybridization using labelled DNA from SY9113T were conducted as described elsewhere (Bouchek-Mechiche et al., 2000a). The 16S rRNA gene sequences were either generated in our previous study (Bouchek-Mechiche et al., 2000a) or obtained from the EMBL database. GenBank was scanned for related sequences using the BLAST algorithm (Altschul et al., 1997) and related sequences of representative Streptomyces species were included in subsequent analyses. Sequences were aligned using CLUSTAL_X (Thompson et al., 1997), excluding indel-containing regions. Matrix pairwise comparisons of nucleic acid sequences were corrected for multiple base substitutions with Kimura's two-parameter method (1980) and phylogenetic trees were constructed with the neighbour-joining (Saitou & Nei, 1987) and parsimony (Kluge & Farris, 1969) methods. A bootstrap confidence analysis was performed with 1000 replicates to determine the reliability of the distance tree topologies obtained (Felsenstein, 1985). The resulting trees were graphically represented using NJPLOT and PHYLO_WIN software (Perrière & Gouy, 1996).
The presence or absence of nec1 homologues was assessed in all strains included in this study by amplification of the gene using the specific PCR primers Nf-Nr (Bukhalid et al., 1998). A 720 bp amplicon was detected in strains possessing nec1. PCR was carried out in a final volume of 50 µl containing 50 ng template DNA, reaction buffer (10 mM Tris/HCl, pH 8.3; 1.5 mM MgCl2, 50 µM KCl and 10 % w/v gelatin), 200 mM of each deoxynucleotide triphosphate, 0.2 µM oligomers and 1 U TaqI DNA polymerase (Gibco-BRL). Thirty cycles of amplification (denaturation of DNA at 95 °C for 1 min, annealing at 60 °C for 1 min and elongation at 72 °C for 2 min) were carried out in a Perkin Elmer thermal cycler. The reaction products were then run on 1 % agarose gels. In parallel, a 16S rRNA gene fragment for all of the strains was amplified as described in Bouchek-Mechiche et al. (2000a) with universal primers FGPS5-281 and FGPS1509'-153 (Normand et al., 1996) to check DNA quality. Amplification experiments were repeated twice from two independent cultures for each strain.
The pathogenicity of S. turgidiscabies (SY9113T and Car 8), S. reticuliscabiei (CFBP 9531T and CFBP 4530), S. scabiei (ATCC 49173T) and S. europaeiscabiei (CFBP 4497T) was assessed in a greenhouse as described in Bouchek-Mechiche et al. (2000b) on the potato cultivars Bintje and Désirée (susceptible to both common and netted scabs) and on radish cultivar Polka (susceptible only to common scab). Artificial soil inoculation, pathogenicity testing and potato tuber scoring were performed as described in Bouchek-Mechiche et al. (2000b). Radish disease scoring was performed on a scale of 0 to 5 as described by Wanner (2004).
The phenogram of phenotypic distances allows the delineation of Streptomyces species at a distance of 0.15 (see Supplementary Fig. S1 in IJSEM Online). On this phenogram, the two strains of S. turgidiscabies isolated in Japan from common scab lesions cluster together with the S. reticuliscabiei strains causing netted scab in France (phenon 4). This phenon is fully differentiated from S. scabiei and from other phytopathogenic Streptomyces species causing common scab (S. europaeiscabiei, S. stelliscabiei and S. acidiscabies). S. reticuliscabiei and S. turgidiscabies share almost all the morphological and biochemical characteristics that are important in the identification of Streptomyces species. Both are tyrosinase-negative and characterized by sparse aerial mycelium forming flexuous spore chains and light grey spores. They do not grow in the presence of crystal violet or streptomycin, but use the nine ISP sugars (Shirling & Gottlieb, 1966). They utilize 
-(+)-D-melibiose, mucate, D-saccharate and 5-keto-D-gluconate. They do not assimilate trans-aconitate or ethanolamine (Table 1).
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). The level of 16S rRNA gene sequence similarity between these two species and the other pathogenic strains of Streptomyces tested was relatively low.
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). In addition, a nec1 homologue was successfully amplified from S. europaeiscabiei CFBP 4497T, S. stelliscabiei CFBP 4521T, S. scabiei ATCC 49173T, S. acidiscabies ATCC 49003T and the two strains of S. turgidiscabies, SY9113T and SY9103, using the originally described primers (Nf-Nr). No amplicon was obtained from the three pathogenic strains of S. reticuliscabiei (CFBP 4530, CFBP 4531T or CFBP 4532) (Fig. 2).
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and Supplementary Fig. S2 in IJSEM Online). S. reticuliscabiei strains produces netted scab symptoms on potato which are clearly different from those of common scab (Supplementary Fig. S2). As expected, this species was not pathogenic on radish (Fig. 3 and Supplementary Fig. S2), nor on other root plants (Bouchek-Mechiche et al., 2000b).
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), S. reticuliscabiei and S. turgidiscabies form a homogeneous group and should constitute a single species. However, all the results obtained (in this study and previously) concerning the aetiology of common and netted scab clearly indicate that the two types of scabs are two different diseases according to the symptoms produced on tubers and roots, potato cultivar behaviour, host range and response to soil temperature (Bouchek-Mechiche et al., 2000b). This concept was further strengthened by the amplification of nec1 homologues in S. turgidiscabies strains and in the other common scab species (S. scabiei, S. acidiscabies, S. europaeiscabiei and S. stelliscabiei), but not in S. reticuliscabiei strains. Although PCR amplification of the nec1 gene is not sufficient in itself to make conclusions about pathogenicity determinants, our results are in accordance with previously published data (Bukhalid et al., 1998, 2002) which showed that the nec1 region and its 5'-transposase pseudogene ORFtnp are highly conserved in common scab-causing species (S. scabiei, S. acidiscabies and S. turgidiscabies).
For the pathologist, the fusion of S. reticuliscabiei and S. turgidiscabies under a single species denomination would cause confusion of separate diseases and create a discrepancy between taxonomists and pathologists. Therefore, we think that the two groups should continue to carry their current denominations, i.e. S. reticuliscabiei for the strains inducing netted scab and S. turgidiscabies for those causing common scab. Although this proposal does not fully conform with internationally agreed taxonomic rules, which are based on phenotypic and genomic relatedness, it is in accordance with the pathogenicity characteristics of the two taxa. A similar situation was described recently concerning Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis, which are genetically closely related and should be considered as belonging to one and the same species, but retain separate names because of differences in virulence and pathogenicity due to differences in plasmid-borne genes (Helgason et al., 2000). This issue was discussed in the report of the ad hoc committee for the re-evaluation of the species definition in bacteriology (Stackebrandt et al., 2002) which specifies that the ecological role can, in certain cases, decide on the species status.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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