International Journal of Systematic and Evolutionary Microbiology (2014), 64, 2264–2266
DOI 10.1099/ijs.0.058693-0
Dickeya aquatica sp. nov., isolated from waterways Neil Parkinson,1 Paul DeVos,2 Minna Pirhonen3 and John Elphinstone1 Correspondence
1
Neil Parkinson
2
[email protected]
Food and Environment Research Agency (Fera), Sand Hutton, York, YO41 1LZ, UK BCCM/LMG Bacteria Collection Ghent University Laboratory of Microbiology, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium
3
Department of Agricultural Sciences, PO Box 27, 00014 University of Helsinki, Finland
Pectinolytic Gram-negative bacteria were isolated from different waterways in the UK and Finland. Three strains (174/2T, 181/2 and Dw054) had the same 16S rRNA gene sequences which shared 99 % sequence similarity to species of the genus Dickeya, and a phylogeny of related genera confirmed attribution to this genus. Fatty acid profile analysis of all three strains found a high proportion of C16 : 1v7c/C16 : 1v7c and C16 : 0 fatty acids, and library profile searches found closest matches to Dickeya chrysanthemi. Production of a concatenated phylogeny using six loci, recA, gapA, atpD, gyrB, infB and rpoB, provided a high-resolution phylogeny which placed strains 174/2T and 181/2 as a distinct clade, separated from the other species of the genus Dickeya by a relatively long branch-length. DNA–DNA hybridization analysis with a limited number of reference species also supported the distinctiveness of strains 174/2T and 181/2 within the genus Dickeya. All three strains could be phenotypically distinguished from other species of the genus by fermentation of melibiose and raffinose but not D-arabinose or mannitol. The name Dickeya aquatica sp. nov. is proposed for the new taxon; the type strain is 174/2T (5NCPPB 4580T5LMG 27354T).
The genus Dickeya was first described by Samson et al. (2005), and six species were discriminated as D. dadantii, D. dieffenbachiae, D. chrysanthemi, D. paradisiaca, D. zeae and D. dianthicola. Recently, D. dieffenbachiae has been reported to belong to the same species as D. dadantii and has been reclassified as D. dadantii subsp. dieffenbachiae (Brady et al., 2012). Furthermore, some isolates from European potatoes belonging to the genus Dickeya (Slawiak et al., 2009) have recently been classified as representing a novel species, Dickeya solani, with IPO 2222T as the type strain (van der Wolf et al., 2014). As part of a survey for members of the genus Dickeya, river water was plated onto Crystal Violet Pectate agar (Janse & Ruissen, 1988) and pectolytic colonies were isolated and purified. Dickeya strains were tentatively identified using a Abbreviation: NCPPB, National Collection of Plant Pathogenic Bacteria. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene, dnaJ, recA, gapA, atpD, gyrB, infB and rpoB sequences of Dickeya aquatica sp. nov. 174/2T are JX273704, JX273684, JX273695, KC238447, KC238451, KC238455 and KC238459, respectively; those for the gyrB, infB and rpoB sequences of Dickeya aquatica sp. nov. 181/2 are KC238452, KC238456 and KC238460, respectively. The GenBank/EMBL/DDBJ accession numbers for the dnaJ and gapA sequences of strains of other species of the genus Dickeya determined in this study are listed in Table S1, available in the online Supplementary Material. Two supplementary tables and two supplementary figures are available with the online version of this paper.
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real-time PCR test (Nassar et al., 1996). Alignments and production of the neighbour-joining phylogenies used in the study were done using programs within the MEGA version 4 software package (Tamura et al., 2007). Two English strains (174/2T, 181/2) and a third (Dw054) which was isolated from a Finnish river (Laurila et al., 2008), shared identical recA sequences and were placed as a distinct new clade (identified as SLC2) within this phylogeny. To confirm that the SLC2 isolates belonged to the genus Dickeya, 16S rRNA gene sequences were determined using the protocol described by Nhung et al. (2007) and a phylogeny was produced which included all related genera within the family Enterobacteriaceae (Fig. S1, available in the online Supplementary Material). This phylogeny found the three SLC2 strains shared identical sequences and were placed as a distinct clade relative to all the other related taxa. To further confirm the distinctiveness of SLC2 strains within the genus Dickeya, sequence analysis on strains 174/ 2T and 181/2, and all species of the genus Dickeya with validly published names was done using six loci: recA, (Parkinson et al., 2009) gapA (Brown et al., 2000), atpD, gyrB, infB and rpoB (Brady et al., 2012). A concatenated phylogeny (Fig. S2) was produced and found to be similar to a previously reported phylogeny for species of the genus Dickeya (Brady et al., 2012). Both SLC2 strains (174/2T and 181/2) had identical sequences. This high-resolution phylogeny placed the two SLC2 sequences as a distinct clade separated by a long branch-length from all the other species 058693 G 2014 British Crown Copyright 2014
Printed in Great Britain
Dickeya aquatica sp. nov.
of the genus Dickeya, which provides further evidence for the genetic distinctiveness of SLC2. DNA–DNA hybridization analysis was used to compare strains 174/2T and 181/2 to D. zeae LMG 2505T, D. chrysanthemi LMG 2804T and D. solani IPO 2222T. Hybridization experiments were performed using the method of Ezaki et al. (1989) including the adaptation given by Willems et al. (2001). The hybridization temperature was 46 uC in the presence of 50 % formamide. Reciprocal reactions (e.g. A6B and B6A) were performed for every DNA pair and the variation was within the limits of this method (Goris et al., 1998). All data (Table S2) are mean values of four measurements. The DNA–DNA hybridization data confirms high similarity (approx. 90 %) between SLC2 strains 174/2T and 181/2. Hybridization values of SLC2 to the other Dickeya taxa was 32 % or less. The DNA G+C contents were measured by HPLC as described by Mesbah et al. (1989). The value of 53.65 mol% determined for the SLC2 strains is lower than the reported range of 56.4–59.5 mol% for members of the genus Dickeya (Samson et al., 2005). However, reference to the genome sequence of Dickeya strain Ech586 (GenBank accession no.CP001836) provides a direct sequence-based G+C value of 53.64 mol%, which is very similar to our estimate for the SLC2 strains. Biochemical activities were determined to indicate the phenotypic distinctiveness of SLC2 strains within the genus Dickeya using the GN2 MicroPlate (Biolog), which provides 94 test substrates with metabolism to reduced products indicated by a blue to yellow redox dye colour change. Plates were inoculated according to the manufacturer’s recommendations and incubated at 28 uC, development of a yellow colour was recorded. Metabolism of the following reagents (using strains174/2T, 181 and Dw054) was observed: Tween 40, N-acetyl-D-glucosamine, L-fucose, D-psicose, L-rhamnose, trehalose, citric acid, D-galactonic acid lactone, D-glucosaminic acid, a-ketobutyric acid,
a-ketovaleric acid, propionic acid, glucuronamide,
Dalanine, L-alanyl glycine, L-asparagine, L-leucine, L-phenylalanine, L-pyroglutamic acid and phenylethylamine. Further tests were completed using bromo-cresol purple pH indicator in 96-well microtitre plates according to the method described by Slawiak et al. (2009). The biochemical tests (Table 1) have previously been selected for differential identification of species of the genus Dickeya (Samson et al., 2005; Slawiak et al., 2009; van der Wolf et al., 2014). SLC2 biochemical reactions were determined from all three strains. Casein solubilization was determined by streak inoculation onto nutrient agar containing 10 % skimmed milk powder. Clear zones were recorded after incubation for 48 h at 28 uC. Additional tests used API 20NE strips (bioMe´rieux) according to the manufacturer’s instructions. The results of the biochemical analyses indicated that the SLC2 strains were distinct from all species of the genus Dickeya with validly published names in their ability to metabolize melibiose and raffinose but not D-arabinose or mannitol. Fatty acid profile analysis (Stead et al., 1992) was done on all three SLC2 strains following incubation for 48 h, using a Hewlett Packard chromatography machine (HP6890), as part of the Sherlock Microbial Identification System (MIDI). Protocols were defined by the supplier. All three SLC2 strains contained high proportions (approx. 63 %) of C16 : 1v7c/C16 : 1v7c and C16 : 0, fatty acids. Profile library searches of plant pathogens produced by National Collection of Plant Pathogenic Bacteria (NCPPB), which contained representatives of the genera Erwinia, Pectobacterium, Brenneria, Pantoea, Enterobacter and Serratia, found all three SLC2 strains matched most closely to D. chrysanthemi, further supporting attribution of SLC2 within the genus Dickeya (Weller et al., 2000).
Previous recA analysis of 188 Dickeya strains maintained by the NCPPB (Parkinson et al., 2009), did not find any strains similar to SLC2. Additionally, a Dutch survey of 41
Table 1. Phenotypic differentiation of species of the genus Dickeya based on data from this study (TS) and previous studies (PS) Species (strain numbers for this study and references to previous studies are indicated where applicable): 1, D. aquatica sp. nov. SLC2 (174/2T, 181/2 and DwW054); 2, D. dadantii subsp. dadantii (TS, NCPPB 898T; PS, Samson et al. 2005); 3, D. zeae (TS, NCPPB 2538T; PS, Samson et al. 2005); 4, D. chrysanthemi (TS, NCPPB 402T; PS, Samson et al. 2005); 5, D. dadantii subsp. dieffenbachiae (TS, NCPPB 2976T; PS, Samson et al. 2005); 6, D. dianthicola (TS, NCPPB 453T; PS, Samson et al. 2005); 7, D. paradisiaca (TS, NCPPB 2511T; PS, Samson et al. 2005); 8, D. solani IPO 2222T (van der Wolf et al., 2014; Slawiak et al. 2009 W5weak reaction, NT5not tested)). +, Positive; 2, negative; V, variable; d, different (percentage of positive strains). Characteristic
Growth at 39 uC (2)-D-Arabinose (+)-Melibiose (+)-Raffinose Mannitol b-Gentiobiose Casein
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1
2
3
4
5
6
7
8
TS
PS
TS
PS
TS
PS/TS
PS
TS
PS
TS
PS
TS
PS
+ 2 + + 2 2 +
+ + + + +
+ + + + + + 2
+ + + + + 2 +
+ + + + + 2 +
+ 2 + + + 2 +
+ + 2 2 + + d(80)
+ + 2 2 + + 2
2 2 d(44) d(44) + 2 d(75)
2 2 + + + 2 2
+ + d(83) d(83) 2 + 2
+ + + + 2 + 2
W+
V
+
+ + + + W
NT
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N. Parkinson and others
strains of Erwinia chrysanthemi (Janse & Ruissen, 1988) from potato and ornamental hosts found none with the biochemical profiles matching SLC2. Therefore, waterways are the only known source of SLC2 strains.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric
On the basis of phylogenetic, phenotypic and chemotaxonomic analyses and DNA–DNA hybridization data, the SLC2 strains represent a novel species of the genus Dickeya for which the name Dickeya aquatica sp. nov. is proposed.
Goris, J., Suzuki, K., De Vos, P., Nakase, T. & Kersters, K. (1998). Evaluation of a microplate DNA-DNA hybridization method
Dickeya aquatica (a.qua9ti.ca. L. fem. adj. aquatica living, growing, or found in the water, aquatic). Shares the following characteristics of the genus (Samson et al., 2005). Growth occurs in the presence and absence of oxygen. On nutrient agar, cells are Gram-negative rods 0.5–1.061.0–3.0 mm with rounded ends. Pectinolytic on pectate-layered media. Produces indole and grows at 36 uC. Catabolizes (+)-myo-inositol, D-mannose, malate and saccharate, but not (+)-trehalose, (+)-D-arabitol or sorbitol. 16S rRNA gene phylogenies group strains of D. aquatica with other species of the genus Dickeya, distinct from other genera in the family Enterobacteriaceae. Strains of D. aquatica are differentiated phenotypically from other species of the genus Dickeya by catalysis of melibiose and raffinose but not D-arabinose or mannitol. Casein is solubilized. At 28 uC on potato dextrose agar, colonies are rounded, dry, wrinkled and cream; a diffusible bluish pigment is often present. Sequence comparison of any of the following gene loci: recA, gapA, atpD, gyrB, infB and rpoB can be used to differentiate D. aquatica from other species of the genus. T
compared with the initial renaturation method. Can J Microbiol 44, 1148–1153. Janse, J. D. & Ruissen, M. A. (1988). Characterization and classification of Erwinia chrysanthemi strains from several hosts in The Netherlands. Phytopathology 78, 800–808.
Description of Dickeya aquatica sp. nov.
T
deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Evol Microbiol 39, 224–229.
T
The type strain, 174/2 (5NCPPB 4580 5LMG 27354 ), was isolated from river water. The DNA G+C content of the type strain is 53.65 mol%. Two additional strains of the species are 181/2 and Dw054.
Acknowledgements This research was funded by the Potato Council and DEFRA.
Laurila, J., Ahola, V., Lehtinen, A., Joutsjoki, T., Hannukkala, A., Rahkonen, A. & Pirhonen, M. (2008). Characterization of Dickeya
strains isolated from potato and river water samples in Finland. Eur J Plant Pathol 122, 213–225. Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise
measurement of the G+C content of deoxyribonucleic acid by highperformance liquid chromatography. Int J Syst Bacteriol 39, 159–167. Nassar, A., Darrasse, A., Lemattre, M., Kotoujansky, A., Dervin, C., Vedel, R. & Bertheau, Y. (1996). Characterization of Erwinia
chrysanthemi by pectinolytic isozyme polymorphism and restriction fragment length polymorphism analysis of PCR-amplified fragments of pel genes. Appl Environ Microbiol 62, 2228–2235. Nhung, P. H., Ohkusu, K., Mishima, N., Noda, M., Monir Shah, M., Sun, X., Hayashi, M. & Ezaki, T. (2007). Phylogeny and species identification
of the family Enterobacteriaceae based on dnaJ sequences. Diagn Microbiol Infect Dis 58, 153–161. Parkinson, N., Stead, D., Bew, J., Heeney, J., Tsror (Lahkim), L. & Elphinstone, J. (2009). Dickeya species relatedness and clade structure
determined by comparison of recA sequences. Int J Syst Evol Microbiol 59, 2388–2393. Samson, R., Legendre, J. B., Christen, R., Fischer-Le Saux, M., Achouak, W. & Gardan, L. (2005). Transfer of Pectobacterium
chrysanthemi (Burkholder et al. 1953) Brenner et al. 1973 and Brenneria paradisiaca to the genus Dickeya gen. nov. as Dickeya chrysanthemi comb. nov. and Dickeya paradisiaca comb. nov. and delineation of four novel species, Dickeya dadantii sp. nov., Dickeya dianthicola sp. nov., Dickeya dieffenbachiae sp. nov. and Dickeya zeae sp. nov. Int J Syst Evol Microbiol 55, 1415–1427. Slawiak, M., van Beckhoven, J. R. C. M., Speksnijder, A. G. C. L., Czajkowski, R., Grabe, G. & van der Wolf, J. M. (2009). Biochemical
and genetical analysis reveal a new clade of biovar 3 Dickeya spp. strains isolated from potato in Europe. Eur J Plant Pathol 125, 245–261. Stead, D. E., Sellwood, J. E., Wilson, J. & Viney, I. (1992). Evaluation
References
of a commercial microbial identification system based on fatty acid profiles for rapid, accurate identification of plant pathogenic bacteria. J Appl Microbiol 72, 315–321.
Brady, C. L., Cleenwerck, I., Denman, S., Venter, S. N., Rodrı´guezPalenzuela, P., Coutinho, T. A. & De Vos, P. (2012). Proposal to
Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA4: molecular
reclassify Brenneria quercina (Hildebrand and Schroth 1967) Hauben et al. 1999 into a new genus, Lonsdalea gen. nov., as Lonsdalea quercina comb. nov., descriptions of Lonsdalea quercina subsp. quercina comb. nov., Lonsdalea quercina subsp. iberica subsp. nov. and Lonsdalea quercina subsp. britannica subsp. nov., emendation of the description of the genus Brenneria, reclassification of Dickeya dieffenbachiae as Dickeya dadantii subsp. dieffenbachiae comb. nov., and emendation of the description of Dickeya dadantii. Int J Syst Evol Microbiol 62, 1592– 1602. Brown, E. W., Davis, R. M., Gouk, C. & van der Zwet, T. (2000).
evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24, 1596–1599. van der Wolf, J. M., Nijhuis, E. H., Kowalewska, M. J., Saddler, G. S., Parkinson, N., Elphinstone, J. G., Pritchard, L., Toth, I. K., Lojkowska, E. & other authors (2014). Dickeya solani sp. nov., a pectinolytic
plant-pathogenic bacterium isolated from potato (Solanum tuberosum). Int J Syst Evol Microbiol 64, 768–774. Weller, S. A., Aspin, A. & Stead, D. E. (2000). Classification and
identification of plant-associated bacteria by fatty acid profiling. EPPO Bulletin 30, 375–380.
Phylogenetic relationships of necrogenic Erwinia and Brenneria species as revealed by glyceraldehyde-3-phosphate dehydrogenase gene sequences. Int J Syst Evol Microbiol 50, 2057–2068.
Willems, A., Doignon-Bourcier, F., Goris, J., Coopman, R., de Lajudie, P., De Vos, P. & Gillis, M. (2001). DNA-DNA hybridization study of
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Bradyrhizobium strains. Int J Syst Evol Microbiol 51, 1315–1322.
DOI 10.1099/ijs.0.058693-0
Dickeya aquatica sp. nov., isolated from waterways Neil Parkinson,1 Paul DeVos,2 Minna Pirhonen3 and John Elphinstone1 Correspondence
1
Neil Parkinson
2
[email protected]
Food and Environment Research Agency (Fera), Sand Hutton, York, YO41 1LZ, UK BCCM/LMG Bacteria Collection Ghent University Laboratory of Microbiology, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium
3
Department of Agricultural Sciences, PO Box 27, 00014 University of Helsinki, Finland
Pectinolytic Gram-negative bacteria were isolated from different waterways in the UK and Finland. Three strains (174/2T, 181/2 and Dw054) had the same 16S rRNA gene sequences which shared 99 % sequence similarity to species of the genus Dickeya, and a phylogeny of related genera confirmed attribution to this genus. Fatty acid profile analysis of all three strains found a high proportion of C16 : 1v7c/C16 : 1v7c and C16 : 0 fatty acids, and library profile searches found closest matches to Dickeya chrysanthemi. Production of a concatenated phylogeny using six loci, recA, gapA, atpD, gyrB, infB and rpoB, provided a high-resolution phylogeny which placed strains 174/2T and 181/2 as a distinct clade, separated from the other species of the genus Dickeya by a relatively long branch-length. DNA–DNA hybridization analysis with a limited number of reference species also supported the distinctiveness of strains 174/2T and 181/2 within the genus Dickeya. All three strains could be phenotypically distinguished from other species of the genus by fermentation of melibiose and raffinose but not D-arabinose or mannitol. The name Dickeya aquatica sp. nov. is proposed for the new taxon; the type strain is 174/2T (5NCPPB 4580T5LMG 27354T).
The genus Dickeya was first described by Samson et al. (2005), and six species were discriminated as D. dadantii, D. dieffenbachiae, D. chrysanthemi, D. paradisiaca, D. zeae and D. dianthicola. Recently, D. dieffenbachiae has been reported to belong to the same species as D. dadantii and has been reclassified as D. dadantii subsp. dieffenbachiae (Brady et al., 2012). Furthermore, some isolates from European potatoes belonging to the genus Dickeya (Slawiak et al., 2009) have recently been classified as representing a novel species, Dickeya solani, with IPO 2222T as the type strain (van der Wolf et al., 2014). As part of a survey for members of the genus Dickeya, river water was plated onto Crystal Violet Pectate agar (Janse & Ruissen, 1988) and pectolytic colonies were isolated and purified. Dickeya strains were tentatively identified using a Abbreviation: NCPPB, National Collection of Plant Pathogenic Bacteria. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene, dnaJ, recA, gapA, atpD, gyrB, infB and rpoB sequences of Dickeya aquatica sp. nov. 174/2T are JX273704, JX273684, JX273695, KC238447, KC238451, KC238455 and KC238459, respectively; those for the gyrB, infB and rpoB sequences of Dickeya aquatica sp. nov. 181/2 are KC238452, KC238456 and KC238460, respectively. The GenBank/EMBL/DDBJ accession numbers for the dnaJ and gapA sequences of strains of other species of the genus Dickeya determined in this study are listed in Table S1, available in the online Supplementary Material. Two supplementary tables and two supplementary figures are available with the online version of this paper.
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real-time PCR test (Nassar et al., 1996). Alignments and production of the neighbour-joining phylogenies used in the study were done using programs within the MEGA version 4 software package (Tamura et al., 2007). Two English strains (174/2T, 181/2) and a third (Dw054) which was isolated from a Finnish river (Laurila et al., 2008), shared identical recA sequences and were placed as a distinct new clade (identified as SLC2) within this phylogeny. To confirm that the SLC2 isolates belonged to the genus Dickeya, 16S rRNA gene sequences were determined using the protocol described by Nhung et al. (2007) and a phylogeny was produced which included all related genera within the family Enterobacteriaceae (Fig. S1, available in the online Supplementary Material). This phylogeny found the three SLC2 strains shared identical sequences and were placed as a distinct clade relative to all the other related taxa. To further confirm the distinctiveness of SLC2 strains within the genus Dickeya, sequence analysis on strains 174/ 2T and 181/2, and all species of the genus Dickeya with validly published names was done using six loci: recA, (Parkinson et al., 2009) gapA (Brown et al., 2000), atpD, gyrB, infB and rpoB (Brady et al., 2012). A concatenated phylogeny (Fig. S2) was produced and found to be similar to a previously reported phylogeny for species of the genus Dickeya (Brady et al., 2012). Both SLC2 strains (174/2T and 181/2) had identical sequences. This high-resolution phylogeny placed the two SLC2 sequences as a distinct clade separated by a long branch-length from all the other species 058693 G 2014 British Crown Copyright 2014
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Dickeya aquatica sp. nov.
of the genus Dickeya, which provides further evidence for the genetic distinctiveness of SLC2. DNA–DNA hybridization analysis was used to compare strains 174/2T and 181/2 to D. zeae LMG 2505T, D. chrysanthemi LMG 2804T and D. solani IPO 2222T. Hybridization experiments were performed using the method of Ezaki et al. (1989) including the adaptation given by Willems et al. (2001). The hybridization temperature was 46 uC in the presence of 50 % formamide. Reciprocal reactions (e.g. A6B and B6A) were performed for every DNA pair and the variation was within the limits of this method (Goris et al., 1998). All data (Table S2) are mean values of four measurements. The DNA–DNA hybridization data confirms high similarity (approx. 90 %) between SLC2 strains 174/2T and 181/2. Hybridization values of SLC2 to the other Dickeya taxa was 32 % or less. The DNA G+C contents were measured by HPLC as described by Mesbah et al. (1989). The value of 53.65 mol% determined for the SLC2 strains is lower than the reported range of 56.4–59.5 mol% for members of the genus Dickeya (Samson et al., 2005). However, reference to the genome sequence of Dickeya strain Ech586 (GenBank accession no.CP001836) provides a direct sequence-based G+C value of 53.64 mol%, which is very similar to our estimate for the SLC2 strains. Biochemical activities were determined to indicate the phenotypic distinctiveness of SLC2 strains within the genus Dickeya using the GN2 MicroPlate (Biolog), which provides 94 test substrates with metabolism to reduced products indicated by a blue to yellow redox dye colour change. Plates were inoculated according to the manufacturer’s recommendations and incubated at 28 uC, development of a yellow colour was recorded. Metabolism of the following reagents (using strains174/2T, 181 and Dw054) was observed: Tween 40, N-acetyl-D-glucosamine, L-fucose, D-psicose, L-rhamnose, trehalose, citric acid, D-galactonic acid lactone, D-glucosaminic acid, a-ketobutyric acid,
a-ketovaleric acid, propionic acid, glucuronamide,
Dalanine, L-alanyl glycine, L-asparagine, L-leucine, L-phenylalanine, L-pyroglutamic acid and phenylethylamine. Further tests were completed using bromo-cresol purple pH indicator in 96-well microtitre plates according to the method described by Slawiak et al. (2009). The biochemical tests (Table 1) have previously been selected for differential identification of species of the genus Dickeya (Samson et al., 2005; Slawiak et al., 2009; van der Wolf et al., 2014). SLC2 biochemical reactions were determined from all three strains. Casein solubilization was determined by streak inoculation onto nutrient agar containing 10 % skimmed milk powder. Clear zones were recorded after incubation for 48 h at 28 uC. Additional tests used API 20NE strips (bioMe´rieux) according to the manufacturer’s instructions. The results of the biochemical analyses indicated that the SLC2 strains were distinct from all species of the genus Dickeya with validly published names in their ability to metabolize melibiose and raffinose but not D-arabinose or mannitol. Fatty acid profile analysis (Stead et al., 1992) was done on all three SLC2 strains following incubation for 48 h, using a Hewlett Packard chromatography machine (HP6890), as part of the Sherlock Microbial Identification System (MIDI). Protocols were defined by the supplier. All three SLC2 strains contained high proportions (approx. 63 %) of C16 : 1v7c/C16 : 1v7c and C16 : 0, fatty acids. Profile library searches of plant pathogens produced by National Collection of Plant Pathogenic Bacteria (NCPPB), which contained representatives of the genera Erwinia, Pectobacterium, Brenneria, Pantoea, Enterobacter and Serratia, found all three SLC2 strains matched most closely to D. chrysanthemi, further supporting attribution of SLC2 within the genus Dickeya (Weller et al., 2000).
Previous recA analysis of 188 Dickeya strains maintained by the NCPPB (Parkinson et al., 2009), did not find any strains similar to SLC2. Additionally, a Dutch survey of 41
Table 1. Phenotypic differentiation of species of the genus Dickeya based on data from this study (TS) and previous studies (PS) Species (strain numbers for this study and references to previous studies are indicated where applicable): 1, D. aquatica sp. nov. SLC2 (174/2T, 181/2 and DwW054); 2, D. dadantii subsp. dadantii (TS, NCPPB 898T; PS, Samson et al. 2005); 3, D. zeae (TS, NCPPB 2538T; PS, Samson et al. 2005); 4, D. chrysanthemi (TS, NCPPB 402T; PS, Samson et al. 2005); 5, D. dadantii subsp. dieffenbachiae (TS, NCPPB 2976T; PS, Samson et al. 2005); 6, D. dianthicola (TS, NCPPB 453T; PS, Samson et al. 2005); 7, D. paradisiaca (TS, NCPPB 2511T; PS, Samson et al. 2005); 8, D. solani IPO 2222T (van der Wolf et al., 2014; Slawiak et al. 2009 W5weak reaction, NT5not tested)). +, Positive; 2, negative; V, variable; d, different (percentage of positive strains). Characteristic
Growth at 39 uC (2)-D-Arabinose (+)-Melibiose (+)-Raffinose Mannitol b-Gentiobiose Casein
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2
3
4
5
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8
TS
PS
TS
PS
TS
PS/TS
PS
TS
PS
TS
PS
TS
PS
+ 2 + + 2 2 +
+ + + + +
+ + + + + + 2
+ + + + + 2 +
+ + + + + 2 +
+ 2 + + + 2 +
+ + 2 2 + + d(80)
+ + 2 2 + + 2
2 2 d(44) d(44) + 2 d(75)
2 2 + + + 2 2
+ + d(83) d(83) 2 + 2
+ + + + 2 + 2
W+
V
+
+ + + + W
NT
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N. Parkinson and others
strains of Erwinia chrysanthemi (Janse & Ruissen, 1988) from potato and ornamental hosts found none with the biochemical profiles matching SLC2. Therefore, waterways are the only known source of SLC2 strains.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric
On the basis of phylogenetic, phenotypic and chemotaxonomic analyses and DNA–DNA hybridization data, the SLC2 strains represent a novel species of the genus Dickeya for which the name Dickeya aquatica sp. nov. is proposed.
Goris, J., Suzuki, K., De Vos, P., Nakase, T. & Kersters, K. (1998). Evaluation of a microplate DNA-DNA hybridization method
Dickeya aquatica (a.qua9ti.ca. L. fem. adj. aquatica living, growing, or found in the water, aquatic). Shares the following characteristics of the genus (Samson et al., 2005). Growth occurs in the presence and absence of oxygen. On nutrient agar, cells are Gram-negative rods 0.5–1.061.0–3.0 mm with rounded ends. Pectinolytic on pectate-layered media. Produces indole and grows at 36 uC. Catabolizes (+)-myo-inositol, D-mannose, malate and saccharate, but not (+)-trehalose, (+)-D-arabitol or sorbitol. 16S rRNA gene phylogenies group strains of D. aquatica with other species of the genus Dickeya, distinct from other genera in the family Enterobacteriaceae. Strains of D. aquatica are differentiated phenotypically from other species of the genus Dickeya by catalysis of melibiose and raffinose but not D-arabinose or mannitol. Casein is solubilized. At 28 uC on potato dextrose agar, colonies are rounded, dry, wrinkled and cream; a diffusible bluish pigment is often present. Sequence comparison of any of the following gene loci: recA, gapA, atpD, gyrB, infB and rpoB can be used to differentiate D. aquatica from other species of the genus. T
compared with the initial renaturation method. Can J Microbiol 44, 1148–1153. Janse, J. D. & Ruissen, M. A. (1988). Characterization and classification of Erwinia chrysanthemi strains from several hosts in The Netherlands. Phytopathology 78, 800–808.
Description of Dickeya aquatica sp. nov.
T
deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Evol Microbiol 39, 224–229.
T
The type strain, 174/2 (5NCPPB 4580 5LMG 27354 ), was isolated from river water. The DNA G+C content of the type strain is 53.65 mol%. Two additional strains of the species are 181/2 and Dw054.
Acknowledgements This research was funded by the Potato Council and DEFRA.
Laurila, J., Ahola, V., Lehtinen, A., Joutsjoki, T., Hannukkala, A., Rahkonen, A. & Pirhonen, M. (2008). Characterization of Dickeya
strains isolated from potato and river water samples in Finland. Eur J Plant Pathol 122, 213–225. Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise
measurement of the G+C content of deoxyribonucleic acid by highperformance liquid chromatography. Int J Syst Bacteriol 39, 159–167. Nassar, A., Darrasse, A., Lemattre, M., Kotoujansky, A., Dervin, C., Vedel, R. & Bertheau, Y. (1996). Characterization of Erwinia
chrysanthemi by pectinolytic isozyme polymorphism and restriction fragment length polymorphism analysis of PCR-amplified fragments of pel genes. Appl Environ Microbiol 62, 2228–2235. Nhung, P. H., Ohkusu, K., Mishima, N., Noda, M., Monir Shah, M., Sun, X., Hayashi, M. & Ezaki, T. (2007). Phylogeny and species identification
of the family Enterobacteriaceae based on dnaJ sequences. Diagn Microbiol Infect Dis 58, 153–161. Parkinson, N., Stead, D., Bew, J., Heeney, J., Tsror (Lahkim), L. & Elphinstone, J. (2009). Dickeya species relatedness and clade structure
determined by comparison of recA sequences. Int J Syst Evol Microbiol 59, 2388–2393. Samson, R., Legendre, J. B., Christen, R., Fischer-Le Saux, M., Achouak, W. & Gardan, L. (2005). Transfer of Pectobacterium
chrysanthemi (Burkholder et al. 1953) Brenner et al. 1973 and Brenneria paradisiaca to the genus Dickeya gen. nov. as Dickeya chrysanthemi comb. nov. and Dickeya paradisiaca comb. nov. and delineation of four novel species, Dickeya dadantii sp. nov., Dickeya dianthicola sp. nov., Dickeya dieffenbachiae sp. nov. and Dickeya zeae sp. nov. Int J Syst Evol Microbiol 55, 1415–1427. Slawiak, M., van Beckhoven, J. R. C. M., Speksnijder, A. G. C. L., Czajkowski, R., Grabe, G. & van der Wolf, J. M. (2009). Biochemical
and genetical analysis reveal a new clade of biovar 3 Dickeya spp. strains isolated from potato in Europe. Eur J Plant Pathol 125, 245–261. Stead, D. E., Sellwood, J. E., Wilson, J. & Viney, I. (1992). Evaluation
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