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Pseudomonas Cannabina Pv. Cannabina Pv. Nov., and Pseudomonas Cannabina Pv. Alisalensis (Cintas Koike and Bull, 2000) Comb. Nov

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Pseudomonas Cannabina Pv. Cannabina Pv. Nov., and Pseudomonas Cannabina Pv. Alisalensis (Cintas Koike and Bull, 2000) Comb. Nov ARTICLE IN PRESS Systematic and Applied Microbiology 33 (2010) 105–115 Contents lists available at ScienceDirect Systematic and Applied Microbiology journal homepage: www.elsevier.de/syapm Pseudomonas cannabina pv. cannabina pv. nov., and Pseudomonas cannabina pv. alisalensis (Cintas Koike and Bull, 2000) comb. nov., are members of the emended species Pseudomonas cannabina (ex Suticˇ ˇ & Dowson 1959) Gardan, Shafik, Belouin, Brosch, Grimont & Grimont 1999$ Carolee T. Bull a,n, Charles Manceau b, John Lydon c, Hyesuk Kong c,1, Boris A. Vinatzer d, Marion Fischer-Le Saux b a United States Department of Agriculture, Agricultural Research Service (USDA/ARS), 1636 E. Alisal St., Salinas, CA 93905, United States b INRA UMR077 Pathologie Ve´ge´tale, F-49070 Beaucouze, France c USDA/ARS, Sustainable Agricultural Systems Laboratory, Belstville, MD, 20705-2350, United States d 551 Latham Hall, PPWS Department, Virginia Tech, Blacksburg, VA 24061, United States article info abstract Article history: Sequence similarity in the 16S rDNA gene confirmed that crucifer pathogen Pseudomonas syringae pv. Received 29 September 2009 alisalensis belongs to P. syringae sensu lato. In reciprocal DNA/DNA hybridization experiments, DNA relatedness was high (69–100%) between P. syringae pv. alisalensis strains and the type strain of Keywords: P. cannabina (genomospecies 9). In contrast, DNA relatedness was low (below 48%) between P. syringae Cannabis sativa pv. alisalensis and reference strains from the remaining genomospecies of P. syringae including the type Hops strain of P. syringae and reference strain of genomospecies 3 (P. syringae pv. tomato) although the well- Marijuana known crucifer pathogen, P. syringae pv. maculicola, also belongs to genomospecies 3. Additional Host range evidence that P. syringae pv. alisalensis belongs to P. cannabina was sequence similarity in five gene Brassica rapa fragments used in multilocus sequence typing, as well as similar rep-PCR patterns when using the BOX- Broccoli raab A1R primers. The description of P. cannabina has been emended to include P. syringae pv. alisalensis. Broccoli Pseudomonas syringae pv. maculicola Host range testing demonstrated that P. syringae pv. alisalensis strains, originally isolated from broccoli, Pseudomonas syringae pv. tomato broccoli raab or arugula, were not pathogenic on Cannabis sativa (family Cannabinaceae). Additionally, P. cannabina strains, originally isolated from the C. sativa were not pathogenic on broccoli raab or oat while P. syringae pv. alisalensis strains were pathogenic on these hosts. Distinct host ranges for these two groups indicate that P. cannabina emend. consists of at least two distinct pathovars, P. cannabina pv. cannabina pv. nov., and P. cannabina pv. alisalensis comb. nov. Pseudomonas syringae pv. maculicola strain CFBP 1637 is a member of P. cannabina. Published by Elsevier GmbH. Introduction syringae is further delineated into pathovars (an infrasubspecific designation for phytopathogenically distinct members of a Pseudomonas syringae is a heterogeneous species consisting of species; [18]). A comprehensive genetic analysis grouped plant pathogens and epiphytes with broad pathogenic capabilities many pathovars of P. syringae into nine genomospecies based on and taxonomic characteristics [11,19,22,35,44,55]. Pseudomonas DNA/DNA hybridization and ribotyping [19]. However, only two of the nine genomospecies, P. cannabina and P. tremae,were proposed as authentic species due to a lack of distinguishing Abbreviations: CFBP, Collection Franc-aise de Bacte´ries Phytopathogenes; G1–G9, phenotypic characteristics among the strains in the other genomospecies 1–9; KMB, King’s medium B; LMG, Laboratorium voor Microbiologie genomospecies. University of Gent; LOPAT, levan production, oxidase reaction, potato rot, arginine Pseudomonas syringae pv. maculicola has long been known as dihydrolase production, tobacco hypersensitivity $Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank an important pathogen of crucifers world-wide [30].Itisa databases under the accession number(s): GQ470207-GQ470215; GQ870338- heterogeneous taxon with strains identified as P. syringae GQ870341; GQ859258-GQ859264. pv. maculicola assigned to three different groups by multilocus n Corresponding author. Tel.: +1 831 755 2889; fax: +1 831 755 2814. sequence typing analysis (MLST [35]). The pathotype of P. syringae E-mail address: [email protected] (C.T. Bull). 1 Current address: U.S. Food and Drug Administration, Center for Biologics pv. maculicola was assigned to genomospecies 3 (the P. syringae Evaluation and Research, Rockville, MD 20852, United States pv. tomato group) in the analysis of Gardan et al. [19]. 0723-2020/$ - see front matter Published by Elsevier GmbH. doi:10.1016/j.syapm.2010.02.001 ARTICLE IN PRESS 106 C.T. Bull et al. / Systematic and Applied Microbiology 33 (2010) 105–115 Subsequently a novel P. syringae (sensu lato) isolated from the Materials and methods cruciferous plant broccoli raab (Brassica rapa subsp. rapa) was designated as P. syrinage pv. alisalensis because it is genetically Bacterial strains and media and pathogenically distinct from P. syringae pv. maculicola [14]. Pseudomonas syringae pv. alisalensis has a unique and broad host All strains used in these studies came from the Collection Franc- range including crucifers and monocots, a distinctive rep-PCR aise de Bacte´ries Phytopathogenes (CFBP) or Laboratorium voor pattern, and is uniquely sensitive to a bacteriophage isolated from Microbiologie University of Gent (LMG) unless otherwise stated a diseased broccoli raab field. This pathogen has been isolated (Table 1). The pathotype strain of Pseudomonas syringae pv. from symptomatic plants from disease outbreaks in convention- alisalensis, CFBP 6866Pt (BS91Pt; from broccoli raab, Brassica rapa ally and organically managed crucifer production fields across subsp. rapa [14]), and additional P. syringae pv. alisalensis strains the US [9,10,12,13,25,26]. The host ranges for P. syringae pv. from broccoli (CFBP 6867 and 6873, Brassica olearacea var botrytis maculicola and P. syringae pv. alisalensis overlap [14] and disease [14]) and arugula (CFBP 6869 and 6875, Eruca sativa [9,10])in outbreaks caused by P. syringae pv. alisalensis have been California and New Jersey, USA, were used in these experiments. incorrectly attributed to P. syringae pv. maculicola (Bull et al., Additional strains from more recent disease outbreaks in rutabaga unpublished). (Brassica napus var. napobrassica; CFBP 7253 [26]), Brussels sprouts Although there are phenotypic and pathogenic similarities (Brassica oleracea L. var. gemmifera; CFBP 7254; Bull unpublished), between P. syringae pv. maculicola and P. syringae pv. alisalensis,it cauliflower transplants (Brassica oleracea var. botrytis; CFBP 7251; is not clear whether they are closely related. This study was Bull unpublished), and Romanesca (Brassica oleracea var. botrytis; undertaken to determine if P. syringae pv.maculicolaand P. syringae CFBP 7252 [25]) were included in some analyses. pv. alisalensis belong to the same or different genomospecies and Reference or other representative strains from each of the if not to determine the appropriate taxonomic placement of eight valid genomospecies (G1–G9) proposed by Gardan et al. [19] P. syringae pv. alisalensis. were used as controls in some experiments (G1, Pseudomonas Table 1 Species and Pathovars used in this study. Species and Pathovars Strain Genomospecies 16S rDNA accession Location of Host of origin Source no. isolation Pseudomonas cannabina CFBP G9 AJ492827 Hungary Hemp, Cannabis sativa Klement 1957 2341T Pseudomonas cannabina CFBP G9 GQ870340 Hungary Hemp, Cannabis sativa Klement 1957 1619 Pseudomonas cannabina CFBP G9 GQ470211 Yugoslavia Hemp, Cannabis sativa Dowson 1968 1631 Pseudomonas cannabina LMG 5540 GQ870338 Yugoslavia Hemp, Cannabis sativa Sutic 1958 Pseudomonas cannabina LMG 5650 GQ870339 Hemp, Cannabis sativa Klement 1957 P. viridiflava CFBP G6 Z76671 Switzerland Phaseolus sp. 2107T P. syringae CFBP G1 Z76669 United Kingdom Syringa vulgaris 1392T P. syringae pv. phaseolicola CFBP G2 AB001448 Phaseolus vulgaris Pt Canada 1390 P. syringae pv. tomato CFBP G3 GQ470214 United Kingdom Lycopersicon esculentum 2212Pt Pseudomonas syringae pv. CFBP G3 GQ470210 New Zealand Brassica oleracea var. botrytis maculicola 1657Pt Pseudomonas syringae pv. CFBP Determined GQ470209 CA, USA Radish, Raphanus sativus [51] maculicola 1637 here P. syringae pv. porri CFBP G4 France Allium porrum 1908Pt P. syringae pv. helianthi CFBP G7 GQ870341 Mexico Helianthus annuus 2067Pt P. syringae pv. theae CFBP G8 AB001450 Japan Thea sinensis 2353Pt Pseudomonas syringae pv. CFBP Determined GQ470207 CA, USA Broccoli raab, Brassica rapa subsp. rapa [14] alisalensis 6866Pt here Pseudomonas syringae pv. CFBP Determined GQ470212 CA, USA Broccoli, Brassica olearacea var botrytis [14] alisalensis 6867 here Pseudomonas syringae pv. CFBP Determined GQ470215 CA, USA Arugula [9] alisalensis 6869 here Pseudomonas syringae pv. CFBP Determined GQ470208 NJ, USA Broccoli raab [14] alisalensis 6873 here Pseudomonas syringae pv. CFBP Determined GQ470213 NJ, USA Arugula [9] alisalensis 6875 here Pseudomonas syringae pv. CFBP CA, USA Cauliflower, Brassica oleracea var. botrytis Bull et al., alisalensis 7251 unpublished Pseudomonas syringae pv. CFBP CA, USA Romanesca, Brassica oleracea var. botrytis [24] alisalensis 7252 Pseudomonas syringae pv. CFBP CA, USA Rutabega, Brassica napus var. napobrassica [25]
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