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 ˇSuticˇ & 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
baUnited States Department of Agriculture, Agricultural Research Service (USDA/ARS), 1636 E. Alisal St., Salinas, CA 93905, United States
bINRA UMR077 Pathologie Ve´ge´tale, F-49070 Beaucouze, France
cUSDA/ARS, Sustainable Agricultural Systems Laboratory, Belstville, MD, 20705-2350, United States
d551 Latham Hall, PPWS Department, Virginia Tech, Blacksburg, VA 24061, United States
a r t i c l e i n f o
Article history:
Received 29 September 2009
Keywords:
Cannabis sativa Hops Marijuana Host range Brassica rapa Broccoli raab Broccoli
Pseudomonas syringaepv.maculicola Pseudomonas syringaepv.tomato
a b s t r a c t
Sequence similarity in the 16S rDNA gene confirmed that crucifer pathogenPseudomonas syringaepv.
alisalensisbelongs 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 P. cannabina(genomospecies 9). In contrast, DNA relatedness was low (below 48%) betweenP. syringae pv.alisalensisand reference strains from the remaining genomospecies ofP. syringaeincluding the type strain ofP. syringaeand reference strain of genomospecies 3 (P. syringaepv.tomato) although the well- known crucifer pathogen, P. syringae pv. maculicola, also belongs to genomospecies 3. Additional evidence thatP. syringaepv.alisalensisbelongs toP. cannabinawas sequence similarity in five gene fragments used in multilocus sequence typing, as well as similar rep-PCR patterns when using the BOX- A1R primers. The description ofP.cannabinahas been emended to includeP. syringaepv.alisalensis.
Host range testing demonstrated thatP. syringaepv.alisalensisstrains, originally isolated from broccoli, broccoli raab or arugula, were not pathogenic onCannabis sativa(family Cannabinaceae). Additionally, P. cannabinastrains, originally isolated from theC. sativawere not pathogenic on broccoli raab or oat whileP. syringaepv.alisalensisstrains were pathogenic on these hosts. Distinct host ranges for these two groups indicate thatP. cannabinaemend. consists of at least two distinct pathovars,P. cannabinapv.
cannabinapv. nov., andP. cannabinapv.alisalensiscomb. nov.Pseudomonas syringaepv.maculicolastrain CFBP 1637 is a member ofP. cannabina.
Published by Elsevier GmbH.
Introduction
Pseudomonas syringaeis a heterogeneous species consisting of plant pathogens and epiphytes with broad pathogenic capabilities and taxonomic characteristics [11,19,22,35,44,55]. Pseudomonas
syringaeis further delineated into pathovars (an infrasubspecific designation for phytopathogenically distinct members of a species; [18]). A comprehensive genetic analysis grouped many pathovars ofP. syringaeinto nine genomospecies based on 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 phenotypic characteristics among the strains in the other genomospecies.
Pseudomonas syringaepv.maculicolahas long been known as an important pathogen of crucifers world-wide [30]. It is a heterogeneous taxon with strains identified as P. syringae pv. maculicola assigned to three different groups by multilocus sequence typing analysis (MLST[35]). The pathotype ofP. syringae pv.maculicolawas assigned to genomospecies 3 (theP. syringae pv. tomato group) in the analysis of Gardan et al. [19].
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Systematic and Applied Microbiology
0723-2020/$ - see front matter Published by Elsevier GmbH.
doi:10.1016/j.syapm.2010.02.001
Abbreviations:CFBP, Collection Franc-aise de Bacte´ries Phytopathogenes; G1–G9, genomospecies 1–9; KMB, King’s medium B; LMG, Laboratorium voor Microbiologie University of Gent; LOPAT, levan production, oxidase reaction, potato rot, arginine dihydrolase production, tobacco hypersensitivity
$Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number(s): GQ470207-GQ470215; GQ870338- GQ870341; GQ859258-GQ859264.
nCorresponding author. Tel.: + 1 831 755 2889; fax: + 1 831 755 2814.
E-mail address:Carolee.Bull@ars.usda.gov (C.T. Bull).
1Current address: U.S. Food and Drug Administration, Center for Biologics Evaluation and Research, Rockville, MD 20852, United States
Subsequently a novel P. syringae (sensu lato) isolated from the cruciferous plant broccoli raab (Brassica rapa subsp. rapa) was designated asP. syrinage pv.alisalensis because it is genetically and pathogenically distinct from P. syringaepv. maculicola[14].
Pseudomonas syringaepv.alisalensishas a unique and broad host range including crucifers and monocots, a distinctive rep-PCR pattern, and is uniquely sensitive to a bacteriophage isolated from a diseased broccoli raab field. This pathogen has been isolated from symptomatic plants from disease outbreaks in convention- ally and organically managed crucifer production fields across the US [9,10,12,13,25,26]. The host ranges for P. syringae pv.
maculicolaandP. syringaepv.alisalensisoverlap[14]and disease outbreaks caused by P. syringae pv. alisalensis have been incorrectly attributed to P. syringae pv. maculicola (Bull et al., unpublished).
Although there are phenotypic and pathogenic similarities betweenP. syringaepv.maculicolaandP. syringaepv.alisalensis, it is not clear whether they are closely related. This study was undertaken to determine ifP. syringaepv. maculicolaandP.syringae pv.alisalensisbelong to the same or different genomospecies and if not to determine the appropriate taxonomic placement of P.syringaepv.alisalensis.
Materials and methods
Bacterial strains and media
All strains used in these studies came from the Collection Franc- aise de Bacte´ries Phytopathogenes (CFBP) or Laboratorium voor Microbiologie University of Gent (LMG) unless otherwise stated (Table 1). The pathotype strain of Pseudomonas syringae pv.
alisalensis,CFBP 6866Pt(BS91Pt; from broccoli raab,Brassica rapa subsp.rapa[14]), and additionalP. syringaepv. alisalensisstrains from broccoli (CFBP 6867 and 6873,Brassica olearaceavarbotrytis [14]) and arugula (CFBP 6869 and 6875, Eruca sativa[9,10]) in California and New Jersey, USA, were used in these experiments.
Additional strains from more recent disease outbreaks in rutabaga (Brassica napusvar.napobrassica; CFBP 7253[26]), Brussels sprouts (Brassica oleraceaL. var.gemmifera; CFBP 7254; Bull unpublished), cauliflower transplants (Brassica oleracea var. botrytis; CFBP 7251;
Bull unpublished), and Romanesca (Brassica oleracea var. botrytis;
CFBP 7252[25]) were included in some analyses.
Reference or other representative strains from each of the eight valid genomospecies (G1–G9) proposed by Gardan et al.[19]
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 no.
Location of isolation
Host of origin Source
Pseudomonas cannabina CFBP 2341T
G9 AJ492827 Hungary Hemp,Cannabis sativa Klement 1957
Pseudomonas cannabina CFBP 1619
G9 GQ870340 Hungary Hemp,Cannabis sativa Klement 1957
Pseudomonas cannabina CFBP 1631
G9 GQ470211 Yugoslavia Hemp,Cannabis sativa Dowson 1968
Pseudomonas cannabina LMG 5540 GQ870338 Yugoslavia Hemp,Cannabis sativa Sutic 1958
Pseudomonas cannabina LMG 5650 GQ870339 Hemp,Cannabis sativa Klement 1957
P. viridiflava CFBP
2107T
G6 Z76671 Switzerland Phaseolussp.
P. syringae CFBP
1392T
G1 Z76669 United Kingdom Syringa vulgaris
P. syringaepv.phaseolicola CFBP 1390Pt
G2 AB001448
Canada Phaseolus vulgaris P. syringaepv.tomato CFBP
2212Pt
G3 GQ470214 United Kingdom Lycopersicon esculentum
Pseudomonas syringaepv.
maculicola
CFBP 1657Pt
G3 GQ470210 New Zealand Brassica oleraceavar. botrytis Pseudomonas syringaepv.
maculicola
CFBP 1637
Determined here
GQ470209 CA, USA Radish,Raphanus sativus [51]
P. syringaepv. porri CFBP 1908Pt
G4 France Allium porrum
P. syringaepv.helianthi CFBP 2067Pt
G7 GQ870341 Mexico Helianthus annuus
P. syringaepv.theae CFBP 2353Pt
G8 AB001450 Japan Thea sinensis
Pseudomonas syringaepv.
alisalensis
CFBP 6866Pt
Determined here
GQ470207 CA, USA Broccoli raab,Brassica rapasubsp.rapa [14]
Pseudomonas syringaepv.
alisalensis
CFBP 6867
Determined here
GQ470212 CA, USA Broccoli,Brassica olearaceavarbotrytis [14]
Pseudomonas syringaepv.
alisalensis
CFBP 6869
Determined here
GQ470215 CA, USA Arugula [9]
Pseudomonas syringaepv.
alisalensis
CFBP 6873
Determined here
GQ470208 NJ, USA Broccoli raab [14]
Pseudomonas syringaepv.
alisalensis
CFBP 6875
Determined here
GQ470213 NJ, USA Arugula [9]
Pseudomonas syringaepv.
alisalensis
CFBP 7251
CA, USA Cauliflower,Brassica oleracea var. botrytis Bull et al., unpublished Pseudomonas syringaepv.
alisalensis
CFBP 7252
CA, USA Romanesca,Brassica oleracea var. botrytis [24]
Pseudomonas syringaepv.
alisalensis
CFBP 7253
CA, USA Rutabega,Brassica napusvar.napobrassica [25]
Pseudomonas syringaepv.
alisalensis
CFBP 7254
CA, USA Brussels sprouts,Brassica oleraceaL. var.
gemmifera
Bull et al., unpublished
syringaeCFBP 1392T; G2, Pseudomonas syringae pv. phaseolicola CFBP 1390Pt; G3,Pseudomonas syringaepv. tomatoCFBP 2212Pt; G4,Pseudomonas syringaepv. porriCFBP 1908Pt; G6,Pseudomonas viridiflava CFBP 2107Pt; G7, Pseudomonas syringae pv. helianthi CFBP 2067Pt; G8,Pseudomonas syringaepv. theaeCFBP 2353Pt; and G9,P. cannabinaCFPB 2341T). The type strain fromP. tremae(G5) was not used in this study, because we have evidence that it belongs to genomospecies 2 (Fischer-LeSaux et al., unpublished data). In some cases additional representatives from an individual genomospecies were included. For example,P. cannabinastrains CFBP 1619, CFBP 1631, LMG 5540, and LMG 5650 andPseudomo- nas syringaepv. maculicolaCFBP 1657Ptand CFBP 1637 (strain B-70 [51]) were included in some analyses. The bacteria were stored at 801C in a solution of 50% glycerol and 50% nutrient broth (NB) and were routinely cultured on King’s medium B agar (KMB[23]).
16S rDNA
For amplification of the 16S rDNA gene, genomic DNA was extracted using DNeasy Tissue Kits (Qiagen, Valencia, CA). An MJ Research DNA-Engine thermo-cycler (MJ Research, Waltham, MA) with a heated lid in the ‘block’ mode was used for all polymerase chain reactions (PCR). Amplicons for the 16S rDNA were generated using universal primers 27F and 1492R [28] using published reaction conditions [50]. After visually checking for amplification by gel electrophoresis, amplicons were purified using Ultra Clean PCR Clean-Up kits (MoBio, Carlsbad, CA).
Amplicons were sequenced directly by an outside vendor (McLab, South San Francisco, CA). Geneious software (http://www.
geneious.com) was used to align sequences from forward and reverse strands, generate sequence alignments and trees and conduct phylogenetic analysis using a neighbour-joining algo- rithm and bootstrapping (n=1000 simulations).
DNA–DNA similarity
Strains representing each of the nine genomospecies defined by Gardan et al.[19]were compared toP. syringaepv.alisalensisin DNA–DNA hybridization experiments. Genomic DNA for DNA–
DNA hybridization experiments was extracted and purified following published methods[7]. Native DNAs fromP. syringae pv.alisalensis strains CFBP 6866Ptand CFBP 6867 and the type strain ofP. cannabina(CFBP 2341T) were labeledin vitroby nick translation with 3H-labeled nucleotides (Amersham) using a previously published S1 nuclease trichloroacetic acid procedure [16]. The reassociation temperature was 701C. Each hybridization was conducted at least three times. DNA relatedness is reported as the average percent reassociation relative to the reassociation of the probe DNA to itself.
Rep-PCR
The BOX-A1R primer, designed to prime DNA synthesis from the boxA subunit of the BOX element, was used in the PCR of repetitive bacterial sequences on purified genomic DNA using published methods [14,34]. Amplified DNA fragments were examined by agarose gel electrophoresis in 1.5% agarose gels in 0.5X Tris acetic acid EDTA buffer, TAE. Gels were stained with ethidium bromide or GelRed and photographed on a UV transilluminator using a digital camera and Kodak Molecular Imaging software (v. 4.5.1, Carestream Health, Inc., Rochester, NY). DNA fragment banding patterns generated from different strains were compared visually.
Multilocus sequence typing (MLST)
Gene fragments of the four locigap1,gltA,gyrB, andrpoDused in the MLST scheme developed by Hwang et al. [22] and a fragment of thekup gene from the MLST scheme developed by Yan et al.[54]were amplified by PCR with primers described in the respective papers and PCR products were sequenced as described in Yan et al. [54]. Sequences were aligned with the allele sequences ofP. syringaepv.tomatofor all loci, cut to size, and single nucleotide polymorphisms (SNPs) were identified using Seqman (DNAstar, Madison, WI, USA). Sequence distances were determined in MegAlign (DNAstar, Madison).
Fatty acid profiles
Strains were grown and extracted for fatty acid methyl ester analysis using a previously published method [14]. Fatty acid methyl esters were analyzed with the Sherlock Microbial Identification System Version 6.1 (MIDI Inc., Newark, DE) using an automated GC 6890 Hewlett-Packard gas chromatograph fitted with a 250.2 mm2phenyl methyl silicone-fused silica capillary column, an HP 7683 automatic sampler, and Agilent ChemStation Software (Ver. B.03.02). The mean and standard deviation of the area for each named peak from three independent replications was reported as a percentage of the total area of all peaks in the chromatogram not including the solvent peak.
Phenotypic characters
The Biotype 100 (bioMerieux, Marcy l’Etoile, France) system was used to determine the ability of the strains to utilize 99 carbon sources. Biotype medium 1 was used to inoculate the cupules of the strips according to manufacturer’s recommenda- tions. The strips were then incubated at 281C and growth was recorded after 2, 4 and 6 days for each cupule. Additional phenotypic characterization included evaluation of fluorescence on KMB, the KOH test to determine Gram character, levan production, oxidase reaction, potato rot, arginine dihydrolase production, tobacco hypersensitivity (LOPAT), reduction of nitrate, hydrolysis of gelatine, DNA and Tween 80, pectinolytic activity at pH 5 and pH 8, acidification of sucrose, sorbitol, erythritol and mannitol, alcalinisation of DL lactate and L( + ) tartrate, sensitivity to bacteriophage PBS1, and ice nucleating ability using published methods[1,14,29,37].
To determine if coronatine biosynthesis genes were present in the pathogens, primers to the cfl gene encoded within the coronatine gene cluster were used to amplify fragments using published methods [57]. DNA from Pseudomonas syringae pv. maculicola (CFBP 1657Pt) andP. syringae pv. syringae (CFBP 1392T) were used, respectively, as the positive and negative controls. To determine if ethylene biosynthesis genes were present in the pathogens, primers to theefegene were used to amplify fragments using published methods [36] with minor modifications. We used purified DNA and a touchdown PCR protocol in which annealing temperatures were 60, 58 and 561C for two cycles each followed by the standard reaction conditions.
Three negative (Pseudomonas syringae pv.syringae CFBP 1392T; P. syrinage pv. maculicola CFBP 1657Pt; Pseudomonas syrinage pv. tomatoCFBP 2212Pt) and one positive control (Pseudomonas cannabinaCFBP 2341T) were used in these experiments. Numer- ical taxonomy analysis and identification of discriminative characters were performed as described by Achouack et al.[1].
Pathogenicity
For pathogenicity tests on Cannabis sativa (L.) (accession 910972 [32]), 24-day-old plants were inoculated. Cells from overnight cultures of bacteria grown in KMB broth were centrifuged, the broth decanted, and the resulting bacterial pellet resuspended in sterile, distilled H2O and adjusted to an OD600 nm
of 0.6. Three milliliters of the bacterial suspension were applied to leaves using a TLC sprayer (Sigma, St. Lewis, MO) at a pressure of 138 Kpa. Plants sprayed with sterile distilled water served as negative controls. Immediately after treatment, plants were placed into plastic bags and held at 231C without light. After 48 h, plants were removed from the plastic bags and grown with light. Plants were evaluated for foliar symptoms at 11 and 21 days after treatment.
Pathogenicity was also evaluated on broccoli (Brassica olear- acea var botrytis cv. ‘Greenbelt’), broccoli raab (Brassica rapa subsp. rapacvs. ‘Spring’ and ‘Sorento’), and oat (Avena sativacv.
‘Montezuma’) seedlings, using previously published methods [14]. The pathogens were grown in nutrient broth with shaking at 200 rpm for 24 h at 271C. Nutrients were removed from cells by centrifugation and cultures were prepared in sterile 0.01 M phosphate buffer as described above except that Tween 20 (0.05%) was added to the suspensions. Each suspension was sprayed until runoff using a hand mister. After inoculation, plants were placed in a humidity chamber for 48 h. Plants were then maintained at 20—251C and evaluated for symptoms after 14 days. For negative controls, plants were sprayed with sterile distilled water amended with Tween 20 (0.05%). An experimental unit was six plants in a six-pack container.
For all pathogenicity tests, lesions or symptomatic tissues from leaves were excised two or three weeks after treatment, surface-disinfested by soaking the leaf tissue in 0.5% sodium hypochlorite for 1 min (with or without a 1 min pretreatment in 70% ethanol) and rinsed with sterile distilled water. The leaf tissue was then macerated in sterile distilled water. The resulting tissue suspensions or dilutions were streaked on KMB supple- mented with boric acid, cephalexin, and cycloheximide [37]
and incubated at 281C. After 4 to 5 days, single colonies were purified, rep-PCR using the BOX-A1R primer performed, and DNA banding patterns were compared to the strains used to inoculate plants. Pathogenicity experiments were conducted at least twice.
The results for pathogenicity tests were recorded as positive for plants from which the appropriate pathogen (as determined by rep-PCR) was isolated from the margins of typical leaf symptoms.
Results
16S rDNA sequences
DNA sequences of 16S rDNA of fiveP. syringaepv. alisalensis strains and of strain CFBP 1637 were obtained and compared to each other and to reference strains. The 1326 nucleotide bases sequenced represent the majority of the primary structure of the 16S rDNA from positions 97 to 1423 based on Escherichia coli nomenclature[8].
The sequences of the 16S rDNA differed slightly among P. syringaepv. alisalensisstrains depending on the hosts (arugula vs. other hosts) from which the strains were isolated but not by region (New Jersey or California). At nucleotide 264 (according to E. coli numbering) the P. syringae pv. alisalensis strains from broccoli raab and broccoli possessed the nucleotide thymine, while the strains from arugula had a cytosine residue at this location, but were otherwise identical. The 16S rDNA sequence of
P. syringae pv. maculicola from radish strain (CFBP 1637) was identical to strains of P. syringae pv. alisalensis from arugula, having a cytosine residue at base 264.
A basic local alignment search tool (BLAST [3]) comparison with sequences in public databases and phylogenetic analysis including type strains of some of the main Pseudomonas phylogenetic clusters previously defined revealed unequivocally that P. syringae pv. alisalensis strains are members of the P. syringae cluster (Fig. 1. [5,33]). The tree presented includes representative strains from each of the genomospecies defined within theP. syringaecluster.
All P. syringae pv. alisalensis strains were nearly identical (99.2%) to the type strains of either P. cannabina (genomo- species 9) or P. syringae (CFBP 1392, genemospecies 1) and (499.9 %) to the pathotype strains ofP. syringaepv. tomatoand P. syringaepv. maculicola, (genomospecies 3). Additionally, there was only moderate or low statistical support (bootstrap values below 60) for separating P. syringae pv. alisalensis from either P. cannabina or P. syringae type strains. Because of the high similarity between all sequences from members of P. syringae sensu latoand low bootstrap values, we were unable to assign P. syringaepv.alisalensisto a genomospecies based on 16S rDNA sequence analysis. All sequences were submitted to Genbank and were given accession numbers GQ470207-GQ470215 and GQ870338-GQ870341.
DNA—DNA hybridizations
Representative strains ofP. syringaepv.alisalensisisolated from different hosts and originating from distant geographic origins (California and New Jersey) belonged to the same genomospecies, with DNA relatedness values ranging from 90% to 100% in experiments using theP. syringaepv.alisalensispathotype (CFBP 6866Pt) as the labeled probe (Table 2).
Hybridization experiments were conducted between selected P. syringaepv.alisalensisstrains and representative strains of the 8 genomospecies defined within theP. syringaecluster. Hybridiza- tion of probes made fromP. syringaepv.alisalensisstrains CFBP 6866Ptor CFBP 6867 toP. cannabinatype strain CFBP 2341T(the reference strain for genomospecies 9) averaged 97% and 100%, respectively, indicating thatP. syringae pv.alisalensisstrains are members ofP. cannabina(genomospecies 9).
In reciprocal experiments usingP. cannabinaCFBP 2341Tas the labeled probe, the percent hybridization to CFBP 6866Ptand CFBP 6867 averaged 81% and 79%, respectively. The average hybridiza- tion of theP. cannabinaprobe to additional strains ofP. syringae pv. alisalensisranged from 69% to 84%. An additional P. syringae strain, CFBP 1637, isolated in the US in 1965 from radish (Raphanus sativus) and previously identified as P. syringae pv. maculicola [51], was evaluated. Strain CFBP 1637 had 83%
DNA relatedness to the probe of the type strain ofP. cannabina.
These data clearly indicated thatP.syringaepv. alisalensisstrains and CFBP 1637 (formerly designatedP. syringaepv.maculicola) are members of genomospecies 9 and should be transferred to P. cannabina.
Moreover, hybridization of CFBP 6867 probe DNA to representative strains of the seven other genomospecies ranged from 34% to 48%. In particular, hybridization of P. syringae pv. alisalensisprobes to the reference strain of genomospecies 3, P. syringae pv. tomato strain CFBP 2212, averaged 42—43%.
This value is far below the threshold value of 70% [47] and indicates that unlike P. syringae pv. maculicola and P. syringae pv. tomato, P. syringae pv. alisalensis is not a member of genomospecies 3.
Rep-PCR patterns
The rep-PCR DNA fragment banding patterns forP. cannabina andP. syringaepv.alisalensisstrains were similar but formed three distinct DNA fragment banding patterns based on the presence or absence of four significant bands (Fig. 2; Table 2). The rep-PCR patterns for the fiveP. cannabinastrains (CFBP 2341T, CFBP 1619, CFBP 1631, LMG 5540, and LMG5650) were identical. Within the nine P. syringae pv. alisalensis strains there were two different banding patterns. The P. syringae pv. alisalensis strains from arugula differ in that they are missing a 1284 kb band that is present in all of the other P. syringae pv. alisalensis and P. cannabina strains and a 1092 kb band that is present in all otherP. syringaepv.alisalensisstrains.Overall, the majority of the bands are shared between P. cannabina and P. syringae pv. alisalensisstrains. However, there is a 799 kb band present in patterns fromP. cannabinabut not in patterns fromP. syringae pv. alisalensis and a 271 kb band that is present in P. syringae pv. alisalensis but not in P. cannabina. Additionally, the P. cannabina strains, like the P. syringae pv. alisalensis from arugula, do not have the 1092 kb band. The rep-PCR patterns for the pathotypes ofP. syringae pv. syringae, P. syringaepv. tomato
andP. syringaepv. maculicolawere significantly different from the patterns forP. cannabinaandP. syringaepv.alisalensisstrains.
MLST
The sequences for the loci gap1, gltA, gyrB, and rpoD were identical for allP. syringaepv.alisalensisstrains (data not shown) while one SNP was found in thekuplocus between the arugula strains and all the other strains tested (Table 2). StrainP. syringae pv.maculicolaCFBP 1637 was identical in all loci to theP. syringae pv. alisalensis strains from arugula. The P. cannabina allele sequences were different for all five loci when compared to the P. syringaepv.alisalensisalleles. However, sequence identity was very high:acnB (98.8%),gap1(99.0%),gyrB(99.9%),kup(95.9%), pgi(99.6%),rpoD(98.3%), andgltA(98.8%). Sequence identity was significantly lower when comparing P. syringae pv. alisalensis alleles to the corresponding alleles of the pathotypes ofP. syringae pv. tomatoand ofP. syringaepv. maculicolaand to the alleles of P. syringae pv. syringae B728a (between 88.7% and 93.4%). All allele sequences have been submitted to Genbank with accession numbers GQ859258 to GQ859264 and have been deposited in the PAMDB database ([4], andwww.pamdb.org).
Fig. 1.Phylogenetic analysis of the 16S rDNA gene of P. syringaepv. alisalensis,P. cannabina, and otherP. syringaestrains. The trees were constructed from evolutionary distance data generated by neighbour joining for 16S rDNA sequences. Numbers by the nodes are bootstrap frequencies expressed as percentages of 1000 replicates (o50%
are not shown). Numbers in parentheses are accession numbers for the sequences used in the analyses. G# refers to the genomospecies published by Gardan et al.[19]to which the bacteria belong. In the cases where the sequence was available, no strain designation is given and all butPseudomonas syringaepv.tomatoDC3000 are from the type (T) or pathotype (Pt) strains.
Analysis of fatty acids
All of the P. cannabina and P. syringae strains evaluated produced 10:0 3OH, 12:0, 12:0 2OH, 12:3OH, 16:0, 16:1
o
7c, 18:0, and 18:1o
7c fatty acids (Table 3). All strains evaluated except the two P. cannabina strains also produced 18:1o
7c 11-methyl. Additionally,P. syringaepv. syringae, and P. syringae pv. maculicola produced 17:0 iso which was not produced by P. cannabinaorP. syringaepv. alisalensis.Trace amounts of 17:0 iso were produced in each of three independent analyses of P. syringaepv. tomatostrain CFBP 2212.Phenotypic analysis
Phenotypic characters for the strains evaluated are given in Table 4, Supplemental Table 1 and in species and pathovar descriptions. AllP. cannabinaandP. syringaepvalisalensisstrains were KOH positive, i.e. Gram negative, and produced a hypersensitive response on tobacco. The strains were oxidase and arginine dihydrolase negative and did not rot potato slices, indicating that they all belonged to Lelliot’s LOPAT group 1[29].
Levan production was variable for individual P. syringae pv. alisalensis strains, whereas levan was consistently positive Table 2
DNA–DNA homology, MLST for thekuplocus and rep-PCR pattern types forPseudomonas syringaestrains and related species. species.
Target DNA or test organism Genomospecies CFBP number DNA–DNA Hybridization (percent) Source of probe DNA Additional genetic characterization P. syringaepv.alisalensis P. cannabina MLSTkupallelea Rep-PCR typeb
CFBP 6867 CFBP 6866 CFBP 2341
P. syringaepv. syringae G1c 1392Pt 41 NTd NT NT NT
P. syringaepv. phaseolicola G2 1390Pt 48 NT NT NT NT
P. syringaepv. tomato G3 2212Pt 43 42 NT NT NT
P. syringaepv. porri G4 1908Pt 48 NT NT NT NT
P. viridiflava G6 2107Pt 35 NT NT NT NT
P. syringaepv. helianthi G7 2067Pt 43 NT NT NT NT
P. syringaepv.theae G8 2353Pt 34 NT NT NT NT
P. cannabina G9 2341T 97 100 100 17 3
P. syringaepv. alisalensis 6866Pt 100 100 81 18 1
P. syringaepv. alisalensis 6867 100 90 79 18 1
P. syringaepv. alisalensis 6869 NT 94 69 19 2
P. syringaepv.alisalensis 6873 NT 100 84 18 1
P. syringaepv.alisalensis 6875 NT 93 73 19 2
P. syringaepv.alisalensis 7251 NT NT NT 18 1
P. syringaepv.alisalensis 7252 NT NT NT 18 1
P. syringaepv.alisalensis 7253 NT NT NT 18 1
P. syringaepv.alisalensis 7254 NT NT NT 18 1
P. syringaepv.maculicola 1637 NT NT 83 19 2
aAllele sequences for thekuplocus were deposited in the PAMDB database ([4], andwww.pamdb.org).
bRep-PCR type refers to the corresponding rep-PCR fingerprint pattern (Fig. 2).
cG1—9 designate reference strains or representative strains of genomonspecies delineated by Gardan et al.[19].
dNT= not tested.
Band sizes (kb)
3054
2036
1636
1018
517, 506
396344298
220
1 KB ladder
P. cannabina LMG 5650 P. cannabina LMG 5540 P. cannabina CFBP 1619 P. cannabina CFBP 1631 P. cannabina CFBP 2341T
P. syringae pv. alisalensis CFBP 6866Pt P. syringae pv. alisalensis CFBP 6867 P. syringae pv. alisalensis CFBP 6873 P. syringae pv. alisalensis CFBP 7251 P. syringae pv. alisalensis CFBP 7252 P. syringae pv. alisalensis CFBP 7253 P. syringae pv. alisalensis CFBP 7254 P. syringae pv. alisalensis CFBP 6869 P. syringae pv. alisalensis CFBP 6875 P. syringae pv. maculicola CFBP 1637 P. syringae pv.maculicola CFBP 1657Pt P. syringae pv. tomato CFBP 2212Pt P. syringae CFBP 1392T
Fig. 2.Rep-PCR fragment banding patterns obtained using the BOX-A1R primers.
for P. cannabina strains as well as the P. syringae pv. syringae, P. syringaepv. tomato andP. syringaepv. maculicolapathotypes.
Additional phenotypic traits common to bothP. cannabina and P. syringaepv.alisalensisare given in the emended description of the P. cannabina species. Among them presence of ethylene production genes is a common trait amongP. cannabinaemend.
within the studied collection. Seventeen carbon sources evaluated by the Biotype 100 system were assimilated by bothP. cannabina and P. syringae pv. alisalensis strains tested, whereas 50 other carbon sources did not allow growth of these strains (Supplemental Table 1).
Pseudomonas cannabinaemend. (including pathogens ofCan- nabis sativa and crucifers, i.e. P. cannabina and P. syringae
pv.alisalensis) can be differentiated from P. syringaestrain CFBP 1392T (G1) by the presence of coronatine production genes, hydrolysis of Tween 80, lack of gelatinase and lack of assimilation of trans-aconitate, DL-glycerate, i-erythritol, DL-lactate and
DL-beta-hydroxybutyrate.Pseudomonas cannabinaemend. can be differentiated fromP. syringaepv.maculicolastrain CFBP 1657Pt and P. syringae pv. tomato strain CFBP 2212Pt (G3) by ice nucleation ability and lack of assimilation of meso-tartrate, trans-aconitate, andDL-glycerate.
TheP. cannabinastrains tested (CFBP 2341T, CFBP 1619, CFBP 1631, LMG 5540, and LMG 5650) reacted identically in 89.5% of the 124 phenotypic tests conducted. The type strain CFBP 2341T differed from at least one of the other P. cannabina strains for Table 3
Fatty acids fromP. cannabina, P. syringaepv. alisalensis, and related strains.
Feature Bacterial strains
Pseudomonas cannabina Pseudomonas syringae
pv.cannabina pv.alisalensis pv.tomato pv.syringae maculicola
CFBP 1631 CFBP 2341T CFBP 6866Pt CFBP 6867 CFBP 6869 CFBP 6873 CFBP 6875 CFBP 2212Pt CFBP 1392T CFBP 1657Pt 10:0 3OH 2.7370.66 2.5870.30 4.3670.23 4.3070.41 4.3370.50 4.1570.39 4.2870.65 4.0070.3 2.7570.29 4.1570.21 12:0 4.2370.27 4.3070.09 4.4570.27 4.3770.10 4.3070.03 4.2970.12 4.4470.06 4.2870.07 4.5870.13 4.5570.21 12:0 2OH 2.6470.15 2.7570.15 2.6670.12 2.6370.19 2.7770.11 2.6770.13 2.9170.09 2.3870.05 2.8870.10 2.5670.23 12:0 3OH 3.3670.53 3.5570.23 3.9470.34 3.8070.52 3.9870.32 3.6970.33 3.9370.25 3.6070.25 3.9570.35 3.9270.49
14:0 Tra Tr Tr Tr – 0.3270.02 Tr Tr Tr Tr
16:0 25.972.66 25.672.51 27.970.97 26.672.09 24.271.22 28.271.39 24.871.33 26.371.74 26.471.47 25.571.48
17:0 iso – – – – – – – Trb 0.4370.08 0.2670.05
18:0 0.8470.03 1.0070.00 1.0570.13 0.6570.23 1.0770.31 0.6970.20 0.9570.29 1.3570.23 1.5170.15 1.0770.42 18:1o7c 11-methyl – – 1.6270.01 0.8270.29 0.5970.54 1.2370.22 0.9270.41 0.6470.09 1.1370.15 0.6570.11 18:1o7c 21.2371.87 21.871.89 13.870.50 15.670.92 18.970.43 14.070.16 18.070.11 18.970.87 20.670.76 18.570.76 Summed Feature 3
(16:1o7c/16:1o6c)
38.870.60 38.470.07 39.871.17 41.070.79 39.4071.27 40.5370.90 39.7470.98 38.070.58 35.271.15 38.671.61 Summed Feature 8
(18:1o7c/18:1o6c)
21.2371.87 21.871.89 13.870.50 15.670.92 18.970.43 14.070.16 18.070.11 18.970.87 20.670.76 18.570.76
aTr, trace amounts detected in at least one of three assays.
bFor this strain, trace amounts of 17:0 iso were produced in each of three independent analyses of CFBP 2212.
Table 4
Characteristics relevant for distinguishingPseudomonas cannabina emend.and its pathovars from closely relatedP. syringaepathovars.
Species P. cannabinaemend. P. syringae
Genomospecies G9a G3 G1
Pathovars P. cannabinapv.cannabina P. cannabinapv.alisalensis pv.
tomato pv.
maculicola pv.
syringae
Phenotypic characters CFBP
2341T CFBP 1619
CFBP 1631
LMG 5540
LMG 5650
CFBP 6866Pt
CFBP 6867
CFBP 6869
CFBP 6873
CFBP 6875
CFBP 1637
CFBP 2212Pt
CFBP 1657Pt
CFBP 1392T
Ethylene production genes ++b ++ ++ ++ ++ + + + + + +
Coronatine production genes + + + + + + + + + + + + +
Brown pigment on KMB + + + + +
Sensitivity to PBS1 + + + + + NTc
Pathogenicity on
Broccoli raaba, Brassica rapa subsp.
rapa, cv. ‘Spring’ or ‘Sorento’
d NT NT NT +e + + + + NT
Oat, Avena sativa,cv. ‘Montezuma’ NT NT NT + + + + + NT
Broccoli, Brassica olearacea var botrytis, cv. ‘Greenbelt’
NT NT NT NT NT + + + + + NT +
Cannnabis sativa, accession 91097 + + NT NT NT NT NT NT NT
aG1, G3, and G9 refer to genomonspecies as delineated by Gardan et al.[19].
bWhile the bands present forP. syringaepv.alisalensisstrains (+ ) clearly indicated the presence of ethylene production genes as specified by theethprimers of Sato et al.[36], significantly more product was produced in reactions containingP. cannabinastrains (++). No amplicons ( ) were detected among the other strains.
cNT= not tested.
dThe data represent uniform results from three replications in at least two independent experiments.
eIdentity of the pathogens associated with symptoms were verified after reisolation by rep-PCR.
assimilation of esculine,L( + ) arabinose,D( + ) malate,D-galactur- onate,D-alanine,D( +)-xylose,D-tagatose, betain,DL-alpha-amino- n-butyrate, succinate, fumarate, and malonate. Additionally, the reactions varied for polypectate hydrolysis at pH 5.
Among the 99 carbon sources tested with the biotype 100, Pseudomonas syringae pv. alisalensis CFBP 6866Pt was able to assimilate 41 carbon sources whereas P. cannabina CFBP 2341T was only able to assimilate 22 carbon sources. Overall, these two strains differed by 28 characters (23 biotype 100 tests and 5 other tests). In contrast,P. syringaepv. alisalensisCFBP 6866Ptdiffered fromP. syringaepv.maculicolaCFBP 1657Ptin only 13 phenotypes (9 biotype 100 tests and 4 other tests). Differences in the assimilation of numerous carbon sources can differentiate these two pathogens.
Characters that differentiateP. cannabinaand P. syringae pv.
alisalensisare given in pathovar descriptions and inTable 4. All five strains of P. cannabina produce a brown pigment on KMB which is not produced by strains ofP. syringaepv. alisalensisor otherP. syringaestrains evaluated. Of the organisms tested, only strains ofP. syringaepv. alisalensis(both broccoli raab and arugula strains) were sensitive to the bacteriophage PBS1 (Table 4). None of thePseudomonas cannabina strains evaluated, nor pathotype strains of P. syringae pv. syringae, P. syringae pv. tomato, and P. syringaepv. maculicolawere sensitive to the PBS1.
Pathogenicity
The host range ofPseudomonas cannabinastrain CFBP 2341T was significantly different from the host range ofP. syringaepv.
alisalensis (Table 4, Supplemental Fig. 1). Whereas strain CFBP 2341Tcaused multiple 1–2 mm necrotic lesions with yellow halos on all inoculated leaves ofCannabis sativa,as verified by rep-PCR, no disease was apparent on leaves inoculated withP. syringaepv.
alisalensisCFBP 6866Pt. LikewiseP. syringaepv. alisalensisstrains CFBP 6867, CFBP 6869, CFBP 6873, and CFBP 6875 were not pathogenic toC. sativain the two experiments conducted.
AllP. syringaepv.alisalensisstrains cause a bacterial blight on broccoli raab (cv ‘Sorento’ and cv ‘Spring’) and individual small lesions on oats (cv ‘Montezuma’; Table 4, Supplemental Fig. 1 [14]). Minor symptoms including diffuse yellowing and small lesions were occasionally detected on broccoli raab or oat plants inoculated withP. cannabinastrains (CFBP 2341Tand CFBP 1631), but P. cannabina was never isolated from these symptoms and was therefore not considered pathogenic on these hosts. None of the pathotype strains of P. syringae pv. syringae, P. syringae pv. tomato, and P. syringaepv. maculicola caused symptoms on broccoli raab or oats[14].
Discussion
Analysis of 16S rDNA sequences is useful for the development of evolutionary inferences at the genus level but has proven to be less useful for the resolution of relationships at the species and pathovar levels in the genusPseudomonas[33,53]. Specifically, the limited sequence variation in the 16S rDNA gene among species and pathovars within theP. syringaegroup of Anzai et al.[5]and rRNA group I of Palleroni [17]) is not particularly useful for resolving the relationships among these taxa[53]. Reliance on a single locus or single method for deducing phylogeny is less sound than analysis of multiple types of data and proposed phylogenetic relationships among pseudomonads can be signifi- cantly different depending on the genes or regions used to infer the relationships [2,53]. Moreover, 16S rDNA has less resolving power than eitherrpoD orgyrB[53]. In this study, the 16S rDNA sequences of P. syringae pv. alisalensis clearly fell within the
P. syringaeand rRNA I groups. However, the 16S rDNA analysis failed to clearly resolve the relationships betweenP. syringaepv.
alisalensis and other organisms in this group. Ribotyping or sequencing of the 16S—23S gene cluster may have been more useful in resolving these relationships [19,27]. Strains within a single species rarely differ in their 16S rDNA sequences by more than 1.3%. The 16S rDNA sequences ofP. syringaepv.alisalensis andP. cannabina, differed by only 0.8%. Although these data would have been insufficient to propose the transfer of P. syringae pv.alisalensistoP. cannabina, they do not contradict it.
The genetic data presented here clearly indicate that the crucifer pathogen P. syringae pv. alisalensis is a member of P. cannabina. DNA/DNA hybridization levels were consistently greater than 70% in pair-wise and reciprocal comparisons between P. syringae pv. alisalensis and P. cannabina. In all but one case, in which the hybridization level was 69%, the average hybridization levels were above the convention of 70% or greater for delineating bacterial species [39,47]. The 70 % DNA/DNA hybridization threshold was among the criteria used by Gardan et al.[19]to support the elevation ofP. syringaepv.cannabinato P. cannabina.
Additional genetic data support the transfer of P. syringae pv. alisalensis to P. cannabina. MLST is particularly useful in understanding phylogeny because genetic distance can be analyzed based on several independent loci. The sequences of the genes used in MLST ofP. syringaepv.alisalensisstrains and P. cannabinawere very similar ranging from 95.9% (kup) to 99.9 % (gyrB) DNA identity. Specifically, the DNA identity of 99.9% in the gyrBfragment corresponds well to relationships determined by DNA/DNA hybridization [45,52]. Among species in the Bacillus subtilisgroup,gyrBsequences of 95.5–100% identity corresponded to DNA/DNA hybridization levels of 70–100%[45].
In addition to sequence data, relationships among rep-PCR DNA fragment banding patterns forP. cannabinaand P. syringae pv.alisalensisindicated that these organisms are more similar to each other than to other members ofP. syringae. Three distinct but similar rep-PCR DNA fragment banding patterns were obtained forP. cannabinastrains andP. syringaepv.alisalensisstrains. DNA banding patterns fromP. cannabinaandP. syringaepv.alisalensis strains had little similarity to those ofP. syringae pv. syringae, P. syringae pv. tomato or P. syringae pv. maculicola. The P. cannabinaandP. syringaepv.alisalensisDNA banding patterns corresponded to the MLST sequence types assigned to these strains. Correlation between rep-PCR and MLST are rarely reported. However, similar clades have been reported from analysis of rep-PCR and MLST data forP. syringae pv.avellanae strains from Italy and Greece[38,46]. Moreover, thekuplocus that distinguished strains within P. syringae pv. alisalensis and displayed the lowest percentage of DNA identity between P. cannabinaand P. syringaepv. alisalensisis more diverse than other MLST loci[54]and thus gives an unusually high resolution to differentiate between very similar strains.
Additional genetic data support the transfer ofP. syringaepv.
alisalensis to P. cannabina. Pseudomonas syringae pv. alisalensis grouped withP. cannabinawhen relationships were analyzed by AFLP (Manceau et al., unpublished).
The fatty acid profiles for all strains tested were indicative of fluorescent pseudomonads in subgroup 1a as described by Stead [40]. Subgroup 1a is characterized by profiles having 12:0 2-OH and 12:0 3-OH, and less than 6% 10:0 3-OH. Similar to the findings of others, in this study the fatty acid profiles were not discriminatory at subspecific ranks [40,43] and did not differ- entiateP. cannabina and P. syringae pv. alisalensisfrom related species and pathovars in this study. The fatty acid profiles of the two P. cannabina strains tested (CFBP 2341T and CFBP 1631) lacked 18:1
o
7c 11-methyl, thus differed from P. syringae.Additionally, the absence of 17:0 iso in the profiles ofP. cannabina and P. syringae pv. alisalensis strains distinguished these from P. syringae pv. syringae and P. syringae pv. maculicola which produced 17:0 iso.Pseudomonas syringae pv. tomato also lacked this fatty acid and could not be distinguished from the strains of interest by this criterion.
Pathovars ofP. syringae sensu latoare not yet identifiable by means of routine biochemical tests[6,19]and it was suspected that many of the pathovars were synonyms[11,18,55].Of the nine genomospecies designated from pathovars ofP. syringae, only two genomospecies were elevated to species because they could be clearly distinguished using routine biochemical tests.Pseudomo- nas cannabinawas elevated fromP. syringaepv.cannabinabecause it differed phenotypically from the other genomospecies of P. syringaeby means of not assimilating fumarate,D(+ )-malate, succinate, andDL-lactate. In this study, we tested four additional strains of P. cannabina and confirmed lack of assimilation of
DL-lactate although we found that assimilation of D(+ )-malate, fumarate, and succinate was variable among these strains.
However, P. syringae pv. alisalensis assimilated fumarate, succi- nate, andD(+ )-malate. Although the previous description of the speciesP. cannabinaindicates otherwise,P. cannabinastrains from C. sativa assimilate L(-)malate and D-glucuronate and did not assimilatesL-histidine. Additionally, the inclusion ofPseudomonas syringae pv. alisalensis strains in P. cannabina will significantly expand the number of carbon sources utilized by at least some strains of this pathogen because Pseudomonas syringae pv. alisalensis assimilated 74% more of the carbon sources evaluated thanP. cannabinaassimilated.
Based on the genetic evidence discussed above we propose to transferP. syringaepv.alisalensisfromP. syringaetoP. cannabina and emend the description ofP. cannabina(below).
Pseudomonas cannabina (ex ˇSuticˇ & Dowson 1959) Gardan, Shafik, Belouin, Brosch, Grimont & Grimont 1999 (formerly P. syringaepv.cannabina), the causal agent of bacterial leaf spot and ulcer stripe of hemp was originally isolated from hemp (Cannabis sativa L.) in ex-Yugoslavia in 1955 [41] and subse- quently from Italy, Germany, Hungary, Bulgaria, Rumania and the ex-USSR during the 1950s and 1960s[20,21,24,31]. There have been no new reports of the disease or isolations of the pathogen from this host since the 1960s. Thus, the diversity of strains ofP.
cannabina available for research is limited to the five strains available in publicly accessible culture collections. More recent research has not increased the number of strains available because the strains reported are not available, were misidentified or are one of the five strains available in public culture collections (although alternatively labeled[42,48,49]).
BecauseP. cannabinaandP. syringaepv.alisalensishave been isolated from different hosts, yet are similar based on genetic and phenotypic data, it was important to investigate the host range of these organisms in order to complete the analysis of their pathovar status. Pathovars are ‘‘a set of strains with the same or similar characteristics, differentiated at the infrasubspecific level from other strains of the same species or subspecies on the basis of distinctive pathogenicity to one or more plant hosts’’ according to The International Standards for Naming Pathovars of Phyto- pathogenic Bacteria [11,18,56]. Here we demonstrated that P. cannabina and P. syringae pv. alisalensis have significantly different host ranges.Pseudomonas cannabinacauses leaf spots on C. sativawhereasP. syringaepv.alisalensisdoes not. Additionally, P. syringaepv.alisalensiscauses disease on broccoli raab and oats, whereasP. cannabinadoes not. Neither of the pathogens produced lesions on hops (Bull and Gent, unpublished), confirming the findings of ˇSuticˇ and Dowson [41] that P. cannabina is not a pathogen on hops or other members of the Cannabinaceae.
BecauseP. syringaepv.alisalensishas a significantly different host
range than P. cannabina we propose that the emended P. cannabina be split into the two pathovars P. cannabina pv.cannabinapv. nov. andP. cannabinapv.alisalensiscomb. nov (see descriptions and pathotype strains below).
Prior to the publication by Cintas et al. [14] describing P. syringae pv. alisalensis, all new bacterial diseases on crucifers caused by fluorescent pseudomonads were reported to be caused by P. syringae pv. maculicola. However, Pseudomonas syringae pv. alisalensis has been reported from crucifers across the US [9,10,12,13,25,26]. Distinguishing between these two pathogens is important because leaf symptoms caused by P. syringae pv.alisalensisare more severe than those caused byP. syringaepv.
maculicolaand the significant differences in the host ranges of the pathogens affect disease management strategies involving crop rotation practices[14,15].Additionally,P. syringaepv.alisalensishas not yet been reported from outside the USA, and accurate identification is important to plant protection agencies. Although Pseudomonas syringaepv.alisalensiscan be easily distinguished from Pseudomonas syringae pv. maculicola in standard biochemical and genetic bioassays [14], P. syringae pv. alisalensis is phenotypically more similar to P. syringae pv. maculicola than P. cannabina.
Pseudomonas syringae pv. alisalensis differs from P. syringae pv.
maculicolain only 10.5% of the characters tested whereas it differs fromP. cannabinain 22.6 % of the phenotypic characters (Supple- mental Fig. 2). This helps to explain whyP. syringaepv.alisalensishad not previously been differentiated from P. syringae pv. maculicola [14]. Consequently, some historical strains including CFBP 1637, identified asP. syringaepv.maculicolain the literature, are identical toP. syringaepv.alisalensis(Bull et al., unpublished).
The data from this manuscript support the hypothesis that P. syringaepv.alisalensisis a member ofP. cannabina. Previously, only five strains ofP. cannabinawere accessible in international public culture collections and these strains appear to be identical according to rep-PCR analysis. There are currently 137P. syringae pv.alisalensisstrains from a variety of hosts and locations in the culture collection at the USDA/ARS in Salinas, CA and a representative subset in international public culture collections.
This research significantly expands our understanding of the speciesP. cannabina and evolution within this species. The data also indicate the need for further research to elucidate the phylogenetic relationships between these organisms and geno- mospecies 3 ofP. syringaeas defined by Gardan et al.[19].
Emended description of Pseudomonas cannabina (Gardan et al., 1999).
Pseudomonas cannabina (can.na’bi.na. L. fem. Adj. cannabina pertaining to Cannabis, the generic name of the host plant, Cannabis sativa L.)
Cells are Gram-negative rods that are l.1–3.0
m
m wide3.0–4.0
m
m long and motile by means of one to four polar flagella.Colonies have a grey color and are slightly convex on YPGA and produces a fluorescent pigment on King’s B medium. Metabolism is respiratory. Results of LOPAT tests are + (v), –. –. – and +[29].
Cell wall fatty acids include 12:0 2-OH and 12:0 3-OH, and less than 6% 10:0 3-OH, but 17:0isois absent.Nitrate is not reduced.
Hydrolysis of starch is negative. Arginine test (Thornley), indole production, DNase activity, and gelatin hydrolysis are negative.
The species hydrolyses tween 80, nucleates ice and assimilates
D-glucose, D-fructose, D-galactose, D-mannose, D-ribose, glycerol,
D-saccharate, mucate, L(-)malate, citrate, D-glucuronate, D-gluco- nate, L-aspartate, L-glutamate, L-proline, L-alanine and L-serine.
Assimilation varied or was negative, respectively, for 32 and 50 of the remaining carbon sources of the Biotype 100 strips (bioMe´rieux). These organisms contain genes for the production
