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Keywords:

  • multilocus sequence typing;
  • nutritional characterization;
  • pathogenicity;
  • pea bacterial blight;
  • Pisum sativum;
  • rep-PCR

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Pseudomonas syringae pv. pisi is a seedborne pathogen distributed worldwide that causes pea bacterial blight. Previous characterization of this pathogen has been carried out with relatively small and/or geographically limited samples. Here, a collection of 91 strains are examined that include strains from recent outbreaks in Spain (53 strains) and from 14 other countries, and that represent all races and the new race 8, including the type race strains. This collection was characterized on the basis of 55 nutritional tests, genetic analysis (rep-PCR, amplification of AN3 and AN7 specific markers, and multilocus sequence typing (MLST)) and pathogenicity on the differential pea cultivars to identify races. Principal component analysis and distance dendrograms confirm the existence of two genetic lineages within this pathovar, which are clearly discriminated by the AN3/AN7 markers, rep-PCR and MLST. Strains from races 1 and 7 amplified the AN3 marker; those from races 2, 6 and 8 amplified AN7, while strains of races 3, 4 and 5 amplified either AN3 or AN7. Nevertheless, strains were not grouped by race type by any of the genetic or biochemical tests. Likewise, there was no significant association between metabolic and/or genetic profiling and the geographical origin of the strains. The Spanish collection diversity reflects the variability found in the worldwide collection, suggesting multiple introductions of the bacteria into Spain by contaminated seed lots.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Pseudomonas syringae pv. pisi (Ppi) is one of the 60 pathovars belonging to the highly heterogeneous species P. syringae and causes pea (Pisum sativum) bacterial blight, a potentially devastating disease that results in serious reduction in yield and seed quality under favourable weather conditions (Lawyer & Chun, 2001). Disease symptoms are more evident after frost periods, heavy rains or mechanical damage to the crop, all of which cause plant injuries that facilitate entry of the pathogen and infection initiation. Disease usually starts with the appearance of small water-soaked areas on any aerial plant part that, favoured by humid and warm conditions, extend and can eventually determine plant wilting and death (Lawyer & Chun, 2001). The disease has been described in all countries in which pea production is important (Lawyer & Chun, 2001), and it is particularly aggressive in autumn-sown fields in several European countries and in Australia (Schmit et al., 1992; Hollaway & Bretag, 1995; Reeves et al., 1996; Martín-Sanz et al., 2011). Control of Ppi is difficult and is mainly based on cultivation of resistant cultivars, crop rotation and, because the pathogen is transmitted by seed, the use of pathogen-free seed (Lawyer & Chun, 2001). The use of pea cultivars combining bacterial blight resistance and frost tolerance has been described as a useful preventive control strategy (Martín-Sanz et al., 2012). On the basis of the differential response of eight pea genotypes, seven pathogenic races (1–7) of Ppi were described (Bevan et al., 1995), and a new race (race 8) was recently described by Martín-Sanz et al. (2011). Race 2 seems to be the most frequent worldwide, although race frequencies vary with region, the predominant pea varieties cultivated and the sowing date (winter or spring) (Lawyer & Chun, 2001). Thus, for instance, race 3 was most frequently found in Australia (Hollaway & Bretag, 1995); race 2 in the UK (Taylor et al., 1989; Reeves et al., 1996); race 2 in spring-sown pea but race 6 in autumn-sown pea in France (Schmit et al., 1992); and race 4 in Spain (Martín-Sanz et al., 2011).

Information about the structure and diversity of Ppi populations is an important task for epidemiological studies, and few studies have reported comparative analyses of genetic variability and population diversity of this pathogen. Acquiring knowledge of the genetic structure of Ppi populations within each country will help to define quarantine risks posed by exotic strains. Moreover, it will provide valuable information that can help to establish efficient strategies for pea bacterial blight control.

Pathovars of the P. syringae complex have traditionally been characterized biochemically, and Schaad et al. (2001) described several biochemical-nutritional tests to differentiate between pathovars of this bacterial species. However, such tests did not accurately discriminate the two pathovars that are pathogenic to pea: Ppi and P. syringae pv. syringae (Martín-Sanz et al., 2011).

Repetitive DNA PCR-based genomic fingerprinting (rep-PCR) with REP, ERIC and BOX primer sets is a sensitive, reproducible, and highly discriminatory method to assess bacterial diversity at the strain and pathovar level. It was used to carry out identification and characterization studies of pathovars belonging to P. syringae and other genera of phytopathogenic bacteria (Louws et al., 1999). Dawson et al. (2002) compared different methods to determine the diversity of fluorescent pseudomonads, concluding that rep-PCR is suitable for the analysis of highly clonal isolates because it is more discriminatory than other DNA fingerprinting techniques and metabolic profiling. Several studies have since successfully used rep-PCR for genetic characterization of different pathovars of P. syringae (Scortichini et al., 2003; Oguiza et al., 2004; Ferrante & Scortichini, 2010).

The rep-PCR procedure has been used to analyse Ppi diversity and strain identification; thus for instance, Hollaway et al. (1997) used rep-PCR to identify pathogenic races in a collection of 41 isolates from Australia and the type races of this pathovar as previously characterized by Bevan et al. (1995), and Cirvilleri et al. (1998) analysed the differentiation of Ppi type races and of Italian isolates. Suzuki et al. (2003) characterized some isolates from Japan that, in addition to the water-soaked spots, also produced a yellowing in the pea plant apex (Ppi White Top isolates). The rep-PCR technique grouped White Top and non-White Top isolates from Japan into two different groups, and White Top isolates shared the same fingerprint pattern. These two groups also differed in some biochemical characteristics. Unfortunately no information on the pathogenic races was included in this report.

Several other molecular techniques have been used to characterize strains and races of Ppi, such as AFLP (Cirvilleri et al., 2007), or specific primers (Arnold et al., 1996). DNA fingerprinting by RAPD-PCR of diverse Ppi and other P. syringae pathovars allowed the identification of two bands that were specific for Ppi and associated with pathogenic races (Arnold et al., 1996); each isolate amplified only one of these two markers, depending on their race assignation. The marker named AN3 (132 bp) was amplified from races 1, 5 and 7 whereas the AN7 marker (272 bp) was amplified from races 2 and 6; additionally, different isolates of both races 3 and 4 amplified one or another. These results suggested the existence of two phylogenetic groups within Ppi: group I (marker AN3) and group II (marker AN7) (Arnold et al., 1996).

Multilocus sequence typing (MLST) is a strain-typing system that focuses strictly on the bacterial core genome. This highly accurate and reproducible approach uses the DNA sequences from four to seven housekeeping genes to differentiate strains and clonal lineages (Enright & Spratt, 1999). MLST has been used to study genetic diversity and evolution in P. syringae and to identify pathovars and strains within this species (Sarkar & Guttman, 2004; Hwang et al., 2005; Ferrante & Scortichini, 2010).

The objectives of this study were: (i) to assess the biochemical, genetic and pathogenic diversity of a collection of Ppi strains from different countries, including the type races and a representative set of Spanish isolates; (ii) to analyse the relationship between the biochemical and genetic profiles and the Ppi race classification; and (iii) to clarify whether the strains isolated in Spain originate from one or several inoculum sources.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Bacterial strains

A collection of 91 Ppi isolates and strains was used, including 53 isolates obtained from infected fields in the main pea producing area of Spain (Martín-Sanz et al., 2011) and 38 strains from international collections representing 14 countries from the five continents, and which included the type race strains (Table S1); type race refers to previously characterized strains for each of the pathogenic races generally used as reference. The 53 Spanish isolates came from an epidemic in Central-North Spain which started in 2002 (Martín-Sanz et al., 2011), 2 years after the land area devoted to pea crop in this region greatly increased, making pea bacterial blight an important limiting factor. Bacteria were routinely grown on King’s B medium (KB) plates (King et al., 1954) and preserved in media with 15% glycerol at −75°C for long-term storage. For simplicity, all strains from collections and Spanish isolates will hereafter be referred to as strains.

Strain identification and pathogenicity tests

Strains were identified according to the methodology described by Schaad et al. (2001), including grouping by Gram reaction using the KOH test, Hugh–Leifson oxidation/fermentation test, fluorescence on KB medium, LOPAT test (Levan production, oxidase reaction, potato soft rot, arginine dihydrolase activity and tobacco hypersensitivity), and the use of homoserine as a carbon source. PCR was carried out using primer pairs specific for the AN3 and AN7 markers of Ppi (Arnold et al., 1996), and using cells from single colonies as template. PCR reactions and visualization of the amplified fragments in agarose gels were carried out according to Arnold et al. (1996). Race identification was done by inoculating strains onto the eight pea differential cultivars classically used to identify the pathogenic races of Ppi (Kelvedon Wonder, Early Onward, Belinda, Hurst’s Greenshaft, Partridge, Sleaford Triumph, Vinco and Fortune), following a previously described methodology (Bevan et al., 1995).

Nutritional characterization

Strains were evaluated for their ability to hydrolyse gelatin and esculin, and to utilize betaine, l-lactate, d-tartrate and homoserine as the sole carbon source according to Schaad et al. (2001). Furthermore, 49 other carbon sources were assayed with the standardized API 50 CH (Biomérieux) test, using the Ayers liquid medium (pH 7·0) without carbon source as the basal medium, as previously described (Palomo et al., 2006). Briefly, a suspension of 5 mL (approximately 1010 CFU mL−1 determined by dilution plating) from each strain culture were mixed with 15 mL of the Ayers basal medium, and 300 μL of this suspension were added to each API test tube. The sets of API test tubes were incubated at 25°C during 5 days, recording colour changes every 12 h. Responses were considered positive when the original green colour changed to yellow or blue, depending upon the compound. Negative controls were inoculated with sterile water, and the HRI strains (type race strains; Table S1) were used as positive references. Each strain was analysed three times for every test in independent replicas.

Rep-PCR

Strains were grown on KB plates at 25°C for 48 h before DNA isolation using the DNeasy Tissue Kit (QIAGEN), following the manufacturer’s instructions. The concentration of DNA was estimated by electrophoresis of 2 μL of DNA preparations, together with a DNA marker, on a 1% agarose gel with 1× TAE buffer and stained with ethidium bromide. Total genomic DNA was adjusted to a final concentration of 20 ng μL−1 and stored at −20°C before use. Rep-PCR with REP-PCR, ERIC-PCR and BOX-PCR primers was performed according to Versalovic et al. (1994) and Rademarker & de Bruijn (1997). PCR products were separated by electrophoresis in 1·5% agarose gels in 0·5× TAE and visualized under UV light after staining with ethidium bromide. Strains were tested at least three times with each rep-PCR method to ensure reproducibility of the DNA fingerprints.

Multilocus sequence typing

Multilocus sequence typing (MLST) was performed with partial sequences of gap1, gltA, gyrB and rpoD genes coding for glyceraldehyde-3-phosphate dehydrogenase, citrate synthase, DNA gyrase B and sigma factor 70, respectively. Gene fragments were amplified from genomic DNA of 15 Ppi strains, which represented the rep-PCR and nutritional variability of the collection (Table S1), with the primers described by Sarkar & Guttman (2004) according to the PCR procedure described by Ferrante & Scortichini (2010). PCR products were purified with the QIAquick PCR purification kit (QIAGEN) following the manufacturer’s instructions, and sequenced using a Megabace 500 (Amersham) sequencer. Sequences were edited, aligned, and compared with other Pseudomonas species sequences (Sarkar & Guttman, 2004) obtained from the NCBI database by using the mega4 program (Tamura et al., 2007).

Data analysis

The sizes of the fragments amplified by rep-PCR were estimated using a DNA 100 bp size-ladder (DNA Molecular Weight Marker XIV, Roche). Band presence/absence was scored as a 1 or 0, respectively. Only clear and repeatable (in three replicas) bands were scored. Biochemical-metabolic responses were also scored as 1 (positive) or 0 (negative). The strains were grouped in dendrograms using distance derived from Jaccard similarity indices. Distances were calculated as 1-similarity. UPGMA clustering method was used to obtain dendrograms by means of the phylip 3.63 package (Felsenstein, 1989), and confidence was estimated by a bootstrap with 1000 resamplings. Principal component analysis was carried out from the variable correlation matrix using the statistical R package (http://www.R-project.org). For MLST analysis a concatenated data set (1874 aligned nucleotides) was used to construct dendrograms in mega4 using the Kimura 2-Parameter distance (Kimura, 1980) and the neighbour joining method; confidence levels of the branching points were determined using 1000 bootstrap replicates. Pseudomonas syringae pv. pisi sequences were registered in the EMBL GenBank under accession numbers HE575254 to HE575313.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Identification and pathogenic characterization

The 91 strains were identified as P. syringae pv. pisi on the basis of their metabolic profiles, amplification of the Ppi specific DNA markers AN3 and AN7, and pathogenic tests on pea tester genotypes, as follows: all of the strains were Gram-negative, had an oxidative metabolism, and belonged to the LOPAT Ia group (LOPAT reactions +−−−+). Their identification as members of pathovar pisi was confirmed because all utilized homoserine as the sole carbon source (Table 1) and also, except isolate Uc021, produced typical pea bacterial blight symptoms after artificial inoculation on Kelvedon Wonder, a pea cultivar susceptible to all known Ppi races. These last two tests are routinely used to differentiate Ppi from P. syringae pv. syringae, which can also infect pea (Martín-Sanz et al., 2011). A total of 62 isolates (68·1%) showed fluorescence on KB medium; all of these, plus four fluorescence-negative isolates, amplified the AN7 marker (272 bp), and were therefore ascribed to Ppi group II (Arnold et al., 1996). The remaining 25 fluorescence-negative isolates belonged to the Ppi group I because they amplified the AN3 marker (132 bp). Inoculation in the pea tester cultivars allowed five isolates to be assigned to race 1, 18 to race 2, four to race 3, 33 to race 4, 10 to race 5, 15 to race 6, two to race 7 and three to race 8 (Table 2). Uc021 was non-pathogenic for all pea tester lines. This strain was isolated in 1953 and it is possible that it has lost its pathogenicity as a consequence of long-term storage.

Table 1.   Response of a collection of 91 Pseudomonas syringae pv. pisi strains to nutritional-biochemical characterization
Test Ppi a Response Ppi Ppi HRI 229A Ppi HRI 202
+
  1. a+, 80% or more of strains are positive; −, 80% or more of strains are negative; V, 19–79% of strains are positive. Strains Ppi HRI 229A and Ppi HRI 202 were used as controls.

Fluorescence on KB68·131·9V+
Esculin hydrolysis42·657·4V+
Gelatin liquefaction18·781·3
Utilization of
 α-methyl-d-glucoside 100
 α-methyl-d-manoside 100
 β-gentobiose 100
 β-methyl-d-xyloside 100
 2-keto-gluconate 100
 5-keto-gluconate 100
 Adonitol 100
 Amygdalin 100
 Arbutin 100 
 Betaine57·142·9V+
 Cellobiose11·089·0+
 d-arabinose18·281·8++
 d-arabitol100 +++
 d-fructose100 +++
 d-fucose100 +++
 d-glucose100 +++
 d-lyxose 100
 dl-lactate16·583·5
 d-mannose100 +++
 d-raffinose100 +++
 d-tagatose 100
 d-tartrate15·484·6
 d-turanose 100
 Dulcitol 100
 d-xylose100 +++
 Erythritol27·572·5V+
 Galactose100 +++
 Glucogen 100
 Gluconate 100
 Glycerol87·912·1+++
 Homoserine100 +++
 Inositol100 +++
 Inuline 100
 Lactose 100
 l-arabinose100 +++
 l-arabitol 100
 l-fucose 100
 l-sorbose 100
 l-xylose 100
 Maltose 100 
 Mannitol100 +++
 Melezitose 100
 Melobiose100 +++
 N-acetyl-glucosamine 100
 Rhamnose 100
 Ribose100 +++
 Salicine 100
 Sorbitol100 +++
 Starch 100
 Sucrose100 +++
 Trehalose 100
 Xylitol 100
Table 2.   Differential characteristics of the two Pseudomonas syringae pv. pisi genetic lineages
Ppi group and raceNumber of strainsCharacteristica
Fluorescence +/−Esculin +/−Erythritol +/−
  1. aFluorescence, production of fluorescent pigments on KB medium; Esculin, esculin hydrolysis; Erythritol, utilization of erythritol as the sole carbon source. Figures indicate the number of positive/negative strains.

Group I (AN3)
 150/55/04/1
 320/22/02/0
 470/77/07/0
 590/99/09/0
 720/22/04/0
Totals Group I250/2525/024/1
Group II (AN7)
 21816/24/140/18
 322/00/20/2
 42625/12/240/26
 511/01/00/1
 61514/16/90/15
 833/01/21/2
 Non-pathogenic11/00/10/1
Totals Group II6662/414/521/65

Nutritional characterization

The Ppi strains displayed biochemical profiles that were congruent with their assignation to the pathovar pisi (Table 1). From the 55 biochemical tests performed, 45 tests produced a clear 100% response, either positive or negative, for all the Ppi strains. Additionally, the majority of the strains utilized glycerol as the sole carbon source (87·9% of the strains), whereas the majority did not utilize cellobiose (89%), d-tartrate (84·6%), dl-lactate (83·5%) and d-arabinose (81·8%), and did not hydrolyse gelatin (81·3%). Likewise, the production of fluorescent pigments on KB medium (68·1% positive strains), esculin hydrolysis (42·6%), and utilization of betaine (57·1%) and erythritol (27·5%) were variable characters among the Ppi population (Table 1). Distance dendrograms obtained using the complete set of biochemical data were not able to discriminate between groups of Ppi strains because all bootstrap supporting values were too low to be reliable (data not shown). Principal component analysis afforded more information (Fig. 1a). The first component (biochemical-nutritional data), which explained 31% of the variance, discriminated most of the group I strains from group II strains, mainly on the basis of the fluorescence, esculin and erythritol responses. Although none of the biochemical tests allowed for a clear-cut differentiation of strains of group I and group II (Table 2, Fig. 1a), a somewhat variable phenotypic profile can be inferred from data from three of these tests: strains from group I (AN3 marker) do not produce fluorescent pigments, hydrolyse esculin and generally can use erythritol as the sole carbon source; conversely, strains from group II (AN7 marker) are generally fluorescent, and normally do not hydrolyse esculin and do not utilize erythritol. No relationship between these responses and geographical origin was found.

image

Figure 1.  Principal component analysis (PCA) and dendrogram based on the biochemical and genetic profiles of Pseudomonas syringae pv. pisi strains. (a) PCA based on metabolic profiling data. (b) PCA based on rep-PCR profiling data. Empty circles, group I strains (AN3 marker); filled circles, group II strains (AN7 marker); underlined strains are included in the AN3 subgroup Ib in (c) (groups I and II as defined by Arnold et al., 1996). Strains in bold-italics are those included in the MLST analysis shown in Fig. 3. Component values are indicated in the left and bottom scales, and vectors indicate the importance of each variable. (c) Condensed dendrogram obtained from rep-PCR data by means of Jaccard distance; only AN7 strains are clearly segregated from the two AN3 subgroups (Ia and Ib; also indicated in b); figures indicate bootstrap support values. Arrows in (a) and (b) indicated the effect of the variables, values at upper and right scales. Variable abbreviations in (a): Bet, betaine; Tar, d-tartrate; Flu, fluorescence; Cel, cellobiose; Gly, glycerol; Ara, d-arabinose; Gel, gelatin; Lac, dl-lactate; Esc, esculin; Ery, erythritol. Variable abbreviations in (b) indicate: B1 to B7, BOX-PCR variable bands of 200, 420, 370, 530, 700, 880 and 1110 bp, respectively; E1 to E5, ERIC-PCR variable bands of 120, 200, 650, 700 and 1450 bp, respectively; R1 to R9, REP-PCR variable bands of 100, 115, 130, 200, 310, 400, 950, 1000 and 1800 bp, respectively. Strain code: the two-first letters indicate the country, and for the Spanish strains the other two the province. The first number indicates the pathogenic race, the second indicates that they amplified the AN3 marker (1) or the AN7 marker (2), and the third is the serial number. Asterisks denote the type race strains. Key for strain names is in Table S1.

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Genetic characterization

The rep-PCR analyses (Fig. 2) yielded a total of 21 clearly observable polymorphic bands and 19 different rep-PCR patterns among the Ppi strains. REP-PCR generated nine R polymorphic bands, ERIC-PCR five E bands, and BOX-PCR seven B bands. The association of some of these markers and AN3 and AN7 markers is straightforward: all strains amplifying the AN3 marker (group I) also amplified bands R-115, R-310, R-1000, E-200, B-420 and B-110 and not R-100, R-130, R-950, R-1800 and B-200, while group II strains, amplifying the AN7 marker, showed the opposite pattern. The remaining bands showed some variability within groups. A distance dendrogram using the data from the rep-PCR analyses allowed for a clear discrimination between group I and group II strains (Figs 1c & S1), and differentiated two subgroups among the group I strains, groups Ia and Ib; all other bootstrap values were too low to be reliable. The first component of a principal component analysis of rep-PCR data (Fig. 1b) discriminated between group I and group II strains, explaining 55·8% of the total variance, probably because it also included the polymorphic band set characteristic of groups I and II mentioned above. The second component mainly discriminated among group II strains on the basis of two polymorphic bands (E-700 and E-1450), which were mostly amplified in strains that did not amplify B-700 (E4, E5 and B5 in Fig. 1b, respectively). Five out of the 19 marker profiles included a majority (69) of the strains, the remaining (all of group II) are scattered along the second axis in the plot representation of the principal component analysis. None of the principal component analyses or the dendrograms (data not shown) showed any evident association between the metabolic or marker profiles and the pathogenic race or the geographical origin, with the exception of some races that were observed only within the AN3 or AN7 strain groups. Likewise, there was also a trend in the distribution of races between the groups, with races 1 and 7 being exclusive of group I and races 2, 6 and 8 exclusive of group II (Table 2), although the limited number of strains examined here cautions against reaching premature conclusions.

image

Figure 2.  Representative rep-PCR profiles of Pseudomonas syringae pv. pisi strains obtained using REP-PCR, ERIC-PCR and BOX-PCR primers. Polymorphic bands are indicated. AN3 and AN7 indicate the patterns included in these two strain groups. The 100 bp ladder is included.

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The four partial gene sequences (gap1, gltA, gyrB and rpoD) generated a concatenated data set of 1874 aligned nucleotides for the MLST analysis. The 15 Ppi strains were clearly discriminated in two groups (Fig. 3). The first group corresponds to four strains (Au 311, EsPa 411, NZ 111 and US 511) amplifying AN3 (group I according to the AN3/AN7 classification by Arnold et al. (1996)) and with the same nucleotide sequences as strain Ppi H5E3, which defines the Ppi2 set of strains (according to the MLST profile defined by Sarkar & Guttman (2004)). The second group amplified AN7 and showed the same (Ch 621, EsNa 421, EsVa 622, EsZa 222, Fr 221, Fr 623, US 222, and US 321) or nearly the same (EsBu 821, Fr 521 and US 422) sequence as strain Ppi H5E1, defining the Ppi1 profile (Sarkar & Guttman, 2004).

image

Figure 3.  Neighbour joining dendrogram showing the similarity of 15 strains of Pseudomonas syringae pv. pisi. Concatenated gap1, gltA, gyrB and rpoD partial sequences were used in the analysis (1874 nucleotides). Pseudomonas fluorescens sequence was used as out-group, GenBank data from two P. syringae pv. pisi (Ppi, H5E1 and H5E3) and two P. syringae pv. syringae (Psy, L 177, B728a) strains were also included (GenBank accession numbers in Table S3). The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. AN3 and AN7 denote the fragment amplified from each strain. Ppi1 and Ppi2 indicate sets of strains with the same sequence and which coincide with the groups indicated by Sarkar & Guttman (2004); Ppi1 included strains H5E1, Ch 621, EsNa 421, EsVa 622, EsZa 222, Fr 221, Fr 623, US 222 and US 321; Ppi2 included strains H5E3, Au 311, Espa 411, NZ 111 and US 511; strain codes as in Fig. 1 (note that the Ppi1 and Ppi2 sets of strains fall into group II and group I, respectively, according to the nomenclature of Arnold et al. (1996)).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This study presents data from the multiphasic characterization of a large collection of Pseudomonas syringae pv. pisi strains, which includes all known pathogenic races and type race strains and encompasses a wide representation of geographical origins. Likewise, it affords the first data on Ppi intra-pathovar diversity using the MLST methodology, and on the use of the standardized API 50 CH profiling method in the characterization of Ppi.

The Ppi strains showed variability in diverse phenotypic characteristics, including fluorescence, gelatin degradation, esculin hydrolysis, and utilization of dl-lactate, d-tartrate, betaine, erythritol, glycerol and d-arabinose. In spite of this variability, no unequivocal relationship was observed between the metabolic profile of the Ppi strains and their race assignation or geographical origin. However, current data support that group I strains (amplifying the AN3 marker) are characterized as fluorescence negative and esculin and erythritol positive while a majority of the group II strains (amplifying AN7) shows the opposite pattern (Table 2). Rep-PCR and MLST of four housekeeping gene sequences also clearly distinguished between group I and group II Ppi strains. The BOX-PCR, REP-PCR and ERIC-PCR sets of the rep-PCR markers afforded a total of 21 polymorphic bands and 19 patterns. BOX-PCR and REP-PCR generated differential profiles that allow group I and II strains to be distinguished. Group I strains were monomorphic. This differentiation was less clear for ERIC-PCR.

The fluorescence positive and fluorescence negative Ppi strains analysed by Suzuki et al. (2003) showed differential rep-PCR profiles, which agree with the results here. Rep-PCR profiles were used to identify Ppi isolates from pea fields in Australia and the seven type race strains (Hollaway et al., 1997). The rep-PCR data described by these authors in relation to the type races coincides with the profiles found in the present work. Thus, for instance, the race type strains 1, 5 and 7 showed the same band pattern in both studies (subgroup Ib in Fig. 1b). Hollaway et al. (1997) indicated that this technique was useful to identify the Ppi races, which disagree with the results here, except for the differentiation between group I and group II strains. This is probably due to a lower number of strains (data from 41 strains), the limited geographical range of the pathogen (all samples were from Australia, except the type strains) and the limited genetic background of the pea host cultivars (cvs Dun, Dundale and Alma, all resistant to races 1, 2, 4, 5 and 7 according to Hollaway & Bretag (1995)). The same geographical and sample-size limitations are observed in the REP- and ERIC-PCR analyses carried out by Cirvilleri et al. (1998) on only 21 strains of the race 6 from Sicily (Italy) and the seven type strains. Again, the results in the present study agree with the results described for the type race strains (e.g. races 1, 5 and 7 showed the same band pattern) and for race 6, although the inclusion of BOX-PCR in this work allows for a further differentiation between strains of this last race.

Since Arnold et al. (1996) described the alternative presence of the AN3 or AN7 markers in the Ppi strains, the existence of two phylogenetic groups within Ppi has been repeatedly supported by experimental data (Hollaway et al., 1997; Cirvilleri et al., 1998; Suzuki et al., 2003). Furthermore, race 4 strains, which amplify either AN3 or AN7, were included in the two phylogenetic groups on the basis of the sequence of the hrpL gene (a putative RNA polymerase sigma factor) (Cournoyer et al., 1996). The MLST analysis reported here supports the existence of these two strain groups within the pathovar pisi of P. syringae and confirms that the genetic variability within them is very limited, because several strains shared common multilocus sequences. Sarkar & Guttman (2004) have already pointed out that different Ppi strains had the same MLST sequences.

Rep-PCR is a valuable method for assessing Ppi diversity. This technique also clearly discriminates between strains of group I and group II of Ppi, although it is apparently not associated with the pathogenic profile, because there is not a single band pattern associated to each race. Some races seem to be exclusively or preferentially included in one of these two groups (1 and 7 exclusive of group I and 2, 6 and 8 of group II). The presence of races 3, 4 and 5 in both phylogenetic lineages (group I and II) cannot easily be explained, in particular because the race 5 phenotype was postulated to depend on the activity of at least three effector genes (Vivian & Arnold, 2000). Although none of the techniques used here allowed the identification of individual races, markers AN3 and AN7 unequivocally identified two groups of races: races 1 and 7 (group I) and races 2, 6 and 8 (group II) (see Table 2). Predictions of the race structure of P. syringae pv. phaseolicola populations was also done by identification of groups of races by PCR (Rico et al., 2003); therefore, further genetic analyses would be needed to develop DNA-markers which allow for an unequivocal identification of Ppi races. The relatively high number of strains analysed in the present study, the dispersion of their geographical origin, the races represented and the pea host cultivars of origin, reveals a larger genetic diversity among Ppi populations than identified in previously published results. Metabolic profiles also allow the differentiation of these two strain groups, although internal variability within groups is higher than that shown with rep-PCR (Fig. 1a,b). For instance, EsBu821 and Uc021 are close to the main group II cluster of strains according to rep-PCR data; however, EsBu821 showed a metabolic profile closer to that of group I strains (Fig. 1a upper-left cluster), and Uc021 showed a particular metabolic pattern intermediate within group II. Again, metabolic profiling is not an unequivocal way to identify Ppi races, even in combination with rep-PCR.

The three strains from the recently described race 8 (Martín-Sanz et al., 2011) analysed here amplified marker AN7. Supporting this, rep-PCR indicated that race 8 strains were included in, or were close to, the main AN7 cluster (Fig. 1b), which in addition included strains of the races 2, 4 and 6. However, these three race 8 strains were clearly separated from each other when metabolic data were considered (Fig. 1a), indicating that genetic variability is present within this race. The race 8 strain (EsBu82) included in the MLST test showed slight sequence differences with the rest of the AN7 group. Nevertheless, it will be necessary to examine more strains of this race from different geographical origins to evaluate the genetic diversity among race 8 strains.

Grondeau et al. (1992) described 13 biochemical patterns in a collection of 150 Ppi strains (most of them isolated in France), although data on the pathogenic race were not included. These patterns were not related to the geographical origin of the Ppi strains. The percentages of Ppi strains showing each response was clearly different in the collection evaluated by Grondeau et al. (1992) and the collection analysed here (Table S2). Percentages in Grondeau et al. (1992) are closer to the group II, with the exception of erythritol use. This could probably result from the fact that most of the Ppi samples in Grondeau et al. (1992) were collected from pea cultivars Belinda, Frijaune, Frilene and Monitor. These four cultivars are resistant to races 1, 3 and 7, and three of them are also resistant to race 5, and races 1, 7 and 5 are exclusively or preferentially included in group I (Arnold et al. (1996), and this study). Thus, considering the Ppi resistance genes present in these pea cultivars the predominant races thriving on them would be 4, 6 and 2, which are exclusively or preferentially included in group II. This would agree with the report that Ppi races 2, 6 and 4 were the most frequent ones among pea cultivars sown in France (Schmit et al., 1992). Benlioglu et al. (2010) reported the occurrence of Ppi in Turkey. They showed that 12 Turkish strains were all fluorescent in KB and erythritol negative which, according to the present study, suggests that all are probably group II strains. Suzuki et al. (2003) distinguished two types of Ppi strains on the basis of the disease symptoms produced in the host: the normal type and the White Top type. This last type produced the classical water soaked symptoms plus yellowing of the pea plant apex. A group of these exclusively Japanese strains was fluorescence negative and gelatin, esculin, erythritol and dl-lactate positive, while the other strains showed the opposite pattern. These characteristics would fit group I and group II, respectively, because the group I strains are fluorescence negative and esculin and erythritol positive, and most of them gelatin and dl-lactate positive, whereas the opposite phenotypes are highly frequent for group II (Fig. 1a).

The relatively wide sample of Spanish strains mirrors the metabolic and genetic variability found in strains collected from other parts of the world (Figs 1 and 3). Thus, although it is not possible to establish with total certainty the origin of the Ppi strains collected in the recent epidemic in Spain, these data point out that, in addition to a possible local Ppi population, the primary inoculum has probably originated from several seed lots from other countries (France, Germany, UK, Denmark, the Netherlands and USA), imported and sown in Spain when the rapid expansion of the pea crop started a decade ago. This would agree with the report by Stead & Pemberton (1987), who indicated that the presence of Ppi in Europe was increasing in parallel with the increase in the land area devoted to pea and the exchange of seed lots between countries without adequate phytosanitary controls. In fact, Ppi was first identified as a pea pathogen in Spain in 1991 in experimental fields in North-Central Spain, sown with cultivars from different countries, where Ppi has been repeatedly found since 2002. Therefore, it is probable that several imported seed lots contaminated with Ppi have contributed to the disease expansion in this country. Subsequently, Ppi populations could have been disseminated throughout the most important pea production areas in Spain, contributing to the high genetic variability that has been revealed in this study.

In conclusion, the characterization of a diverse group of strains confirms the existence of two well-defined phylogenetic lineages in Ppi. The rep-PCR and MLST analyses applied in this study were suitable for the study of Ppi diversity, although they cannot be used for race identification. All these results contribute to a better understanding of the worldwide Ppi population structure and genetic diversity of this important seedborne pathogen, and can help identify appropriate Ppi strains for disease resistance testing as a tool to pea germplasm screening and to breed new resistant cultivars.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This research was supported by the projects ITACYL 2004/845 and GR113 from the Junta de Castilla y León and RTA 2006-00077-00-00 from the INIA, respectively, and by an INIA personal PhD grant to A. Martín-Sanz. The authors thank CFBP (France) and HRI-W (UK) for supplying the collection strains, and also Dr J. L. Palomo (Centro Regional de Diagnóstico, Junta de Castilla y León, Spain) for help with nutritional analyses.

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1. Dendrogram constructed from rep-PCR data generated with the DNA of the 91 Pseudomonas syringae pv. pisi strains characterized in this study. Numbers in the right denote the association to group I and II according to Arnold et al. (1996). Key for strain names is in Table S1.

Table S1. Bacterial strains of Pseudomonas syringae pv. pisi used in this study.

Table S2. Percentage of Ppi AN3-positive and AN7-positive strains which were positive in the variable biochemical tests, and comparison of the metabolic profile results by Grondeau et al. (1992).

Table S3. Additional Pseudomonas sequences used in the neighbour joining dendrogram displayed in Figure 3.

FilenameFormatSizeDescription
PPA_2604_sm_FigtS1.tif1366KSupporting info item
PPA_2604_sm_TableS1.doc154KSupporting info item
PPA_2604_sm_TableS2.doc37KSupporting info item
PPA_2604_sm_TableS3.doc32KSupporting info item

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