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Applied and Environmental Microbiology, August 2003, p. 4979-4982, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4979-4982.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Analysis of the argK-tox Gene Cluster in Nontoxigenic Strains of Pseudomonas syringae pv. phaseolicola

Ana Isabel González, Marcelino Pérez de la Vega, María Luisa Ruiz, and Carlos Polanco*

Área de Genética, Facultad de Biología, Universidad de León, E-24071 León, Spain

Received 25 February 2003/ Accepted 14 May 2003


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ABSTRACT
 
The analysis of 46 isolates obtained directly from different and distant common bean fields from the northwestern part of Spain revealed that they do not produce phaseolotoxin. The isolates were classified as race 5, and their analysis revealed that they do not carry the argK-tox gene cluster involved in the biosynthesis of the phaseolotoxin.


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INTRODUCTION
 
Pseudomonas syringae pv. phaseolicola is a seed-borne bacterial pathogen of beans (Phaseolus spp.) with a worldwide distribution. This pathogen causes the halo blight disease of beans, and even low levels of primary infection can result in severe epidemics due to a fast spread of the pathogen under suitable weather conditions (22). The best practical control for this serious disease is the use of pathogen-free seeds. Thus, the need of a highly sensitive and specific assay to identify the halo-blight pathogen has recently promoted the development of several molecular methods for the detection of P. syringae pv. phaseolicola in bean seeds (1, 8, 13, 17). These methods are based on the PCR amplification of specific DNA sequences, such as the phaseolotoxin gene cluster (1, 13, 17).

Phaseolotoxin [N{delta}(N'-sulfo-diaminophosphinyl)-ornithyl-alanyl-homoarginine (6)] is a non-host-specific toxin, characteristically produced by P. syringae pv. phaseolicola, which inhibits ornithine carbamoyltransferase (OCTase), an enzyme involved in arginine biosynthesis (11). Phaseolotoxin production is regulated by temperature and decreases progressively at temperatures above 18°C (4, 9). The pathogen is resistant to its own toxin because it possesses phaseolotoxin-resistant OCTase (ROCT), which is not inhibited by phaseolotoxin (7). The sequence of the corresponding gene, named argK, has also been used to develop a method of detection (8).

The use of the phaseolotoxin gene cluster has the possible disadvantage that strains defective in toxin production (nontoxigenic strains) may go undetected. The use of the ROCT gene might have the same disadvantage if this gene has somehow been modified in such nontoxigenic strains. However, to date it is thought that nontoxigenic strains have little or no epidemiological importance in bean halo blight (17), although they multiply at rates equal to those of toxigenic strains and they cause lesions indistinguishable from those caused by the toxigenic strains, with the exception of the absence of chlorotic halos (10, 17). No molecular characterization studies of nontoxigenic strains obtained from bean fields have been carried out to date.

In this work we present data indicating that wild nontoxigenic strains of P. syringae pv. phaseolicola are commonly present in northern Spanish bean fields, and their molecular analysis revealed that these strains are undetected by available methods of fast detection.

In order to obtain P. syringae pv. phaseolicola isolates, bean pods from plants showing typical symptoms of the halo blight disease were collected from different bean fields located in León province in the northwestern part of the Spanish Central Plateau during the years 1999, 2000, and 2001 (Table 1). A sterile inoculating loop was used to place exudates from the typical water soaking marks of halo blight-infected pods in MSP (5) semiselective medium at room temperature for 3 days. Suspected colonies of P. syringae pv. phaseolicola were selected on the basis of both colony morphology and surrounding medium characteristics (5) and were confirmed to be P. syringae pv. phaseolicola after growth at room temperature for 2 days in King's B medium (3), in which the organism showed a typical fluorescence and morphology, and at 28°C for 2 days in MT medium (2). MT medium allows the differentiation of P. syringae pv. phaseolicola, P. syringae pv. syringae, Xanthomonas axonopodis pv. phaseoli, and Xanthomonas axonopodis pv. phaseoli var. fuscans, the etiological agents of bacterial halo blight, brown spot, common blight, and fuscous blight diseases of dry beans, respectively (2).


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  		TABLE 1. Bacterial strains used in this study and results obtained for phaseolotoxin production and the presence of the argK-tox gene cluster (phtE locus and argK gene)

The 46 strains isolated in this work were classified into one of the nine races described for this pathogen after controlled infections on pods from differential cultivars (19). Race classification was helped by PCR amplification of the avrPphF locus described as the A1 avirulence gene matching the R1 gene for resistance in Phaseolus (21). This locus is present only in races of P. syringae pv. phaseolicola expressing the A1 phenotype: races 1, 5, 7, and 9. We designed primers AVR1-F (20-mer; 5'-CCGCCGTAGCAGGGCTTCAC-3') and AVR1-R (20-mer 5'-GGACCAATCTCTTTTCTCAA-3') from the corresponding sequence (21) in order to amplify a 1.4-kb fragment which included the two open reading frames (ORFs) present in the avrPphF locus. The thermal profile used was 94°C for 1 min, 58°C for 1 min, and 72°C for 2 min (30 cycles). All the PCRs described in this work were carried out in a Perkin-Elmer (Norwalk, Conn.) 2400 thermocycler with Taq polymerase (Promega, Madison, Wis.) in final volumes of 25 µl containing 2.5 µl of 10x PCR buffer (100 mM Tris-HCl, 500 mM KCl, 1% Triton X-100; pH 9.0), 1.5 µl of MgCl2 (25 mM), 2.5 µl of deoxynucleoside triphosphates (25 mM each), 1 µl of each corresponding primer (5 pmol/µl), 50 ng of template DNA, and 0.1 µl of enzyme (5 U/µl). Genomic DNA was extracted from all the strains by the method of Zhu et al. (25).

The production of phaseolotoxin was analyzed by using the Escherichia coli toxin bioassay (18). Test strains of P. syringae pv. phaseolicola were spotted onto the E. coli test plates prepared according to the directions of Sawada et al. (16) and incubated at 18°C for 3 days. All 46 isolates were classified as race 5 and nontoxigenic (Table 1).

Eighteen other pathogen strains isolated from bean fields located in the northern Spanish Plateau region were kindly donated by C. Asensio (Servicio de Investigaciones Agrarias, Valladolid, Spain). The analysis of the phaseolotoxin production of these strains revealed that all the race 5 strains as well as a single race 1 strain did not show production of phaseolotoxin (Table 1).

We obtained another 35 strains of P. syringae pv. phaseolicola from C. Stevens (University of London, Kent, United Kingdom) and 9 strains from J. D. Taylor (Horticulture Research International, Warwick, United Kingdom). These 44 strains have been maintained in culture for several years and were isolated mainly from African bean growing areas in the 1980s and middle half of the 1990s (19, 20). It was observed that several of them did not produce phaseolotoxin. These nontoxigenic strains had been classified as races 1, 2, 3, 4, 5, 6, 8, and 9 (Table 1).

The presence of the phaseolotoxin gene cluster and the ROCT gene were analyzed for all the strains of the three collections. Peet et al. (12) and Zhang et al. (23) reported that genes involved in phaseolotoxin production are clustered. Zhang et al. (23) described a cosmid (pHK120) with a 25-kb P. syringae pv. phaseolicola genomic insert which complements all Tox- mutants tested and that can be divided into al least eight transcriptional loci (phtA to phtH). The sequence analysis of the region encompassing the phtE locus (24) revealed six putative ORFs transcribed as one large (6-kb) transcript and indicating that the ORFs constitute an operon (Fig. 1). Previously, Peet et al. (12) reported that another genomic cosmid (pRCP17) containing a 24-kb insert harbors the ROCT gene (argK) and restores toxin production to several random Tox- mutants. The comparison of pHK120 with pRCP17 indicated that the two clones have overlapping but nonidentical inserts and confirmed that the argK gene is physically linked to phaseolotoxin genes in the argK-tox cluster (24).



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  		FIG. 1. Map of the amplified regions used to detect the presence of the phaseolotoxin gene cluster and the phaseolotoxin-resistant OCTase gene (argK), also known as the argK-tox gene cluster. Open boxes indicate functional loci for phaseolotoxin production (A to H) and ORFs in the phtE locus (ORF1 to ORF6). Gene argK is located within a 5-kb DNA fragment following the H locus, but its precise location and orientation relative to the phaseolotoxin gene cluster are not known.

Prosen et al. (13) designed the oligonucleotides HM13 and HM6 in order to amplify by PCR a region required for toxin production. This region is 1,901 bp long and includes most of ORF5 and all of ORF6 in the phtE locus. We have designed another primer pair, PHTE-F (21-mer; 5'-AATATAGGCTTCAACTTCCTC-3') and PHTE-R (19-mer; 5'-CCAGGTCAACTCACTTCCG-3'), to amplify most of the remaining region of the phtE locus, including ORF1, ORF2, ORF3, and half of ORF4 in a 3,025-bp fragment (Fig. 1). The thermal profile for the PHTE-F and PHTE-R primers was 1 min at 94°C, 2 min at 55°C, and 4 min at 72°C for 30 cycles. The analysis of the ROCT gene was carried out using primers OCT-F and OCT-R, which were designed by Sawada et al. (16) and which amplify a 1,100-bp fragment, including the whole argK coding sequence (Fig. 1).

The analysis of the three collections of strains by PCR using the above primers revealed the presence of three groups (Table 1): (i) toxigenic strains showing the presence of phtE and argK sequences, (ii) nontoxigenic strains carrying the phtE and argK sequences (argK-tox cluster), and (iii) nontoxigenic strains that did not show the presence of the argK-tox cluster (Fig. 2).



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  		FIG. 2. Analysis of phaseolotoxin production using the E. coli inhibition assay (upper half) and the presence of the 1.9-kb HM band (Fig. 1) obtained from the phaseolotoxin gene clusters (bottom half) from several strains. The presence of the 3.0-kb PHTE band and the 1.1-kb OCT band followed the same pattern (data not shown). The molecular markers in the first lane are for HindIII-digested lambda phage DNA. Lanes: A, strain N1; B, strain 528; C, strain 508; D, strain 1375AT; E, strain 1375AS; F, strain 882S; G, strain 1281S; H, strain 1281T.

The second group included a race 1 strain (strain 557) from Spain and several strains of all races from the collection of C. Stevens or J. D. Taylor, except race 7. The amplification products obtained showed the expected sizes of the three sequences amplified according to the primers used, and no detectable size differences were observed among the analyzed strains. The 1,901-bp fragment amplified using primers HM13 and HM6 was sequenced from strains 1281AS, 1281AT, 1375AS, and 1375AT (where the superscript "S" and "T" indicate the collections of C. Stevens and J. D. Taylor, respectively), since they differ in levels of phaseolotoxin production (Table 1), but no base changes were observed among the 1,901 bp. Three other strains (2671A, 2656AT, and 2660A) revealed very weak phaseolotoxin production, as shown by a very small halo of E. coli growth inhibition, but their amplification products also showed a size identical to that obtained from phaseolotoxin producer strains.

All these strains from the second group were collected from infected plants more than 5 years ago, and they have been maintained in different laboratories, some of them for more than 15 years, such as 1281A, 1301A, 1302A, and 1375A (19). It is probable that during the course of these years they have accumulated random mutations in genes responsible for phaseolotoxin production other that those included in the 1,901-bp sequence. In this way, samples of the same strains (1281A, 1375A, and 2656A) maintained in different laboratories showed a different phenotype.

The third group of strains that did not show the amplification of the argK-tox gene cluster were analyzed by Southern blotting using EcoRI-digested genomic DNA and digoxigenin-labeled probes. Probes included all three fragments obtained from a toxigenic strain by amplification with the three primer pairs previously described. The negative hybridization results confirmed that all the samples of race 5 isolated by us and by C. Asensio in Spain did not carry the phtE locus of the phaseolotoxin gene cluster or the argK gene. As these genes are physically linked in the P. syringae pv. phaseolicola chromosome (23), our results indicate that a large deletion, including the deletion of several genes involved in phaseolotoxin and ROCT production, has occurred in these strains. The presence of this large deletion suggests a possible common origin for all race 5 strains that infect plants in the northern Spanish Plateau, one of the most important areas in common bean production in Spain and Europe today.

Studies by Sawada et al. (14, 15) have revealed that the argK-tox gene cluster is of foreign origin and that it has expanded its distribution by horizontal transmission to P. syringae quite recently from a bacterial species distantly related. The molecular mechanisms that caused the horizontal gene transfer and the genomic rearrangement have not been identified (15). Our results open the question of whether nontoxigenic race 5 is an old form of Pseudomonas before the acquisition of the argK-tox gene cluster or if the same mechanisms involved in such an acquisition have removed this DNA fragment from the genome of this strain.

The presence of wild nontoxigenic strains which cause halo blight but do not carry the argK-tox gene cluster represents a pitfall in current molecular methods to detect P. syringae pv. phaseolicola, since these strains cannot be detected by these methods in dry seeds and halo blight disease would be spread by sowing contaminated bean seeds. In particular, in areas in which such nontoxigenic strains are frequently found or the most frequent strains, such as in Spain, halo blight will be transmitted undetected to new bean fields. New specific DNA sequences are needed in order to develop new molecular methods to detect this pathogen species with a better resolution.


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ACKNOWLEDGMENTS
 
This work was supported by the European Community F.E.D.E.R. research project IF-0308-103-03.


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FOOTNOTES
 
* Corresponding author. Mailing address: Área de Genética, Facultad de Biología, Universidad de León, E-24071 León, Spain. Phone: 34-987291549. Fax: 34-987291501. E-mail: degcpp{at}unileon.es. Back


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Applied and Environmental Microbiology, August 2003, p. 4979-4982, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4979-4982.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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