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University of Florida, Citrus Research and Education Center (CREC), 700 Experiment Station Road, Lake Alfred, FL 33850-2299, USA
Correspondence
James H. Graham
jhg{at}lal.ufl.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Published online ahead of print on 18 September 2003 as DOI 10.1099/ijs.0.02784-0.
The GenBank/EMBL/DDBJ accession numbers for the lrp gene sequences determined in this study are AY227394–AY227435.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Much revision of the classification of xanthomonads into several species and infraspecific pathovars has been proposed in recent years (Dye & Lelliott, 1974; Vauterin et al., 1995, 2000; Vauterin & Swings, 1997; Schaad et al., 2000). Despite the broad host range of and the importance of the diseases caused by xanthomonads, taxonomy and classification of this bacterial group remain controversial (Vauterin & Swings, 1997; Schaad et al., 2000; Vauterin et al., 2000; Young et al., 2001). Accurate classification of Xanthomonas species is not only important for basic knowledge of the phylogeny of this genus, but also has practical implications for global regulation of exotic diseases that are caused by these plant pathogens. Precise discrimination of pathovars and strains of Xanthomonas axonopodis that cause citrus bacterial canker has been particularly important for risk assessment in eradication programmes for this leaf- and fruit-spotting disease in Florida and elsewhere. Misidentification and unclear classification of xanthomonads have occurred in the past, resulting in unnecessary quarantine regulations and economic losses (Schoulties et al., 1987; Gabriel et al., 1989; Graham & Gottwald, 1990; Graham et al., 1992).
Among DNA-based methods for bacterial characterization, DNA–DNA reassociation is considered to be a major determinant for definition of bacterial species; a hybridization value of 70 % or greater has been used to group related pathovars into species of Xanthomonas (Wayne et al., 1987; Vauterin et al., 1995; Louws et al., 1999). Nevertheless, several other criteria have also been applied for differentiation at the species, pathovar or strain levels (Van den Mooter & Swings, 1990; Vauterin et al., 1991, 1995). In particular, comparative sequencing of rDNA subunits has been used to study the phylogeny of Xanthomonas and Pseudomonas, as they are functionally and evolutionarily conserved genes (Louws et al., 1999). However, highly restricted variability has been shown in 16S rDNA within the genus Xanthomonas: all strains that showed 70 % or more DNA–DNA homology exhibited 100 % 16S rDNA sequence similarity (Hauben et al., 1997). Better discrimination was obtained by analysis of the 16S–23S rDNA intergenic spacer (ITS) sequence, which showed approximately ninefold higher diversity than 16S rDNA (Gonçalves & Rosato, 2002). Moreover, combined analysis of ITS and 16S rDNA sequences allowed discrimination of different Xanthomonas spp. that cause citrus diseases (Cubero & Graham, 2002).
To deal with the limitations of rDNA-based methods, amplification of repetitive sequences (rep-PCR) has been used to confirm classification among Xanthomonas species and pathovars (Louws et al., 1994, 1995, 1999; Opgenorth et al., 1996; Pooler et al., 1996; Rademaker et al., 2000; Vauterin et al., 2000).
Previously, fingerprints from enterobacterial repetitive intergenic consensus (ERIC)- and BOX-PCRs were used to define strain genotypes and to trace the origin of strains that cause citrus bacterial canker in Florida (Cubero & Graham, 2002). Among the different fingerprints that were obtained by ERIC-PCR, a characteristic pattern was demonstrated for unusual strains with a limited host range that differed from type A strains, which have a wide host range. These strains from south-west Asia and Florida were designated X. axonopodis pv. citri types A* and Aw, respectively (Vernière et al., 1998; Sun et al., 2000). Recent studies confirmed the coexistence of A* strains with common A strains in Iran (Mohammadi et al., 2001; Khodakaramian & Swings, 2002).
In this study, we report that polymorphisms between the wide- and narrow-host-range strains within X. axonopodis pv. citri are partially related to differences in the sequence of a transcriptional regulator, the leucine-responsive regulatory protein (lrp) gene. The lrp gene was then detected in a selected group of Xanthomonas spp. and pathovars and differential sequence analysis was used to classify and confirm previous concepts of phylogenetic relationships among Xanthomonas spp. The overall aim of this work is to establish a new tool for polyphasic analysis of the genus Xanthomonas. This approach complements current concepts that are based on 16S rDNA sequence and rep-PCR analyses.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Strains were grown on nutrient agar plates for 48 h and bacterial cells were scraped from the plates and suspended in water. Quarantined organisms were processed as heat-killed bacteria in water suspensions, to comply with regulations for phytopathogens. DNA was extracted from bacterial cells with a single phenol/chloroform/isoamyl alcohol step, precipitated in ethanol and resuspended in ultrapure water. DNA was stored at -20 °C until use.
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) and a PCR DIG-labelling mix, according to the manufacturer's instructions (Roche).
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). Hybridization was detected with the chemiluminescent substrate CSPD (Roche) according to the manufacturer's instructions, followed by autoradiography by using medical X-ray film (Fuji).
PCR amplification.
Primers used in this study are listed in Table 2
. DNA from strains shown in Table 1 was used as the target for PCRs. ERIC-PCR was performed as described previously, using X. axonopodis pv. citri strains A, A* and Aw (Cubero & Graham, 2002). PCR with primers J-lrp1 and J-lrp2 was performed in 25 µl mixtures that contained 1x Taq buffer, 3 mM MgCl2, 0·1 µM each primer, 0·2 mM each dNTP and 1 U Taq polymerase (Invitrogen). PCR conditions for amplification consisted of 93 °C for 30 s, 58 °C for 30 s and 72 °C for 45 s for 40 cycles, plus an initial step of 94 °C for 5 min and a final step of 72 °C for 10 min. PCR with primers J-lrp3 or J-lrp6b (upstream) and J-lrp5 (downstream) was performed in 50 µl mixtures with the same reaction concentrations as described above. Amplification conditions in this case consisted of 93 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min for 40 cycles, plus an initial step of 94 °C for 5 min and a final step of 72 °C for 10 min. Products were visualized under UV light in 2 % agarose gels stained with ethidium bromide.
Sequencing of lrp gene.
After ERIC-PCR analysis, a band representing about 375 bp of the lrp gene sequence that varied among the A, A* or Aw strains of X. axonopodis pv. citri was extracted from a 2 % agarose gel by using the Wizard PCR Preps DNA Purification system, following the manufacturer's instructions (Promega). The isolated fragment was reamplified by PCR using primers ERIC1R and ERIC2 (Hulton et al., 1991; Versalovic et al., 1991; Louws et al., 1994) in a 50 µl mixture that contained 1x Taq buffer, 3 mM MgCl2, 0·1 µM each primer, 0·2 mM each dNTP and 1 U Taq polymerase (Invitrogen). Amplification conditions were the same as described above for primers J-lrp3 or J-lrp6b and J-lrp5. Amplicons were purified by the Wizard PCR Preps DNA Purification system (Promega) and sequenced at the DNA sequencing core laboratory of the University of Florida, Gainesville, FL, USA. Results were confirmed by sequencing the complete region in both forward and reverse directions by using primers ERIC1R and ERIC2.
For bacterial strains listed in Table 1, primers J-lrp3 or J-lrp6b and J-lrp5 were used to amplify a fragment of about 600 bp that included the complete sequence of the lrp gene. Both strands of purified PCR products were sequenced as above, by using primers J-lrp3 or J-lrp6b and J-lrp5. The 3' termini of primers J-lrp3 and J-lrp6b correspond to positions upstream of the lrp gene, whilst the 5' terminus of primer J-lrp5 corresponds to the 3' end of the lrp gene. The J-lrp5 sequence and sequences upstream of the start codon of the lrp gene were not included in further analysis.
Sequence analysis.
Sequences were aligned and compared with those available in GenBank for Ralstonia solanacearum (accession no. AL646061), Pseudomonas aeruginosa (AE004943), X. campestris pv. campestris (AE012486) and X. axonopodis pv. citri (AE012019) (Stover et al., 2000; da Silva et al., 2002; Salanoubat et al., 2002) by using the programs BioEdit Sequence Alignment Editor (Hall, 1999) and CLUSTAL W (Higgins & Sharp, 1988; Thompson et al., 1994). Phylogenetic variation in lrp nucleotide and amino acid sequences from different xanthomonads was explored with the program PHYLPRO, which graphically displays phylogenetic correlation over the entire length of a set of aligned sequences (Weiller, 1998). The analytical program MEGA version 2.1 (Kumar et al., 2001) was used to construct a dendrogram by using the neighbour-joining method (Saitou & Nei, 1987). The model of Jukes & Cantor (1969) and ‘p’ distances were used to calculate genetic pairwise distances among sequences at the nucleotide and amino acid levels, respectively. Reliability of clusters was evaluated by bootstrapping with 1000 replicates. Pairwise sequence comparisons were also made by using the procedure of Li et al. (1985) to estimate the number of synonymous substitutions per synonymous site (Ks) and the number of non-synonymous substitutions per non-synonymous site (Ka), by using MEGA 2.1.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The 375 bp product in A strains was identified as an lrp gene sequence, based on alignment with known sequences in GenBank after a BLAST search. The lrp gene was deduced after comparison not only with data available in GenBank for the genus Xanthomonas, such as strains 306 of X. axonopodis pv. citri and ATCC 33913T of X. campestris pv. campestris, but also with species in other genera, such as P. aeruginosa and R. solanacearum. The lrp nucleotide sequence was translated to the amino acid sequence and aligned with those in GenBank. Amino acid sequences were even more conserved, showing affinities with bacteria as distantly related as Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis.
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). Thus, the absence of the 375 bp amplification product in ERIC-PCR fingerprints of Aw and A* strains was not due to absence of the lrp gene, but to differences in lrp sequence between wide- and narrow-host-range strains.
Based on the complete genomic sequence of X. axonopodis pv. citri (da Silva et al., 2002), another set of primers (J-lrp3 or J-lrp6b as upstream primer and J-lrp5 as downstream primer) was designed to obtain the entire lrp gene sequence. Analysis of the entire lrp gene from A and A*/Aw strains of X. axonopodis pv. citri revealed some sequence differences between these strains. However, after translation to amino acids, no differences in protein sequence were detected between wide- and narrow-host-range strains. Therefore, variation in lrp sequences was not related directly to restriction of host range.
Detection of the lrp gene in Xanthomonas species
Representative strains of 15 genomic species, described by Vauterin et al. (1995), within the three phylogenetic lineages of Xanthomonas by analysis of the 16S rDNA sequence (Hauben et al., 1997) were examined by hybridization using the J-lrp1/J-lrp2 probe (Table 1). The lrp sequence was detected in all Xanthomonas strains. No hybridization was detected for an Agrobacterium strain used in the analysis as a negative control, which confirms the absence of a homologous gene; however, Agrobacterium possesses a putR gene with a function that is analogous to that of the lrp gene (Chen & Calvo, 2002).
By using the J-lrp1 and J-lrp2 primer set, the lrp gene was amplified from all Xanthomonas strains examined except Xanthomonas hyacinthi (Table 1). Presence of the lrp gene in X. hyacinthi was corroborated by the hybridization analysis described above (Table 1) and successful amplification was demonstrated with the combination of primers J-lrp1 and J-lrp5 (results not shown). This confirms the presence of the central core of the lrp gene in all Xanthomonas spp. examined in this study (Table 1) and suggests a universal distribution of the lrp gene in this genus.
Although DNA from most xanthomonad strains yielded amplification products with the J-lrp1 and J-lrp2 primer set, some failed to do so with primers J-lrp3 or J-lrp6b and J-lrp5, which were designed to amplify the entire lrp gene (Table 1). Therefore, two groups of xanthomonads are recognized, based on this PCR analysis of the lrp sequence. One group includes most of the species and pathovars of Xanthomonas whose complete lrp sequence could be amplified by using primers J-lrp3 or J-lrp6b and J-lrp5, and which corresponds with the main group that is defined by 16S rDNA sequence similarity [X. campestris core or cluster 1 (Hauben et al., 1997)]. The other groups of strains, which were not amplified by the J-lrp3 or J-lrp6b and J-lrp5 primer set, belong to Xanthomonas sacchari cluster 2 and Xanthomonas albilineans cluster 3 (Hauben et al., 1997). Other primers that were based on sequences at the 5' and 3' termini of the lrp gene in strain 306 of X. axonopodis pv. citri were designed and used unsuccessfully to amplify the complete gene in the X. sacchari and X. albilineans groups (results not shown).
Analysis of the lrp sequence in Xanthomonas species
Phylogenetic correlation over the entire length of aligned lrp sequences was displayed by using the PHYLPRO program (Weiller, 1998) (Fig. 2). Phylogenetic profiles showed high correlation in the central core of the lrp nucleotide sequence, but lower correlation in the 5' and 3' ends of the sequences. Variability in the 5' and 3' ends of the nucleotide sequence probably explains the failure of the primer sets to amplify this gene in other Xanthomonas spp., such as the X. sacchari and X. albilineans groups.
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; Chen & Calvo, 2002). The Lrp protein in E. coli contains three functional domains: an N-terminal domain that has a putative DNA-binding motif, a middle domain that is responsible for transcriptional activation and a C-terminal domain that is required for the response to leucine (Platko & Calvo, 1993). Our findings for xanthomonads confirm that the most conserved region of the sequence encodes the domain for transcriptional activation, whereas changes that appear to be species-specific occur in the sequences that encode the N- and C-termini of the protein. Xanthomonas spp. infect many host plants, each with specific conditions for growth of the bacterium in the plant. The requirement for host-specific species and pathovars of Xanthomonas to respond to different plant environments may be a selection pressure for changes in the N- and C-termini of the Lrp protein.
Classification of Xanthomonas species according to lrp sequences
Sequence analysis of the lrp gene revealed a high level of similarity within the genus Xanthomonas. The mean similarity level (±SD) was 94·2±0·7 %, with a maximum of 47 nucleotide differences over a sequence of 459 nt. Similarities between the xanthomonad group and P. aeruginosa or R. solanacearum were 80·5±2·3 and 61·5±3·5 %, with a maximum of 89 and 136 nucleotide differences, respectively. Similarity of amino acid sequences within the Xanthomonas group was 98·2±0·6 % (maximum number of differences, 10 over 153 aa). Similarity of amino acid sequences between xanthomonads and P. aeruginosa or R. solanacearum was 85·1±2·7 or 65·5±3·7 % (maximum of 28 or 55 amino acid differences), respectively.
In 346 of 459 nucleotide positions, the lrp gene sequences of Xanthomonas strains were identical. At the remaining 113 positions, most of the differences were due to a change in the third position of the codon. Thus, the mean similarity level based on analysis of only the third codon position was 84·9±1·7 %, and in the first and second codon positions, similarity was 97·6±0·7 and 99·2±0·4 %, respectively.
Results of the analysis of synonymous and non-synonymous substitutions in the lrp gene confirmed a high stability of the lrp gene, not only within the genus Xanthomonas, but also compared to other bacteria. Within the genus Xanthomonas, Ks (number of synonymous substitutions per synonymous site) was 0·23±0·03, whilst Ka (number of non-synonymous substitutions per non-synonymous site) was 0·01±0·00; the maximum values of Ks and Ka were 0·48±0·09 and 0·03±0·01, respectively. Stability of lrp gene codons in Xanthomonas spp. was similar to that demonstrated for several enteric bacteria, including E. coli, Enterobacter aerogenes, Klebsiella aerogenes and Salmonella typhimurium (Friedberg et al., 1995).
Variability in the lrp gene was higher than that in 16S rDNA alone or 16S rDNA combined with the rDNA ITS region (Hauben et al., 1997; Cubero & Graham, 2002) and slightly lower than that in the ITS region alone (Gonçalves & Rosato, 2002). Thus, the moderate variability in the lrp gene sequence may be used to investigate phylogenetic relationships and to clarify the classification of xanthomonads. Although rDNA sequences, such as 16S rDNA and the ITS region, have been used successfully to identify and classify xanthomonads (Hauben et al., 1997; Gonçalves & Rosato, 2002; Cubero & Graham, 2002), a more detailed and accurate understanding of the relationships among Xanthomonas species and pathovars may be achieved by analysis of other stable and independent genes, such as lrp.
A dendrogram based on pairwise comparison of all lrp sequences (Fig. 3) showed that Xanthomonas species grouped together, with P. aeruginosa and R. solanacearum as outgroups. The largest cluster within Xanthomonas was cluster 1 (Fig. 3), which encompassed all strains that are presently proposed as pathovars of X. axonopodis (Vauterin et al., 2000). Within cluster 1, two groups were discriminated. One group included all strains that cause citrus bacterial canker and also X. axonopodis pv. malvacearum, the cause of bacterial blight in cotton (group 1, Fig. 3). The close relationship between the genomes of citrus bacterial canker and cotton blight strains was first revealed by DNA–DNA hybridization (Egel et al., 1991). Pathovars citri and aurantifolii, which cause canker in citrus, were in two different subgroups, confirming their somewhat distant phylogenetic relationships based on DNA–DNA hybridization (Egel et al., 1991). Within pathovar citri, two types of strain sequences were distinguished: strains with a narrow host range (A* and Aw) and those with a wide host range (A) on citrus. In pathovar aurantifolii, two types of lrp sequence corresponded to B and C strains. Classification of canker-producing strains based on lrp sequence analysis matched with the groups that were established previously by rDNA, DNA–DNA hybridization and rep-PCR analyses (Egel et al., 1991; Cubero & Graham, 2002). Moreover, lrp gene analysis confirmed that Aw strains are a type of X. axonopodis pv. citri A strain that is related closely to the other restricted-host-range strains from south-west Asia, designated A* (Vernière et al., 1998; Cubero & Graham, 2002).
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), known as pathovar citrumelo, was differentiated clearly from citrus bacterial canker strains. Pathovar citrumelo strains have been postulated to be related closely to Xanthomonas strains that have been isolated from other hosts, including Ficus benjamina and Dieffenbachia spp. (X. axonopodis pv. fici and X. axonopodis pv. dieffenbachiae), based on pathological, phenotypic and genetic traits of these citrus and non-citrus strains (Graham et al., 1990). Group 2 based on the lrp gene supports this hypothesis (Fig. 3).
Based on analysis of lrp sequences, other Xanthomonas species were confirmed to have slightly different sequences from each other and to be related moderately to X. axonopodis. Xanthomonas codiaei from poinsettia showed marked sequence variation compared to other Xanthomonas species, as reflected by a similarity of 92·7±1·3 % to X. axonopodis cluster 1 and by a separate position in the dendrogram (Fig. 3). A unique sequence for Xanthomonas oryzae was 94·0±1·1 % similar to cluster 1. Xanthomonas vesicatoria type B and Xanthomonas gardneri sequences were 92·7±1·2 and 91·4±1·3 % similar to cluster 1 and 90·7±1·4 similar to each other. Two strains of X. campestris were only 91·1±1·4 % similar to the X. axonopodis cluster. Distinct sequences were found for other Xanthomonas species, as shown in Fig. 3.
Within each pathovar, lrp sequences were identical, except for the two strains of X. axonopodis pv. dieffenbachiae and two strains of X. vesicatoria type B. This finding for X. vesicatoria type B confirmed the heterogeneity of this group, which was described by using other approaches (Louws et al., 1995; Jones et al., 1998). Strains of X. axonopodis pv. citrumelo had an identical lrp sequence to that of X. axonopodis pv. fici, which confirmed that there is adaptation of very similar strains to a range of host plants (Graham et al., 1990). This may also be the case for X. axonopodis pv. malvacearum and X. axonopodis pv. citri. Two races of X. oryzae from rice had the same sequence, whereas xanthomonads that attack cucurbit hosts, Xanthomonas melonis and Xanthomonas cucurbitae, showed closely related lrp gene sequences.
Xanthomonas spp. that cause bacterial spot on tomato and pepper were grouped in different clusters. The A and C types of X. axonopodis pv. vesicatoria were placed within the X. axonopodis cluster or cluster 1, and the B and D types (X. gardneri) showed different sequences from each other. These results were in accordance with the recent classification of A and C types on tomato and pepper as X. axonopodis pathovars that belong to the same species, but to different subgroups (Bouzar et al., 1999; Jones et al., 2000), and types B and D as separate species that were named X. vesicatoria and X. gardneri, respectively (Jones et al., 1998).
Overall, the difference between X. campestris and other Xanthomonas species supported the proposed reclassification of the genus (Vauterin et al., 2000). The genus Xanthomonas is composed both of groups of strains that are closely related genetically, but have different host ranges, and by strains with the same host range that are phylogenetically distant. An example of the first case is represented by the species X. axonopodis, which includes strains that infect a diversity of hosts from woody plants, such as citrus, to herbaceous plants, such as tomato or pepper. The second case is represented by strains that produce bacterial spot in tomato and pepper, which comprise at least three genetically distinct species within the genus Xanthomonas. Analysis of lrp gene or 16S rDNA sequences confirmed the concept of a common ancestor of X. axonopodis that evolved to adapt to different hosts, whereas less related strains of Xanthomonas spp. evolved to adapt to the same host, as occurred with pepper and tomato spot strains.
Negative selection pressure that restricts variability in the lrp gene is revealed in the case of the X. axonopodis cluster. Eleven lrp nucleotide sequences were contained in this cluster, but only four different Lrp protein sequences, with a maximum difference of 2 aa, were found. Lrp amino acid sequences have been reported to be highly adapted to their functions, which means that almost all amino acid changes to the protein are less efficient than the wild-type (Friedberg et al., 1995). In addition, Lrp is a major regulatory protein that is involved in the control of at least 75 genes in E. coli (Newman et al., 1992). Genes that encode proteins with a major role in the molecular metabolism of the bacterium may provide more precise evidence for the relationships among closely allied xanthomonads. These essential genes are unlikely to be transferred and the corresponding products experience long-term coevolution, along with the rest of the cellular machinery (Lawrence, 1999).
Analysis of the lrp gene in Xanthomonas provides independent confirmation of the relationships among pathogenic subgroups of this important genus. Because of its universal presence in this genus, as well as in other Gram-negative bacteria, and the fact that horizontal transmission is improbable, analysis of the lrp gene may be a widely adaptable tool for polyphasic taxonomic studies of bacteria. This method should be particularly valuable as a complement to current rDNA- and rep-PCR-based approaches, due to its capability for high resolution at the specific and infraspecific levels.
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ACKNOWLEDGEMENTS |
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