In this study, three Xanthomonas species, XAP (NCPPB 581), XH (NCPPB 205) and XCZ (NCPPB 4326) were obtained from the National Collection of Plant Pathogenic Bacteria (NCPPB). All Xanthomonas species were cultured on LB agar (Difco, Detroit, MI, USA) and incubated at 28°C for 48 h. Yellow colonies of Xanthomonas were subcultured until pure colonies were obtained. Genomic DNA was extracted from bacterial cells using HiGene Genomic DNA Prep Kit (BIOFACT, Daejeon, Korea) according to the manufacturer’s instructions. The extracted DNA was confirmed by agarose gel (1%) electrophoresis and genomic DNA was then quantified by using PicoDrop™ Pico100 (PicoDrop Technologies, Cambridge, UK).

XAP, XH and XCZ genome sequences were determined on the Ion Torrent personal genome machine (Life Technologies, Korea). Libraries were produced using 1 μg of total genomic DNA and an Ion Xpress Plus fragment library kit comprising of the Ion Shear chemistry. During the emulsion PCR, each diluted library was used as the template for clonal amplification on Ion Sphere particles and the enriched amplification products were then loaded onto an Ion 316 chip and sequenced using 500 sequencing cycles. Five hundred sequencing cycles result in an average reading length of approximately 200 nucleotides. The MIRA program (version 4.0.2.1) was then used for de novo assembly. The genome projects for XAP, XH and XCZ are listed in the Genome OnLine Database (GOLD) as projects Gi0074998, Gi0075001 and Gi0074999, respectively. The JGI microbial genome automated annotation pipeline (Mavromatis et al., 2009) was used for the annotation of the selected genome followed by manual curation using the GenePRIMP software program. The predicted coding sequences (CDSs) were then translated and used to search the NCBI non redundant database and the TIGRFam, Uni-Prot, PRIAM, Pfam, COG, KEGG and InterPro databases. Additional functional annotations and gene predictions were performed by using the IMG-ER platform.

The bioinformatics approach was used to select target DNA sequences for Xanthomonas species-specific primer designing from the draft genome sequence DNA sequences were selected randomly and a standard nucleotide BLAST search was performed on the NCBI database (http://www.ncbi.nlm.nih.gov). Through BLAST analysis, unique regions in XAP, XH and XCZ genome were chosen to obtain specific target sequences for primer design. The ClustalW software (http://clustalw.ddbj.nig.ac.jp/top-e.html) was used to design specific primers from the obtained consensus sequences, following multiple alignments of several Xanthomonas sequences.

A total of 36 primer sets (12 primer sets for each species) were tested for species specificity against XAP, XH and XCZ (data not shown). Among them, three primer pairs, XPN: XPN-F (5′-TGT TGG TGT CGA CGT ACA GC-3′)/XPN-R (5′-TGA CAC ACG CAG CGT AGG AG-3′), XHG:XHG-F (5′-ACC ACC ACG TCG CTG GGT TCC-3′)/XHG-R (5′-CGG CGC GCT GGC GAC CCA CCG-3′) and XZRN: XZRN-F(5′-TTC TTC TCC GTC CTC CCT CA-3′)/XZRN-R (5′-ACA GGT GCC CAC CGG TTA CT-3′) were then selected on the basis of their amplification results and specificity to respective strains. The specificity of these primers was assessed on genomic DNA extracted from pure bacterial cultures: 9 strains of XAP (NCPPB 581, 1939 and 3948), XH (NCPPB 205, 1622 and MAFF 301390), XCZ (NCPPB 4326, 2978 and MAFF 301641) from different origins, and 17 strains of allied Xanthomonas species (Table 1). Furthermore, these primers were also tested for specificity against the host DNA and the microbial community of the host surface. PCR amplification was performed in a total volume of 20 μl containing 1× reaction buffer, 0.4 μl dNTP (10 mM), 0.1 μl Taq (Solg™ h-Taq DNA Polymerase), 1 μl (10 pm/μl) primers, and 1 μl (10 ng/μl) of DNA. The PCR program for XHG and XPN primer sets consisted of an initial denaturation at 95°C for 5 min followed by 35 cycles at 95°C for 30 s, 65°C for 20 s and 72°C for 30 s with a final extension step at 72°C for 5 min. For the primer set XZRN, the amplification conditions were as follows: initial denaturation at 95°C for 5 min followed by 35 cycles at 95°C for 30 s, 60°C for 20 s and 72°C for 30 s with a final extension step at 72°C for 5 min. The amplified PCR products were separated by gel electrophoresis using 1.5% agarose. The gel was stained with ethidium bromide and visualized with a UV transilluminator.

Three primer sets XPN, XHG and XZRN were selected based on their high specificity to XAP, XH and XCZ, respectively. To check their sensitivity, a test was performed using a 10 fold dilutions of purified genomic DNA from XAP, XH and XCZ strains. The concentration of the samples used to test the specificity of the primers was 10 ng/μl. The XAP, XH and XCZ genomic DNA was then diluted to 10⁰, 10⁻¹, 10⁻², 10⁻³, and 10⁻⁴ ng/μl with DDW to test primer sensitivity. Furthermore, to check the efficiency of the primers to amplify XAP, XH and XCZ from four symptomless plants (E. pulcherrima, H. orientalis, M. armeniacum, and Z. aethiopica), plant tissues (0.2 g) from four host plants, and their respective pathogenic bacterial cells (1×10⁹ cfu/μl) were mixed and subjected to grinding. Genomic DNA was extracted from these mixtures by using HiGene Genomic DNA Prep Kit (BIOFACT) following the manufacturer’s instruction. The PCR efficiency of the three primer sets, XHG, XPN and XZRN, was checked on these DNA mixtures.

The properties and statistical analyses for the genome of the three Xanthomonas species, XAP, XH and XCZ, are summarized in Table 2. The genes distribution into COG functional categories for each genome is detailed in Table 3 and Fig. 1. The genome of XAP, XH and XCZ consists of single chromosomes and contains 5,221, 4,395 and 7,986 protein coding genes, respectively. Moreover, a total of 3,868, 3,318 and 4,754 protein coding genes were assigned a putative function (Table 2). These species showed genomic similarities with previously reported Xanthomonas species, X. oryzae pv. oryzae, X. campestris pv. vesicatoria and X. fuscans subsp. fuscans (Darrasse et al., 2013; Ochiai et al., 2005; Thieme et al., 2005). Similarly, high G+C contents were found in XAP, XH and XCZ, which is a common characteristic of most genera within the Xanthomonadaceae family (Darrasse et al., 2013). Furthermore, XAP, XH and XCZ genomic information facilitated the selection of species-specific primers based on variable regions in their genome. In addition, previous reports showed that, for the prevention of bacterial disease dissemination, early detection of Xanthomonas species is the most effective measure and conventional protocols carried out by the phytosanitary authorities are laborious, costly and excessively time consuming, which are major drawbacks for extensive surveillance of these pathogens (Lopez et al., 2003). However, over the last two decades, many DNA-based approaches, mostly PCR-based techniques, have been used for the detection of various pathogenic bacteria in general and Xanthomonas species in particular (Palacio-Bielsa, 2009). These PCR assays have been implemented successfully for the detection and identification of economically important Xanthomonas species such as X. axonopodis pv. dieffenbachiae, X. axonopodis pv. citri, X. citri subsp. citri, X. campestris pv. citri and X. fragariae (Adikini et al., 2011). Furthermore, previous studies also reported that Xanthomonas pathovars are difficult to differentiate as they are almost indistinguishable in bacteriological and biochemical traits (Bradbury, 1984).

All 36 primer sets (12 for each strain) tested amplified against their respective strains (data not shown). The primer sets, XPN, XHG and XZRN, were then selected based on their best amplification results and specificity to their respective strains. The expected band of 966 bp, 1,049 bp and 328 bp for the three primer sets XHG, XPN and XZRN, respectively, were obtained (Fig. 1). These primer sets also allowed the amplification of 9 strains of XAP, XH and XCZ (data not shown). However, the evaluation results against 17 allied Xanthomonas species showed negative amplification with all tested strains. Consistent positive amplification was only achieved in their respective strains (XAP, XH and XCZ) (data not shown). Moreover, no amplification was observed when assessing these primer pairs against their respective host DNAs and microbial community of the host surface (Fig. 2).

According to these results, these primers are specific to XAP, XH and XCZ. Thus, they can be used for the detection and routine diagnosis of these Xanthomonas strains in the field. To date, there is no report of species-specific primers for the detection of these species, although, many techniques, including DNA assays, are available for the detection of Xanthomonas species, which are mainly based on 16S ribosomal DNA amplification (Louws et al., 1994). However, these methods are not suitable for specific detection at the species and pathovar level. Therefore, in this study, we successfully developed species-specific primer pairs, which showed high specificity for the detection of these pathogens. The specificity of these primers relies upon the unique features of the template sequences that were used for primer designing. The XPN, XHG and XZRN primers were randomly designed from draft genome sequences. The portions of the sequence unique to XAP, XH and XCZ were identified by multiple sequence alignment and then used for these species-specific primer designing. The respective genes are nucleoside hydrolase for primer XPN, glucosylphosphate transferase for XHG and resistance-Nodulation-Cell Division (RND) for XZRN. The lack of amplification in all allied Xanthomonas spp. demonstrates a high degree of specificity for these primers and their ability to consistently detect their respective species.

For pathogen quarantine and inspection, primer sets are often required to be not only specific to the templates, but also sensitive to small quantities of the pathogens. Therefore, these primers were found to be highly sensitive and could be used to detect small quantity of bacterial DNA. The detection limit for the primers, XPN and XHG, was 10⁻² ng/μl, whereas XZRN was more sensitive and allowed the detection of 10⁻³ ng/μl of bacterial DNA per reaction (Fig. 3). Furthermore, the PCR efficiency in bacterial and host cell artificial mixture was determined. All three primer sets showed positive results and the specific PCR fragment for each primer set, XPN, XHG and XZRN, was amplified from all four hosts and bacterial cell mixture (data not shown). The ability of these primer sets to detect XAP, XH and XCZ pathogen in small quantities suggests that this PCR assay can be used to detect these species in latently infected plants.

Ultimately, the PCR assay described here is a quick, sensitive and more suitable diagnostic tool for the detection of these pathogens. This selective and sensitive method circumvents many problems associated with the current detection techniques, enabling the reliable detection of XAP, XH and XCZ at early stages of infection and help monitor diseases.