Introduction

Bacterial leaf spot, an important disease in cruciferous plants (Brassicaceae), is caused by Pseudomonas syringae pv. maculicola (Psm), and it globally occurs in plants such as cabbage, cauliflower, broccoli, Chinese cabbage, turnip, radish, and Japanese radish (Peters et al. 2004; Takikawa and Takahashi 2014; Zhao et al. 2000). In 1911, McCulloch described the occurrence of a spot disease on cauliflower grown in Virginia, USA, and this was the first report of the disease (McCulloch 1911; Takikawa and Takahashi 2014). The pathogenic bacteria invades the stomata and wounds, creates small chlorotic and necrotic spots with water-soaked regions on the leaves, and rarely causes severe blight symptoms (Peters et al. 2004; Takikawa and Takahashi 2014; Zhao et al. 2000). The pathogenic bacteria are classified into three groups according to bacteriological characteristics and the host that they are isolated from (Takikawa and Takahashi 2014; Zhao et al. 2000). In addition, Psm is considered a heterogeneous pathovar (Peters et al. 2004; Wiebe and Campbell 1993), closely resembling pathovar tomato (Psto) in bacteriological characteristics and genetic similarity. As both pathogens can infect hosts of each other when artificially inoculated, it has been argued that they should be separated (Hendson et al. 1992; Takikawa et al. 1994; Wiebe and Campbell 1993). Gironde and Manceau (2012) showed that there are at least eight genetic lineages among Psm and Psto isolates and considered that there is a relationship between the genetic lineages and isolation sources.

In 2002, bacterial leaf blight was newly reported in broccoli, and P. syringae pv. alisalensis was proposed as the causative bacterium. The species was then changed to P. cannabina (Bull et al. 2010; Cintas et al. 2002). P. cannabina pv. alisalensis (Pca) has been isolated from cabbage, radish, and arugula globally (Bull et al. 2004; Ishiyama et al. 2013; Mauzey et al. 2011; Rubio et al. 2012; Sarris et al. 2010; Takahashi et al. 2013; Wechter et al. 2010). Bacterial leaf blight of cabbage, Chinese cabbage, and Japanese radish has also been reported in Japan (Ishiyama et al. 2013; Takahashi et al. 2013), and some strains isolated and once identified as Psm were reclassified as Pca (Takahashi et al. 2013; Takikawa and Takahashi 2014). Similar to Psm, the pathogen invades stomata and wounds, creates chlorotic and necrotic spots with water-soaked areas in the leaves, but it tends to cause large coalescing blight lesions extending up to several centimeters in length and sometimes causes severe blight symptoms (Takikawa and Takahashi 2014). Pca is classified into two types based on the differences in host virulence and genetic diversity (Sarris et al. 2013; Takikawa and Takahashi 2014). Furthermore, the pathogen has been reported to naturally infect bristle oat cultivated as a green manure crop, which is a problem in the rotation of cruciferous crops and bristle oat (Ishiyama et al. 2013).

In Japan, the occurrence of black-brown discoloration in the core of the stem root and the occurrence of bacterial spots on the surface of the stem root of Japanese radish caused by Psm and Pca is a problem (Horinouchi et al. 2009; Otani 2016; Takeuchi et al. 1989). The discoloration in the core of the stem root is difficult to distinguish externally, but it has been discovered in agricultural products, leading to consumer complaints. An outbreak of black-brown discoloration in the stem root is mainly caused by infection from the petiole base, and the leaf spot symptom does not always lead to black-brown discoloration in the stem root (Otani 2016). Thus, it is difficult to diagnose the occurrence of such symptoms in the field, and farmers are wary of the occurrence of bacterial leaf spots and bacterial leaf bright. Similar to other P. syringae group bacteria, both Pca and Psm are considered seed borne (Schofield et al. 2012; Takimoto 1931), and seed companies encounter difficulty in responding to this disease because seed transmission of the disease is suspected.

Causing disease in host plants and eliciting a hypersensitive response in non-host plants are fundamental features controlled by the hrp genes in P. syringae (Bonas 1996; He et al. 1993; Hueck 1998; Preston et al. 1995). The hrp/hrc-encoded proteins are involved in a type-III secretion pathway (Jin and He 2001; Li et al. 2002). Inoue and Takikawa (2006) reported that P. syringae bacterial group could be classified into five groups based on differences in gene homology within the hrpZ operon. According to the present study, Psm and Pca belong to different groups, and it is possible to distinguish them using designed primers (Takahashi et al. 2013). However, many pathovars are in the same group, and cannot be readily distinguished, and nonspecific amplification may occur when this primer is used for the direct detection of bacterial cells (Yoshioka et al. 2020).

Zaccardelli et al. (2005) reported a primer designed for hrpZ that can be used to detect Psto, but the specificity among pathogenic types of P. syringae has not been sufficiently compared. In P. syringae, the hrp/hrc gene cluster is flanked by a unique exchangeable effector locus (EEL) and a conserved effector locus and comprises a pathogenicity island (Alfano et al. 2000). In this region, there are differences in DNA homology among P. syringae pathovars (Inoue and Takikawa 1999a, 2000, 2003). There are type III effector genes in this region, and the EEL, sandwiched between hrpK and tRNALeu, has different genetic repertoires depending on the strain (Charity et al. 2003; Deng et al. 2003). Our previous studies also confirmed that the EEL region has large differences in gene sequences among strains and low homology among P. syringae group bacteria (Inoue and Takikawa 1999a, b, 2003).

In the present study, for the purpose of developing a gene marker that distinguishes Psm and Pca, DNA sequences of EEL of Psm and Pca were newly determined, and the DNA sequences, including published information, were compared. As a result, we designed primers that distinguished Psm and Pca and confirmed their usefulness.

Materials and methods

Bacterial strains and culture conditions

Seventy-six strains of plant pathogenic bacteria, including eight strains of Pca, 13 strains of Psm, and 30 other P. syringae group strains, were used (Table 1). Xanthomonas strains were cultured on potato semisynthetic medium (PSA) (Wakimoto 1960) at 27 °C, and other plant pathogenic bacteria were cultured on PPGA medium (Nishiyama 1978) at 25 °C or 27 °C. These cultures were suspended in sterile distilled water at an optical density of 0.3 at 600 nm (~108 cfu (colony-forming units)/mL in each case) and appropriately diluted for the subsequent examinations. Rifampicin-resistant strains (KMrR-R03 derived from MAFF [Ministry of Agriculture, Forestry and Fisheries, Japan] 106179 and NMH-R1 derived from MAFF 106156) were obtained by modifying the method of Glandorf et al. (1992). The medium and culture temperature were changed to YPA containing 20 mg/L rifampicin and 25 °C.

Table 1 Bacterial strains
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Artificial inoculation of Japanese radish seeds

We used Japanese radish seeds (“Heian-hayabutori-tokinashi,” Takii, Kyoto, Japan), which have been found to have negligible contamination with Psm and Pca. Rifampicin-resistant strains KMrR-R03 and NMH-R1 were used for inoculation. Bacterial suspensions were prepared as described above. Approximately 5 mL of seeds was placed in a 100-mL beaker, 10 mL of the bacterial suspension was added, and air pressure was reduced at 0.02 MPa for 5 min. Thereafter, the liquid was removed, and the seeds were spread on paper towels and dried for 3 days in a clean room. To confirm the adhesion of the infected strain, an inoculated seed was immersed in 100 μL of sterile distilled water in a PCR tube and shaken for 2.5 h, and 10 μL of the immersion liquid was applied to yeast extract-peptone agar (YPA; 5 g yeast extract, 10 g peptone, 15 g agar, and 1000 mL of distilled water; pH 6.8) medium containing 20 ppm of rifampicin and 100 ppm of cycloheximide. The number of colonies formed was counted after 3 days at 25 °C. It was tested with 24 seeds.

Artificial inoculation of radish plants

In the inoculation test, radish (“Yukikomachi,” Sakata seed, Kanagawa, Japan), which is smaller than Japanese radish and shows similar lesions on the leaves, was used. Radish seeds were sown in a 7-cm pot and cultivated in a greenhouse for approximately 3 weeks. KMrR-R03, NMH-R1, and re-isolated strains from the inoculated seeds were used for inoculation. Six hours before inoculation, the plant was placed in a sealed container and placed in the dark at 22 °C. The plant was sprayed with 10 mL of approximately 107 cfu/mL bacterial suspension with 0.01% Tween20, which was placed in a sealed container again. The plant was kept in the dark at 22 °C for 24 h, and then incubated for 2 days under a 12-h light and 12-h dark photoperiod. Subsequently, it was taken out from the sealed container, and the occurrence of disease was investigated for 5–14 days after inoculation.

DNA manipulation

Genomic DNA for a PCR template was purified using NucleoSpin Tissue (MACHEREY-NAGEL, Düren, Germany) according to the manufacturer’s instructions. The purified DNA was diluted in 10 mM Tris-HCl buffer or 1 mM HEPES buffer and used as a PCR template. Amplification of the EEL in Psm strains MAFF 106179, 90S-4, and R101, and Pca strains MAFF 106156 and R-1 was performed according to the method of Deng et al. (2003). The nucleotide sequence of the DNA fragment amplified by PCR was determined using ABI3130XL (Thermo Fisher Scientific, Waltham, MA) with specific primers according to the manufacturer’s instructions. The EEL of P. syringae pv. spinaciae strain MAFF 211666 was not amplified using the method of Deng et al. (2003). Its DNA fragments were amplified by PCR using the specific primers (K0; 5′-GCAACCCAGGCCATTTACG-3′, Q004; 5′-TTTGGCCTGTGGTGCTTG-3′), and nucleotide sequences were analyzed as described above. DNA sequences have been registered in DNA Data Bank of Japan (Acc. No. LC593618- LC593623).

Primer design and PCR amplification

To design the primers, the DNA sequences derived from draft genome sequences of Psm PMC8301 and Pca MAFF 106179 (in the middle analysis), the DNA sequence of Psm PMC8301 (Acc. No. AB023074), Psto DC3000 (Acc. No. AB166796), and Pca ES4326 (Acc. No. CP047260) obtained from the database and determined above were used. Primers designed in the present study are shown in Table 2. Primers hrpK_fw1 and hrpK_fw2 were designed from the hrpK sequence of Pca. Primers MAC_rv1 and MAC_rv2 were designed for hopPtoB1 of Psm, and the predicted PCR products using hrpK_fw1 and MAC_rv1, and hrpK_fw2 and MAC_rv2 were 591 and 256 bp, respectively. Primers ALS_rv1 and ALS_rv2 were designed for hopX1 of Pca, and the predicted PCR products using hrpK_fw1 and ALS_rv1, and hrpK_fw2 and ALS_rv2 were 897 and 543 bp, respectively. As a PCR template, 5 μL of bacterial suspension or DNA solution was used. PCR amplification with hrpK_fw1, MAC_rv1, and ALS_rv1 (PsmPca_PS1) was performed as follows. The PCR mixture of volume 20 μL contained 0.5 U BIOTAQ DNA Polymerase (Bioline, London, UK), 1× reaction buffer, 1.5 mM MgCl2, 200 μM of each dNTP, 160 nM respective primers, and the template. The PCR was performed using the following thermocycling conditions: 95 °C for 3 min, followed by 30 cycles at 95 °C for 30 s, 58 °C for 25 s, and 72 °C for 30 s, and a final extension step at 72 °C for 5 min. PCR amplification with hrpK_fw2, MAC_rv2, and ALS_rv2 (PsmPca_PS2) was performed as follows. The contents of the mixture were the same as above. The PCR was performed using the following thermocycling conditions: 95 °C for 3 min, followed by 30 cycles at 95 °C for 30 s, 58 °C for 10 s, and 72 °C for 20 s, and a final extension step at 72 °C for 5 min. The amplified PCR products were visualized by electrophoresis on 1.5% or 3% agarose gels and stained with Midori Green advance (Nippon Genetics, Tokyo, Japan) or SYBR Safe DNA Gel Stain (Thermo Fisher Scientific, Waltham, MA, USA). To improve detection sensitivity, nested PCR was also performed. For this, we used PsmPca_PS1 in the first PCR and PsmPca_PS2 in the second PCR. The first PCR was performed as described above. In the second PCR, 1 μL of the first PCR product was added to 20 μL of the reaction mixture as a template.

Table 2 Primers list
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Detection of Psm and Pca by PCR in seeds

Either of one Pca- or Psm-inoculated seed or two (one Pca- and one Psm-inoculated) seeds was added to healthy seeds for a total of 1000 seeds and placed in a 300-mL conical flask. Each seed set was prepared in triplicate. One set was added to 30 mL of wash buffer (0.85% NaCl, 0.02% Tween 20) and shaken for 2.5 h, and approximately 10 mL of the immersion liquid was collected in a conical tube. DNA was purified from 1 mL of the liquid and eluted with 100 μL of 10 mM Tris-HCl buffer. Multiplex PCR with PsmPca_PS1 was performed using DNA solution diluted five volumes as the template. Thereafter, nested PCR with PsmPca_PS2 was performed. The seed immersion liquid was also applied to two YPA plates containing 20 ppm of rifampicin and 100 ppm of cycloheximide in a volume of 100 μL to count the bacterial density in the liquid. The number of colonies formed was counted after 3 days at 25 °C. This test was repeated three times.

Colony PCR

For the colony PCR, 4, 4, and 8 colonies formed on YPA plates, derived respectively from the seed immersion liquid containing one Psm-inoculated seed, one Pca-inoculated seed, and one Psm- and one Pca-inoculated seed in the above test, were used. A colony was picked with the tip of a toothpick and immersed in 25 μL of sterile distilled water. Multiplex PCR with PsmPca_PS1 was performed using 5 μL of the suspension diluted to five volumes as a template.

Detection of Psm and Pca by PCR from inoculated plants

Infected or non-infected parts of an inoculated leaf were cut into approximately 1-cm2 squares and ground in a 1.5-mL tube with 500 μL sterile distilled water. The suspension was centrifuged at 500×g for 1 min, and the supernatant was collected. DNA was purified from the supernatant and eluted with 100 μL of elution buffer. Multiplex PCR with PsmPca_PS1 was performed using DNA solution diluted in five volumes as a template. To confirm the presence of viable bacteria, a part of the supernatant was added to YPA medium. Two replicates were tested.

Results

Nucleotide sequence of EEL

The nucleotide sequences of EEL of Psm strains MAFF 106179, 90S-4, and R101, and Pca strains MAFF 106156 and R-1 were determined. The nucleotide sequence of Psm strain PMC8301 (Acc. No. AB023074) previously determined by us (Inoue and Takikawa 1999b) was also included in the analysis. The structure of EEL of Psm is shown in Fig. 1. In Psm strain 90S-4, an IS3-like sequence was inserted, and it had the same structure as Psto strain DC3000. The BLAST search showed that Psto strains B13-200 (Acc. No. CP019871) and PT23 (Acc. No. AB166796), P. syringae pv. avii strain CFBP3846 (Acc. No. LT963402), and P. syringae pv. persicae strain 5846 (Acc. No. AY147018) have a homologous region of length approximately 1 kbp adjacent to hrpK. In Psto strains B13-200 and PT23, there was an internal deletion of 315 bp in the front part of hopPstB1. In Pca, hopX1 was adjacent to hrpK. The BLAST search showed there was no homologous gene structure other than Pca ES4326 (Acc. No. DQ196428) in the GenBank database. In MAFF106156, an IS66-like sequence was inserted at a position 400 bp away from t-RNALeu, but it was not present in ES4326 and R-1. Based on the analyzed nucleotide sequences described above, hrpK_fw1 and hrpK_fw2 were designed in hrpK of Pca. In Psm, there were a few mismatches in hrpK_fw1 and hrpK_fw2. Psm-specific primers MAC_rv1 and MAC_rv2 were designed in hopPtoB1, and Pca-specific primers ALS_rv1 and ALS_rv2 were designed in hopX1.

Fig. 1
figure 1

Gene map of the exchangeable effector locus (EEL) of Pseudomonas syringae pv. maculicola (Psm)-related pathovars and P. cannabina pv. alisalensis and the location of the specific PCR primers that we designed. The horizontal lines indicate the following: thick black lines are homologous regions in all strains, black double lines are homologous regions to Psm, gray double lines are homologous regions other than Psm, and thin gray lines are not homologous. Thick arrows indicate the open reading frames and their direction and thin arrows indicate the designed primers and their direction

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Specificity of primers

Conventional PCR with hrpK_fw1 and MAC_rv1, and hrpK_fw2 and MAC_rv2 amplified approximately 590- and 260-bp long DNA fragments from Psm, Psto, and P. syringae pv. spinaciae (Supplementary Fig. S1a, b). Therefore, we tried to amplify the EEL of P. syringae pv. spinaciae but could not amplify it. An analysis of the partial nucleotide sequence of the EEL of P. syringae pv. spinaciae revealed homology with P. syringae pv. avii from hrpK to the outside of hopPtoB1 (Fig. 1). Conventional PCR with hrpK_fw1 and ALS_rv1, and hrpK_fw2 and ALS_rv2 amplified approximately 900- and 540-bp DNA fragments from Pca strains among the 76 strains of plant pathogenic bacteria, respectively (Supplementary Fig. S1c, d). Using multiplex PCR with PsmPca_PS1 or PsmPca_PS2, we identified Psm and related pathovars, Pca, and other plant pathogenic bacteria (Fig. 2). In the multiplex PCR using PsmPca_PS1 and PsmPca_PS2, amplification was confirmed up to a 5 × 103-fold dilution of the bacterial suspension (Supplementary Fig. S2). This corresponded to the presence of approximately 100 cfu per reaction. Using the nested PCR approach, the detection limits were limited to a few bacterial cells (Supplementary Fig. S3, Table S1).

Fig. 2
figure 2

Multiplex PCR amplification using the PsmPca_PS1 primer set (a) and PsmPca_PS2 primer set (b). The PCR samples were subjected to electrophoresis on 1.5% (a) and 3.0% (b) agarose gel. Lane numbers correspond to the numbers in Table 1. Lane M, DNA molecular size marker (1 Kb Plus DNA Ladder; Thermo Fisher Scientific, Waltham, MA, USA)

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Pathogen detection from disease lesion on Japanese radish

Purified DNA from lesions showing typical symptoms of Psm and Pca infections (Supplementary Fig. S4) showed specific amplification of Psm and Pca by multiplex PCR using PsmPca_PS1, respectively, but there was no amplification with DNA extracted from healthy leaf sections (Fig. 3). Each pathogen was also isolated on YPA medium from the solution extracted from each lesion, but not from healthy leaf sections.

Fig. 3
figure 3

Detection of Pseudomonas syringae pv. maculicola and P. cannabina pv. alisalensis from radish leaves by Multiplex PCR using the PsmPca_PS1 primer set. Lanes 1 and 2, bacterial leaf spot symptomatic leaves; Lanes 3 and 4, bacterial leaf blight symptomatic leaves; Lanes 5 and 6, symptomless leaves; Lane M, DNA molecular size marker (1 Kb Plus DNA Ladder; Thermo Fisher Scientific, Waltham, MA, USA). The PCR samples were subjected to electrophoresis on 1.5% agarose gel

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Pathogen detection in seed samples contaminated with Psm and Pca

The number of colonies that grew from 10 μL of an inoculated seed immersion liquid varied from 0 to >500, and only one of the 24 seeds did not develop any colonies. The same results were obtained for seeds inoculated with either bacterium. There was no amplification in multiplex PCR with PsmPca_PS1 when purified DNA from the immersion liquid of 1000 seeds was used as the template. Multiplex nested PCR with PsmPca_PS2 was performed using 1 μL of the first PCR product as the template. The results showed that the length of amplified DNA fragments was 260 bp from Psm-contaminated seed samples, 540 bp from Pca-contaminated seed samples, and 260 and 540 bp from samples containing both types of contaminated seeds (Fig. 4). Conversely, no amplification was observed in samples with no contaminated seeds. Thus, multiplex nested PCR can detect the contamination of one seed with Psm and/or Pca out of 1000 seeds and can discriminate the two species. The number of target bacteria that could be cultivated on YPA medium contained in 100 μL of seed immersion liquid was 0–12, and the detection sensitivity by PCR when using this liquid was higher than that when using the bacterial suspension as a template. These colonies were used in subsequent tests.

Fig. 4
figure 4

Detection of Pseudomonas syringae pv. maculicola (Psm) and P. cannabina pv. alisalensis (Pca) in 1000 seeds by multiplex nested PCR. PCR using the PsmPca_PS1 primer set was performed with the DNA purified from the immersion of 1000 seeds as a template, followed by nested PCR using the PsmPca_PS2 primer set. Lanes 1–3, only healthy seed samples; Lanes 4–6, seed samples contained one Pca-inoculated seed; Lanes 7–9, seed samples contained one Psm-inoculated seed; Lanes 10–12, seed samples contained one Psm- and one Pca-inoculated seeds; Lane M, DNA molecular size marker (1 Kb Plus DNA Ladder; Thermo Fisher Scientific, Waltham, MA, USA). The PCR samples were subjected to electrophoresis on 3% agarose gel

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Colony PCR

Following the multiplex PCR with PsmPca_PS1, DNA fragments of length 590 and 900 bp were amplified from colonies (four for each pathovar) isolated from the immersion liquids of Psm- and Pca-contaminated seeds, respectively (Fig. 5). Colonies derived from the seed immersion liquid containing a Psm-inoculated seed were only Psm, whereas those derived from the liquid containing a Pca-inoculated seed were only Pca. Of the eight colonies derived from immersion liquid containing seeds carrying both inoculants, two amplified a 590-bp DNA fragment and six amplified a 900-bp DNA fragment when subjected to multiplex PCR with PsmPca_PS1 (Fig. 5). Therefore, Psm and Pca colonies grew from the sample derived from seeds contaminated with both pathovars, and multiplex PCR with PsmPca_PS1 successfully distinguished the two. Inoculation tests using two colonies each revealed that colonies showing amplification similar to Psm formed typical lesions of bacterial leaf spot, and colonies showing amplification similar to Pca formed typical lesions of bacterial leaf bright on radish (data not shown).

Fig. 5
figure 5

Identification of Pseudomonas syringae pv. maculicola (Psm) and P. cannabina pv. alisalensis (Pca) colonies by multiplex PCR using the PsmPca_PS1 primer set. Lanes 1–4, colonies isolated from seed samples containing one Psm-inoculated seed; Lanes 5–8, colonies isolated from seed samples containing one Pca-inoculated seed; Lanes 9–16, colonies isolated from seed samples containing one Psm- and one Pca-inoculated seeds; Lane M, DNA molecular size marker (1 Kb Plus DNA Ladder; Thermo Fisher Scientific, Waltham, MA, USA). The PCR samples were subjected to electrophoresis on 1.5% agarose gel

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Discussion

EEL is a genomic region where a large number of effector genes exist (Alfano et al. 2000), but the distribution of individual effector genes among pathovars and host range within a pathovar has been noted, and research on the locus has been limited in recent years. We focused on the fact that this region has different gene repertories among pathovars, considering that it is possible to develop genetic markers that distinguish the pathovars of P. syringae group bacteria. By designing primers in the transition site, but not in a specific effector gene, between the common and specific genes, we simultaneously detected and distinguished two pathogens with three primers. Furthermore, we improved the detection sensitivity through nested PCR by designing and using two primer sets, thus enabling simultaneously detection and identification of Psm and Pca. Although Psm and Pca were targeted here, it is possible that EEL can be used to identify other pathovars of P. syringae group bacteria.

The presence of many Type III effectors in P. syringae group has been reported, and their relationship with pathogenicity has been investigated (Dillon et al. 2019a; Laflamme et al. 2020; Lindeberg et al. 2012). Type III-related effector genes are mutated and distributed among various pathovars (Laflamme et al. 2020). Therefore, these distributions and the homology between genes have also been used to consider the acquisition of pathogenicity and evolution of pathogenic bacteria (Dillon et al. 2019b; Lindeberg et al. 2009). Some of the predicted genes in the EEL are of unknown function and may also be involved in plant pathogenicity.

Although hrpK was used as the common gene, it is possible to design a common primer using queA. However, in Pca MAFF 106156, the insertion sequence was inserted adjacent to tRNALeu, and therefore, the queA side could not be used as a gene marker in the present study. Psm 90S-4 also had an IS element in the EEL. EEL has many insertion sequences (Inoue and Takikawa 1999b). This indicates that this region is variable and rich in mutations. Using this region, it may be possible to clarify differences in strains within the same pathovar. In the present analysis, we confirmed that P. syringae pathovars tomato, avii, persicae, and spinaciae have homologous sequences with Psm. Psm has been described as a heterogeneous pathovar (Peters et al. 2004; Wiebe and Campbell 1993). Psto has long been discussed for differences with Psm (Hendson et al. 1992; Takikawa et al. 1994; Wiebe and Campbell 1993). Furthermore, pathovar spinaciae is the same as Psm (Zhao et al. 2000), but this difference must be verified in the future. The primers we designed in the present study could not distinguish Psto and pathovar spinaciae from Psm. Both pathovars avii and persicae have been reported as pathogens of stone fruits (Ménard et al. 2003; Young 1987), and they will also be amplified. Both pathovars were not used in the present study because there are no reports of their occurrences in Japan. Psm and heterogeneous pathogens can be distinguished by focusing on the specific sequence in the EEL.

Japanese radish seeds and plants were used in the present study owing to the occurrence of black-brown discoloration in the core of the stem root in Japanese radish, a major problem in Japan (Otani 2016; Horinouchi et al. 2009; Takeuchi et al. 1989). The pathogen of this disease has been reported on Xanthomonas campestris pv. raphani, the causal agent of bacterial spot, in addition to Psm and Pca (Omi et al. 2015; Otani et al. 2014). They all cause leaf spots and are seed-borne pathogens, and a method for identifying them is required. Psm and Pca can be detected in the lesions on leaves and infected seeds using the genetic markers designed in the present study. Currently, we are developing genetic markers for the detection and discrimination of X. campestris pv. raphani.