Abstract
Bacterial leaf spot and bacterial leaf blight are global threats to the cultivation of cruciferous vegetables, and it is necessary to develop methods to easily detect, identify, and distinguish the causative pathogens Pseudomonas syringae pv. maculicola (Psm) and P. cannabina pv. alisalensis (Pca). Here, we used the sequence specificity of the exchangeable effector loci flanking the hrp gene cluster to design primers that can help detect and discriminate between Psm and Pca. Primers common to both bacteria (hrpK_fw1 and hrpK_fw2) were designed within hrpK at the end of the hrp gene cluster. 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 adjacent to hrpK. PCR using hrpK_fw1 and MAC_rv1 or hrpK_fw2 and MAC_rv2 amplified DNA fragments of only Psm, P. syringae pv. tomato (causal agent of tomato bacterial speck), and P. syringae pv. spinaciae (causal agent of spinach bacterial leaf spot), among 76 strains of phytopathogenic bacteria. PCR using hrpK_fw1 and ALS_rv1 or hrpK_2 and ALS_rv2 amplified DNA fragments of only Pca. Multiplex PCR with these primers could easily distinguish Psm and Pca from bacterial colonies isolated on growth media and detect the pathogen in symptomatic leaves. Multiplex nested PCR with the primers detected contamination in one Psm- and/or one Pca-infected seeds in 1000 seeds. These results suggest that these PCR primers could help detect and discriminate Psm and Pca.
Key points
• We investigated Pseudomonas syringae pv. maculicola and P. cannabina pv. alisalensis.
• Novel primers common to both bacteria were designed following genome comparison.
• Multiplex PCR with new primers could discriminate Psm and Pca.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
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.
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.
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.
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).
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.
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.
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).
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.
Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
References
Alfano JR, Scharkowski AO, Deng WL, Badel JL, Petnicki-Ocwieja T, van Dijk K, Collmer A (2000) The Pseudomonas syringae hrp pathogenicity island has a tripartite mosaic structure composed of a cluster of type III secretion genes bounded by exchangeable effector and conserved effector loci that contribute to parasitic fitness and pathogenicity in plants. Proc Natl Acad Sci U S A 97:4856–4861. https://doi.org/10.1073/pnas.97.9.4856
Bonas U (1996) hrp genes of phytopathogenic bacteria. In: Dangl JL (ed) Bacterial pathogenesis of plants and animals. Springer, Berlin, pp 79–98
Bull CT, Goldman P, Koike ST (2004) Bacterial blight on arugula, a new disease caused by Pseudomonas syringae pv. alisalensis in California. Plant Dis 88:1384. https://doi.org/10.1094/pdis.2004.88.12.1384a
Bull CT, Manceau C, Lydon J, Kong H, Vinatzer BA, Fischer-Le Saux M (2010) Pseudomonas cannabina pv. cannabina pv. nov., and Pseudomonas cannabina pv. alisalensis (Cintas Koike and Bull, 2000) comb. nov., are members of the emended species Pseudomonas cannabina (ex Šutič & Dowson 1959) Gardan, Shafik, Belouin, Brosch, Grimont & Grimont 1999. Syst Appl Microbiol 33:105–115. https://doi.org/10.1016/j.syapm.2010.02.001
Charity JC, Pak K, Delwiche CF, Hutcheson SW (2003) Novel exchangeable effector loci associated with the Pseudomonas syringae hrp pathogenicity island: evidence for integron-like assembly from transposed gene cassettes. Mol Plant-Microbe Interact 16:495–507. https://doi.org/10.1094/mpmi.2003.16.6.495
Cintas NA, Koike ST, Bull CT (2002) A new pathovar, Pseudomonas syringae pv. alisalensis pv. nov., proposed for the causal agent of bacterial blight of broccoli and broccoli raab. Plant Dis 86:992–998. https://doi.org/10.1094/pdis.2002.86.9.992
Deng WL, Rehm AH, Charkowski AO, Rojas CM, Collmer A (2003) Pseudomonas syringae exchangeable effector loci: sequence diversity in representative pathovars and virulence function in P. syringae pv. syringae B728a. J Bacteriol 185:2592–2602. https://doi.org/10.1128/jb.185.8.2592-2602.2003
Dillon MM, Almeida RND, Laflamme B, Martel A, Weir BS, Desveaux D, Guttman DS (2019a) Molecular evolution of Pseudomonas syringae Type III secreted effector proteins. Front Plant Sci 10:418. https://doi.org/10.3389/fpls.2019.00418
Dillon MM, Thakur S, Almeida RND, Wang PW, Weir BS, Guttman DS (2019b) Recombination of ecologically and evolutionarily significant loci maintains genetic cohesion in the Pseudomonas syringae species complex. Genome Biol 20:3. https://doi.org/10.1186/s13059-018-1606-y
Gironde S, Manceau C (2012) Housekeeping gene sequencing and multilocus variable-number tandem-repeat analysis to identify subpopulations within Pseudomonas syringae pv. maculicola and Pseudomonas syringae pv. tomato that correlate with host specificity. Appl Environ Microbiol 78:3266–3279. https://doi.org/10.1128/aem.06655-11
Glandorf DCM, Brand I, Bakker PAHM, Schippers B (1992) Stability of rifampicin resistance as a marker for rot colonization studies of Pseudomonas putida in the field. Plant Soil 147:135–142
He SY, Huang HC, Collmer A (1993) Pseudomonas syringae pv. syringae harpin Pss: a protein that is secreted via the Hrp pathway and elicits the hypersensitive response in plants. Cell 73:1255–1266. https://doi.org/10.1016/0092-8674(93)90354-s
Hendson M, Hildebrand DC, Schroth MN (1992) Relatedness of Pseudomonas syringae pv. tomato, Pseudomonas syringae pv. maculicola and Pseudomonas syringae pv. antirrhini. J Appl Bacteriol 73:455–464. https://doi.org/10.1099/00207713-49-2-469
Horinouchi H, Watanabe H, Shirakawa T, Hasegawa J, Mamiya T, Kuwabara K (2009) Occurrence and control of root browning symptom of Japanese radish at Gifu highland region (in Japanese). Ann Rept Kansai Plant Prot 51:45–47
Hueck CJ (1998) Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 62:379–433
Inoue Y, Takikawa Y (1999a) Grouping Pseudomonas syringae strains by comparing DNA homology at the hrp gene cluster and its neighboring regions. Ann Phytopathol Soc Jpn 65:32–41. https://doi.org/10.3186/jjphytopath.65.32
Inoue Y, Takikawa Y (1999b) Investigation of repeating sequences in hrpL neighboring region of Pseudomonas syringae strains. Ann Phytopathol Soc Jpn 65:100–109. https://doi.org/10.3186/jjphytopath.65.100
Inoue Y, Takikawa Y (2000) Pseudomonas syringae strains are classified into five groups by comparing DNA homology at the hrp neighboring regions. J Gen Plant Pathol 66:238–241. https://doi.org/10.1007/PL00012952
Inoue Y, Takikawa Y (2003) Phylogenic analysis of DNA sequences around the hrpL and hrpZ regions of Pseudomonas syringae group bacteria. In: Iacobellis NS, Collmer A, Hutcheson SW, Mansfield JW, Morris CE, Murillo J, Schaad NW, Stead DE, Surico G (eds) Pseudomonas syringae pathovars and related pathogens. Kluwer, Dordrecht, pp 687–695
Inoue Y, Takikawa Y (2006) The hrpZ and hrpA genes are variable, and useful for grouping Pseudomonas syringae bacteria. J Gen Plant Pathol 72:26–33. https://doi.org/10.1007/s10327-005-0240-1
Ishiyama Y, Yamagishi N, Ogiso H, Fujinaga M, Takahashi F, Takikawa Y (2013) Bacterial brown spot on Avena storigosa Schereb. caused by Pseudomonas syringae pv. alisalensis. J Gen Plant Pathol 79:155–157. https://doi.org/10.1094/pdis.2004.88.12.1384a
Jin Q, He S-Y (2001) Role of the Hrp pilus in Type III protein secretion in Pseudomonas syringae. Science 294:2556–2558. https://doi.org/10.1126/science.1066397
Laflamme B, Dillon MM, Martel A, Almeida RND, Desveaux D, Guttman DS (2020) The pan-genome effector-triggered immunity landscape of a host-pathogen interaction. Science 367:763–768. https://doi.org/10.1126/science.aax4079
Li CM, Brown I, Mansfield JW, Stevens C, Boureau T, Romantschuk M, Taira S (2002) The Hrp pilus of Pseudomonas syringae elongates from its tip and acts as a conduit for translocation of the effector protein HrpZ. EMBO J 21:1909–1915. https://doi.org/10.1093/emboj/21.8.1909
Lindeberg M, Cunnac S, Collmer A (2009) The evolution of Pseudomonas syringae host specificity and type III effector repertoires. Mol Plant Pathol 10:767–775. https://doi.org/10.1111/j.1364-3703.2009.00587.x
Lindeberg M, Cunnac S, Collmer A (2012) Pseudomonas syringae type III effector repertoires: last words in endless arguments. Trends Microbiol 20:199–208. https://doi.org/10.1016/j.tim.2012.01.003
Mauzey SJ, Koike ST, Bull CT (2011) First report of bacterial blight of cabbage (Brassica oleracea var. capitata) caused by Pseudomonas cannabina pv. alisalensis in California. Plant Dis 95:71. https://doi.org/10.1094/pdis-09-10-0642
McCulloch L (1911) A spot disease of cauliflower. Bulletin, Bureau of Plant Industry, United States Department of Agriculture 225:1–15
Ménard M, Sutra L, Luisetti J, Prunier JP, Gardan L (2003) Pseudomonas syringae pv. avii (pv. nov.), the causal agent of bacterial canker of wild cherries (Prunus avium) in France. Eur J Plant Pathol 109:565–576. https://doi.org/10.1023/A:1024786201793
Nishiyama K (1978) Shokubutsu byogen saikin kan-i doteiho no shian (in Japanese). Plant Protection 32:283–288
Omi M, Watanabe H, Otani Y, Inoue Y, Takikawa Y (2015) Xanthomonas campestris pv. raphanini causing infection on surfaces and internal tissues of radish root (Abstract in Japanese). Jpn J Phytopathol 81:300
Otani Y (2016) Notes on the development of root rot and blackening symptoms on Japanese radish infected with Pseudomonas syringae pv. maculicola (in Japanese with English summary). Ann Rept Kansai Plant Prot 58:23–26. https://doi.org/10.4165/kapps.58.23
Otani Y, Etou K, Nakamura H, Omi M, Takikawa Y (2014) Occurrence of root rot and blackening symptoms on Japanese radish in Wakayama Prefecture and reproduction of the symptoms (Abstract in Japanese). Jpn J Phytopathol 80:327
Peters BJ, Ash GJ, Cother EJ, Hailstones DL, Noble DH, Urwin NAR (2004) Pseudomonas syringae pv. maculicola in Australia: pathogenic, phenotypic and genetic diversity. Plant Pathol 53:73–79. https://doi.org/10.1111/j.1365-3059.2004.00946.x
Preston G, Huang HC, He SY, Collmer A (1995) The HrpZ proteins of Pseudomonas syringae pvs. syringae, glycinea and tomato are encoded by an operon containing Yersinia ysc homologs and elicit the hypersensitive response in tomato but not soybean. Mol Plant-Microbe Interact 8:717–732. https://doi.org/10.1094/mpmi-8-0717
Rubio I, Hiddink G, Asma M, Bull CT (2012) First report of crucifer pathogen Pseudomonas cannabina pv. alisalensis causing bacterial blight on radish (Raphanus sativus) in Germany. Plant Dis 96:804. https://doi.org/10.1094/pdis-01-12-0043-pdn
Sarris PF, Karri IV, Goumas DE (2010) First report of Pseudomonas syringae pv. alisalensis causing bacterial blight of arugula (Eruca vesicaria subsp. sativa) in Greece. New Dis Rep 22:22. https://doi.org/10.5197/j.2044-0588.2010.022.022
Sarris PF, Trantas EA, Baltrus DA, Bull CT, Wechter WP, Yan S, Ververidis F, Almeida NF, Jones CD, Dangl JL, Panopoulos NJ, Vinatzer BA, Goumas DE (2013) Comparative genomics of multiple strains of Pseudomonas cannabina pv. alisalensis, a potential model pathogen of both monocots and dicots. PLoS One 8:e59366. https://doi.org/10.1371/journal.pone.0059366
Schofield DA, Bull CT, Rubio I, Wechter WP, Westwater C, Molineux IJ (2012) Development of an engineered bioluminescent reporter phage for detection of bacterial blight of crucifers. Appl Environ Microbiol 78:3592–3598. https://doi.org/10.1128/aem.00252-12
Takahashi F, Ogiso H, Fujinaga M, Ishiyama Y, Inoue Y, Shirakawa T, Takikawa Y (2013) First report of bacterial blight of crucifers caused by Pseudomonas cannabina pv. alisalensis in Japan. J Gen Plant Pathol 79:260–269. https://doi.org/10.1007/s10327-013-0458-2
Takeuchi K, Tsuchiya K, Kagawa H, Kase M (1989) Occurrence of root browning symptom on Japanease radish caused by Pseudomonas syringae pv. maculicola (in Japanese). Proc Kanto-Tosan Plant Prot Soc 36:60–62
Takikawa Y, Takahashi F (2014) Bacterial leaf spot and blight of crucifer plants (Brassicaceae) caused by Pseudomonas syringae pv. maculicola and P. cannabina pv. alisalensis. J Gen Plant Pathol 80:466–474. https://doi.org/10.1007/s10327-014-0540-4
Takikawa Y, Nishiyama N, Ohba K, Tsuyumu S, Goto M (1994) Synonymy of Pseudomonas syringae pv. maculicola and Pseudomonas syringae pv. tomato. In: LeMattre M, Freigoun S, Rudolph K, Swings JG (eds) Plant pathogenic bacteria; Proceedings of 8th International Conference on Plant Pathogenic Bacteria, INRA, Versailles, pp 199–204
Takimoto S (1931) Bacterial black spot of cruciferous plants II (in Japanese with English summary). Bult Sci Fak Terkult Kyushu Imp Univ 4:545–559
Wakimoto S (1960) Classification of strains of Xanthomonas oryzae on the basis of their susceptibility against bacteriophages. Jpn J Phytopathol 25:193–198
Wechter WP, Keinath AP, Farnham MW, Smith JP (2010) First report of bacterial leaf blight on broccoli and cabbage caused by Pseudomonas syringae pv. alisalensis in South Carolina. Plant Dis 94:132. https://doi.org/10.1094/pdis-94-1-0132c
Wiebe WL, Campbell RN (1993) Characterization of Pseudomonas syringae pv. maculicola and comparison with P. s. tomato. Plant Dis 77:414–419
Yoshioka R, Uematsu H, Takikawa Y, Kajihara H, Inoue Y (2020) PCR detection of Pseudomonas syringae pv. syringae, the causal agent of bacterial black node in barley and wheat, using newly designed primer sets. J Gen Plant Pathol 86:387–392. https://doi.org/10.1007/s10327-020-00930-6
Young JM (1987) New plant disease record in New Zealand: Pseudomonas syringae pv. persicae from nectarine, peach, and Japanese plum. N Z J Agric Res 30:235–247. https://doi.org/10.1080/00288233.1987.10430502
Zaccardelli M, Spasiano A, Bazzi C, Merighi M (2005) Identification and in planta detection of Pseudomonas syringae pv. tomato using PCR amplification of hrpZPst. Eur J Plant Pathol 111:85–90. https://doi.org/10.1007/s10658-004-2734-7
Zhao Y, Damicone JP, Demezas DH, Rangaswamy V, Bender CL (2000) Bacterial leaf spot of leafy crucifers in Oklahoma caused by Pseudomonas syringae pv. maculicola. Plant Dis 84:1015–1020. https://doi.org/10.1094/PDIS.2000.84.9.1015
Author information
Authors and Affiliations
Contributions
YI carried out sequence alignment, designed primers, and drafted the manuscript. YT participated in the design of the study and performed a validity verification test. All authors have read and approved the final manuscript.
Corresponding author
Ethics declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
ESM 1
(PDF 505 kb).
Rights and permissions
About this article
Cite this article
Inoue, Y., Takikawa, Y. Primers for specific detection and identification of Pseudomonas syringae pv. maculicola and P. cannabina pv. alisalensis. Appl Microbiol Biotechnol 105, 1575–1584 (2021). https://doi.org/10.1007/s00253-021-11118-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-021-11118-z