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.
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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
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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.
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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
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DOI: https://doi.org/10.1007/s00253-021-11118-z