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Three semi-selective media for Pseudomonas syringae pv. maculicola and P. cannabina pv. alisalensis

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Abstract

Three semi-selective media, DTarTA, SPbc, and SPamt, were developed and tested to isolate Pseudomonas syringae pv. maculicola (Psm) and P. cannabina pv. alisalensis (Pca) from Raphanus sativus seeds. DTarTA contained D-tartaric acid as a carbon source and potassium tellurite, ampicillin sodium, and methyl violet as antibiotics. DTarTA suppressed growth in 19 of the 24 pathovars from the P. syringae complex, whereas Psm and Pca grew and formed gray to black colonies. SPamt contained sucrose and peptone as nutrient sources and was supplemented with bromothymol blue and the same antibiotics present in DTarTA and Psm and Pca formed yellowish to dark brown colonies on the SPamt medium. SPbc contained sucrose and peptone and was supplemented with cephalexin and boric acid as antibiotics and Psm and Pca formed semi-translucent to white colonies on the SPbc medium. SPamt and SPbc suppressed the growth of several plant-associated bacteria (except the P. syringae complex). The growth of saprophytic bacteria in seeds on the different media was compared with that on King’s B medium, using five types of commercially available Raphanus sativus seeds. The suppression rate of DTarTA was 85–99% and was lower for seeds with more saprophytic bacteria. The suppression rates of SPamt and SPbc were 90–99%. In detection tests using 10,000 seed samples mixed with Pca or Psm-contaminated seeds, it was possible to selectively isolate Psm and Pca using SPamt and SPbc, even when the colony numbers of the target bacterium constituted less than 10% of the total colonies.

Key points

• Bacterial leaf spot and blight pathogens were selectively isolated from seeds.

• DTarTA medium distinguishes these pathogens from P. syringae complex pathovars.

• SPamp and SPbc media have different selectivity for plant-associated bacteria.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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References

  • Arino S, Marchal R, Vandecasteele J-P (1996) Identification and production of a rhamnolipidic biosurfactant by a Pseudomonas species. Appl Microbiol Biotechnol 45:162–168

    Article  CAS  Google Scholar 

  • Asaad S, Sands DC, Mohan SK (2017) Detection of Pseudomonas syringae pv. syringae in wheat seeds. In: Fatmi M, Walcott RR, Schaad NW (eds) Detection of plant-pathogenic bacteria in seed and other planting material. APS Press, St. Paul, pp 21–26

    Chapter  Google Scholar 

  • Bazzi C, Gozzi R, Stead D, Sellwood J (1988) A bacterial leaf spot of spinach (Spinacia oleracea L.) caused by a non-fluorescent Pseudomonas syringae van Hall (pathovar). Phytopathol Mediterr 27:103–107

    Google Scholar 

  • Billing E (1970) Pseudomonas viridiflava (Burkholder, 1930; Clara 1934). J Appl Bacteriol 33:492–500

    Article  CAS  Google Scholar 

  • Bull CT, Koike ST (2017) Detection of Pseudomonas pathogens from crucifer seeds. In: Fatmi M, Walcott RR, Schaad NW (eds) Detection of plant-pathogenic bacteria in seed and other planting material. APS Press, St. Paul, pp 165–172

    Chapter  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Export and International Affairs Bureau (2016) Manual of detection method of black rot pathogen from Japanese radish seeds (in Japanese). https://www.maff.go.jp/j/kanbo/tizai/brand/b_syokubut/attach/pdf/index-43.pdf. Accessed 10 February 2022

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Granér G, Persson P, Meijer J, Alström S (2003) A study on microbial diversity in different cultivars of Brassica napus in relation to its wilt pathogen, Verticillium longisporum. FEMS Microbiol Lett 224:269–276

    Article  Google Scholar 

  • 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/10.1111/j.1365-2672.1992.tb05005.x

    Article  CAS  Google Scholar 

  • 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

    Article  Google Scholar 

  • Inoue Y, Takikawa Y (2021) Primers for specific detection and identification of Pseudomonas syringae pv. maculicola and P. cannabina pv. alisalensis. Appl Microbiol Biotechnol 105:1575–1584

    Article  CAS  Google Scholar 

  • Inoue Y (2022) Development of medium for detecting bacterial leaf spot and bacterial leaf blight pathogen (Abstract in Japanese). Proc Kanto Tosan Plant Prot Soc 69 (in press)

  • International Seed Testing Association (2017) International rules for seed testing, Edition 2017, Annexe to Chapter 7: Seed Health Testing Methods, pp. 7–019a: Detection of Xanthomonas campestris pv. campestris on Brassica spp

  • 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

    Article  Google Scholar 

  • King EO, Ward MK, Raney DE (1954) Two simple media for the demonstration of pyocyanin and fluorescin. J Lab Clin Med 44:301–307

    CAS  PubMed  Google Scholar 

  • Koike ST, Azad HR, Cooksey DC (2002) First Report of Bacterial Leaf Spot of Spinach Caused by a Pseudomonas syringae pathovar in California. Plant Dis 86:921. https://doi.org/10.1094/pdis.2002.86.8.921a

    Article  CAS  PubMed  Google Scholar 

  • Lelliott RA, Billing E, Hayward AC (1966) A determinative scheme for the fluorescent plant pathogenic pseudomonads. J Appl Bacteriol 29:470–489

    Article  CAS  Google Scholar 

  • Matsuda D, Takikawa Y (2003) Classification of maculicola/tomato complex in Pseudomonas syringae (abstract in Japanese). Jpn J Phytopathol 69:302–303

    Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • McCulloch L (1911) A spot disease of cauliflower. Bulletin, Bureau of Plant Industry, United States Department of Agriculture 225: 1–15

  • Mohan SK, Schaad NW (1987) An improved agar plating assay for detecting Pseudomonas syringae pv. syringae and P. s. pv. phaseolicola in contaminated been seed. Phytopathology 77:1390–1395

    Article  Google Scholar 

  • Mori M, Sogou K, Inoue Y (2019) Development of a selective medium and antisera for Pseudomonas syringae pv. syringae from seeds of barley and wheat. J Gen Plant Pathol 85:211–220

    Article  Google Scholar 

  • National Agriculture and Food Research Organization, “Method for detection of bacterial leaf spot and bacterial leaf blight pathogens from radish seeds”, National Agriculture and Food Research Organization. https://www.naro.go.jp/project/results/4th_laboratory/carc/2020/20_041.html. Accessed 11 November 2021

  • Nishiyama K (1978) Shokubutsu byogen saikin kan-i doteiho no shian (in Japanese). Plant Prot 32:283–288

    Google Scholar 

  • 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

    Article  Google Scholar 

  • Ozaki K, Kimura T, Matsumoto K (1998) Pseudomonas syringae pv. spinaciae pv. nov., the causal agent of bacterial leaf spot of spinach in Japan. Jpn J Phytopathol 64:264–269

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Randhawa P, Pradhanang P, Schaad NW (2017) Detection of Pseudomonas syringae pv. tomato in tomato seeds. In: Fatmi M, Walcott RR, Schaad NW (eds) Detection of plant-pathogenic bacteria in seed and other planting material. APS Press, St. Paul, pp 119–124

    Chapter  Google Scholar 

  • 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

    Article  Google Scholar 

  • Rybakova D, Mancinelli R, Wikiström M, Birch-Jensen A-S, Postma J, Ehlers R-U, Goertz S, Berg G (2017) The structure of the Brassica napus seed microbiome is cultivar-dependent and affects the interactions of symbionts and pathogens. Microbiome 5:104. https://doi.org/10.1186/s40168-017-0310-6

    Article  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shepherd LM, Vidaver AK (2017) Detection of Pseudomonas savastanoi pv. glycinea in soybean seeds. In: Fatmi M, Walcott RR, Schaad NW (eds) Detection of plant-pathogenic bacteria in seed and other planting material. APS Press, St. Paul, pp 85–88

    Chapter  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Google Scholar 

  • 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. Proc. 8th Int. Conf., Versailles, pp. 199–204

  • 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

    Article  Google Scholar 

  • 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

    Google Scholar 

  • Tyc O, Putra R, Gols R, Harvey JA, Garbeva P (2020) The ecological role of bacterial seed endophytes associated with wild cabbage in the United Kingdom. Microbiol Open 9:e00954. https://doi.org/10.1002/mbo3.954

    Article  Google Scholar 

  • Wakimoto S (1960) Classification of strains of Xanthomonas oryzae on the basis of their susceptibility against bacteriophages. Jpn J Phytopathol 25:193–198

    Article  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Wiebe WL, Campbell RN (1993) Characterization of Pseudomonas syringae pv. maculicola and comparison with P. s. tomato. Plant Dis 77:414–419

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

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Acknowledgements

I thank Dr. Nobutaka Someya (Institute for Plant Protection, NARO) for their technical assistance. I also thank Dr. Yuichi Takikawa (Graduate School of Science and Technology, Shizuoka University, Japan) for distributing the strains and Dr. Kohei Osaki, Dr. Masatoshi Sato (Center for Seeds and Seedlings, NARO), and Mr. Mitsuru Igarashi (Sakata Seeds Corporation, Japan) for providing the radish seeds.

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  1. Institute for Plant Protection, National Agriculture and Food Research Organization (NARO), Kan-nondai, Tsukuba, Ibaraki, 305-8666, Japan

    Yasuhiro Inoue

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YI conceived and designed the research, conducted the experiments, analyzed data, and wrote the manuscript.

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Correspondence to Yasuhiro Inoue.

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Inoue, Y. Three semi-selective media for Pseudomonas syringae pv. maculicola and P. cannabina pv. alisalensis. Appl Microbiol Biotechnol 106, 5741–5755 (2022). https://doi.org/10.1007/s00253-022-12092-w

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