Skip to main content

Loop-mediated isothermal amplification of bacterial effector genes to detect Pseudomonas syringae pv. actinidiae biovars 1 and 3

Abstract

Loop-mediated isothermal amplification (LAMP) was designed to rapidly detect biovars 1 and 3 of Pseudomonas syringae pv. actinidiae (Psa), a serious, global kiwifruit pathogen, using two primer sets targeting biovar-specific type III effector genes. Each primer set was specific for the targeted biovars, and no signals were obtained for any other biovars. Detection limits of the assay were tenfold higher than that of the conventional PCR method for both biovars. The primer set to detect biovar 3 was highly sensitive at detecting this biovar directly from leaf lesions.

This is a preview of subscription content, access via your institution.

References

  1. Balestra GM, Mazzaglia A, Quattrucci A, Renzi M, Rossetti A (2009) Current status of bacterial canker spread on kiwifruit in Italy. Austral Plant Dis Notes 4:34–36

    Google Scholar 

  2. Balestra GM, Taratufolo MC, Vinatzer BA, Mazzaglia A (2013) A multiplex PCR assay for detection of Pseudomonas syringae pv. actinidiae and differentiation of populations with different geographic origin. Plant Dis 97:472–478

    CAS  Article  Google Scholar 

  3. Cameron A, Sarojini V (2014) Pseudomonas syringae pv. actinidiae: chemical control, resistance mechanisms and possible alternatives. Plant Pathol 63:1–11

    CAS  Article  Google Scholar 

  4. Chapman JR, Taylor RK, Weir BS, Romberg MK, Vanneste JL, Luck J, Alexander BJR (2012) Phylogenetic relationships among global populations of Pseudomonas syringae pv. actinidiae. Phytopathology 102:1034–1044

    CAS  Article  Google Scholar 

  5. Chen Q, Benjin L, Peiqing L, Chengzhong L, Zhixiong Z, Qiyong Y (2013) Development and evaluation of specific PCR and LAMP assays for the rapid detection of Phytophthora melonis. Eur J Plant Pathol 137:597–607

    CAS  Article  Google Scholar 

  6. Cunty A, Poliakoff F, Rivoal C, Cesbron S, Fischer-Le Saux M, Lemaire C, Jacques MA, Manceau C, Vanneste JL (2015) Characterization of Pseudomonas syringae pv. actinidiae (Psa) isolated from France and assignment of Psa biovar 4 to a de novo pathovar: Pseudomonas syringae pv. actinidifoliorum pv. nov. Plant Pathol 64:582–596

    CAS  Article  Google Scholar 

  7. European and Mediterranean Plant Protection Organization (2014) PM 7/120(1) Pseudomonas syringae pv. actinidiae. EPPO Bull 44:360–375

    Article  Google Scholar 

  8. Ferrante P, Scortichini M (2009) Identification of Pseudomonas syringae pv. actinidiae as causal agent of bacterial canker of yellow kiwifruit (Actinidia chinensis Planchon) in central Italy. J Phytopathol 157:768–770

    Article  Google Scholar 

  9. Ferrante P, Scortichini M (2011) Molecular and phenotypic variability among Pseudomonas avellanae, P. syringae pv. actinidiae and P. syringae pv. theae: the genomospecies 8 sensu Gardan et al. (1999). J Plant Pathol 93:659–666

    CAS  Google Scholar 

  10. Fujikawa T, Aono M, Nakaune R (2018) New medium for sorting of Pseudomonas syringae pv. actinidiae (abstract in Japanese). Jpn J Phytopathol 84:48–49

    Google Scholar 

  11. Fusayasu S, Sato T, Shimizu H (2006) Detection of citrus greening disease (Candidatus Liberobacter asiaticum) by loop-mediated isothermal amplification (in Japanese). Res Bull Plant Prot Jpn 42:81–87

    Google Scholar 

  12. Gallelli A, Talocci S, Pilotti M, Loreti S (2014) Real-time and qualitative PCR for detecting Pseudomonas syringae pv. actinidiae isolates causing recent outbreaks of kiwifruit bacterial canker. Plant Pathol 63:264–276

    CAS  Article  Google Scholar 

  13. Goto M, Honda E, Ogura A, Nomoto A, Hanaki K (2009) Colorometric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. Biotechniques 46:167–172

    CAS  Article  Google Scholar 

  14. Lalkhen AG, McCluskey A (2008) Clinical tests: sensitivity and specificity. Contin Educ Anaesth Crit Care Pain 8:221–223

    Article  Google Scholar 

  15. McCann HC, Rikkerink EHA, Bertels F, Fiers M, Lu A, Rees-George J, Andersen MT, Gleave A, Haubold B, Wohlers MW, Guttman DS, Wang PW, Straub C, Vanneste J, Rainey PB, Templeton MD (2013) Genomic analysis of the kiwifruit pathogen Pseudomonas syringae pv. actinidiae provides insight into the origins of an emergent plant disease. PLoS Pathog 9:e1003503

    CAS  Article  Google Scholar 

  16. Ministry of Agriculture, Forestry and Fisheries (2018) Manual for control of bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae biovar 3 (Psa3), 3rd ed (in Japanese). Available at https://www.maff.go.jp/j/syouan/syokubo/gaicyu/siryou2/attach/pdf/index-14.pdf. Cited 23 Dec 2020

  17. Miyoshi T, Shimizu S, Sawada H (2012) Occurrence and distribution of a defective non-phaseolotoxin-producing mutant of Pseudomonas syringae pv. actinidiae in Ehime Prefecture, Japan (in Japanese with English summary). Jpn J Phytopathol 78:92–103

    Article  Google Scholar 

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

    Article  Google Scholar 

  19. Mori Y, Nagamine K, Tomita N, Notomi T (2001) Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun 289:150–154

    CAS  Article  Google Scholar 

  20. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucl Acid Res 28:e63

    CAS  Article  Google Scholar 

  21. R Core Team (2018) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna

    Google Scholar 

  22. Ruinelli M, Schneeberger PHH, Ferrante P, Buehlmann A, Scortichini M, Vanneste JL, Duffy B, Pothier JF (2017) Comparative genomics-informed design of two LAMP assays for detection of the kiwifruit pathogen Pseudomonas syringae pv. actinidiae and discrimination of isolates belonging to the pandemic biovar 3. Plant Pathol 66:140–149

    CAS  Article  Google Scholar 

  23. Sawada H, Fujikawa T (2019) Genetic diversity of Pseudomonas syringae pv. actinidiae, pathogen of kiwifruit bacterial canker. Plant Pathol 68:1235–1248

    CAS  Article  Google Scholar 

  24. Sawada H, Miyoshi T, Ide Y (2014) Novel MLSA group (Psa5) of Pseudomonas syringae pv. actinidiae causing bacterial canker of kiwifruit (Actinidia chinesis) in Japan (in Japanese with English summary). Jpn J Phytopathol 80:171–184

    Article  Google Scholar 

  25. Sawada H, Shimizu S, Miyoshi T, Shinozaki T, Kusumoto S, Noguchi M, Naridomi T, Kikuhara K, Kansako M, Fujikawa T, Nakaune R (2015) Characterization biovar 3 strains of Pseudomonas syringae pv. actinidiae isolated in Japan (in Japanese with English summary). Jpn J Phytopathol 81:111–126

    CAS  Article  Google Scholar 

  26. Sawada H, Kondo K, Nakaune R (2016) Novel biovar (biovar 6) of Pseudomonas syringae pv. actinidiae causing bacterial canker of kiwifruit (Actinidia deliciosa) in Japan (in Japanese with English summary). Jpn J Phytopathol 82:101–115

    Article  Google Scholar 

  27. Serizawa S, Ichikawa T (1993) Epidemiology of bacterial canker of kiwifruit 3. The seasonal changes of bacterial population in lesions and of its exudation from lesions. Ann Phytopathol Soc Jpn 59:469–476

    Article  Google Scholar 

  28. Serizawa S, Ichikawa T, Takikawa Y, Tsuyumu S, Goto M (1989) Occurrence of bacterial canker of kiwifruit in Japan: description of symptoms, isolation of the pathogen and screening of bactericides. Ann Phytopathol Soc Jpn 55:427–436

    Article  Google Scholar 

  29. Takikawa Y, Serizawa S, Ichikawa T, Tsuyumu S, Goto M (1989) Pseudomonas syringae pv. actinidiae sp. nov.: the causal bacterium of canker in kiwifruit in Japan. Ann Phytopathol Soc Jpn 55:437–444

    Article  Google Scholar 

  30. Tyson JL, Curtis CL, Manning MA, Dobson SJ, McKenna CE (2016) Preliminary investigation of the risk of plant debris as a Pseudomonas syringae pv. actinidiae inoculum source. N Z Plant Prot 69:11–16

    Google Scholar 

Download references

Funding

This study was supported by a Grant-in-Aid from the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry of the Ministry of Agriculture, Forestry and Fisheries (27008C).

Author information

Affiliations

Authors

Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by KS, HS, and GK. The first draft of the manuscript was written by KS. All authors commented on previous versions of the manuscript and read and approved the final manuscript.

Corresponding author

Correspondence to Koichi Suzaki.

Ethics declarations

Conflict of interest

The authors have no conflicts of interest to declare that are relevant to the content of this article.

Human and animal rights

No human participants or animals were involved in this study.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 24 KB)

About this article

Verify currency and authenticity via CrossMark

Cite this article

Suzaki, K., Sawada, H. & Kisaki, G. Loop-mediated isothermal amplification of bacterial effector genes to detect Pseudomonas syringae pv. actinidiae biovars 1 and 3. J Gen Plant Pathol (2021). https://doi.org/10.1007/s10327-021-01030-9

Download citation

Keywords

  • Biovar
  • Diagnosis
  • Kiwifruit
  • Loop-mediated isothermal amplification (LAMP)
  • Pseudomonas syringae pv. actinidiae