Skip to main content

Advertisement

SpringerLink
Log in

Rapid diagnosis of Ralstonia solanacearum infection sweet potato in China by loop-mediated isothermal amplification

  • Original Paper
  • Published:
Archives of Microbiology Aims and scope Submit manuscript

Abstract

Bacterial wilt of sweet potato is caused by Ralstonia solanacearum, which is distributed in southern China and causes significant economic losses each year. The pathogen is soil- and rhizome-borne, and thus its rapid detection may prevent the occurrence and spread of the disease. R. solanacearum has been listed as a quarantine disease in China. With the advent of molecular biology, many novel tools have been explored for the rapid identification of plant pathogens. In this study, a strain-specific detection method was developed for this specific pathogen that infects sweet potato using loop-mediated isothermal amplification (LAMP). A set of new LAMP-specific primers was designed from the orf428 gene, which can specifically detect the R. solanacearum bacterium that infect sweet potato. The LAMP reaction consisted of 8.0 mmol·L−1Mg2+, 1.4 mmol·L−1 dNTPs, and 0.32U μL−1 Bst 2.0 DNA polymerase and was performed at 65 °C for 1 h. The amplification products were detected by visualizing a mixture of color changes using SYBR Green I dye and assessing ladder-like bands by electrophoresis. Our method has specificity, i.e., it only detected R. solanacearum in sweet potato, and it has high sensitivity, with a detection limit of 100 fg·μL−1 genomic DNA and 103 CFU·mL−1 of bacterial fluid. In addition, R. solanacearum could be directly detected in infected sweet potato tissues without the need for DNA extraction. The LAMP method established in this study is a highly specific, sensitive, and rapid tool for the detection of bacterial wilt in sweet potato caused by R. solanacearum.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Bonizzoni M, Afrane Y, Yan GYJAJOTM (2009) Loop-mediated isothermal amplification (LAMP) for rapid identification of Anopheles gambiae and Anopheles arabiensis mosquitoes. Am J Trop Med Hyg 81:1030

    CAS  PubMed  Google Scholar 

  • Boubourakas IN, Fukuta S, Kyriakopoulou PEJJOVM (2009) Sensitive and rapid detection of peach latent mosaic viroid by the reverse transcription loop-mediated isothermal amplification. J Virol Methods 160:63–68

    CAS  PubMed  Google Scholar 

  • Chandra A, Keizerweerd AT, Que Y, Grisham MP (2015) Loop-mediated isothermal amplification (LAMP) based detection of Colletotrichum falcatum causing red rot in sugarcane. Mol Biol Rep 42:1309–1316

    CAS  PubMed  Google Scholar 

  • Chen Y, Lin Y, Chung W (2011) Creeping stem cuttings, the possible inoculum source for bacterial wilt of vegetable sweetpotato. Phytopathology 101:34

    CAS  Google Scholar 

  • Chen Y-J, Lin Y-S, Chung W-H (2012) Bacterial wilt of sweet potato caused by Ralstonia solanacearum in Taiwan. J Gen Plant Pathol 78:80–84

    Google Scholar 

  • Chen Y-J, Lin Y-S, Tseng K-J, Chung W-H (2014) Vine cuttings as possible initial inoculum sources of Ralstonia solanacearum race 1 biovar 4 on vegetable sweet potato in fields. Eur J Plant Pathol 140:83–95

    Google Scholar 

  • Cheng TY, Liu GHJAP (2017) PCR denaturing gradient gel electrophoresis as a useful method to identify of intestinal bacteria flora in Haemaphysalis flava ticks. Acta Parasitologica 62:269–272

    PubMed  Google Scholar 

  • Clark C, Davis J, Abad J, Cuellar W, Fuentes S, Kreuze J, Rw G, Mukasa S, Tugume A, Tairo F, Jpt V (2012) Sweetpotato viruses: 15 years of progress on understanding and managing complex diseases. Plant Dis 96:168–185

    CAS  PubMed  Google Scholar 

  • Clark CA, Moyer JW (1988) Compendium of sweet potato diseases. Mycologia 81:175

    Google Scholar 

  • Clark CA, Wilder-Ayers JA, Duarte V (1989) Resistance of sweet potato to bacterial root and stem rot caused by Erwinia chrysanthemi. Plant Dis 73:984–987

    Google Scholar 

  • Clark CA, Dukes PD, Moyer JW (1992) Fifty years of cooperative sweetpotato research. Southern Coop Ser Bull 369:88–105

    Google Scholar 

  • Ding S-W, Yang S-H, Wu W-J, Xie H, Xu C-L (2019) Rapid diagnosis of Ditylenchus destructor by loop-mediated isothermal amplification assay based on 28S rRNA sequences. Eur J Plant Pathol 153:1165–1175

    CAS  Google Scholar 

  • Douda O, Marek M, Zouhar M, Ryšánek PJPM (2013) Insights into the structure and phylogeny of the 28S rRNA expansion segments D2 and D3 of the plant-infecting nematodes from the genus Ditylenchus (Nematoda: Anguinidae). Phytopathologia Mediterranea 52:84–97

    CAS  Google Scholar 

  • Du L, Shi W, Li X, Lan Y, Sun F, Fan Y, Zhou T, Zhou Y (2019) A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for detecting the pathogen of maize rough dwarf disease in China. Australas Plant Pathol 48:485–489

    CAS  Google Scholar 

  • Duan Y, Ge C, Zhang X, Wang J, Zhou M (2014) A rapid detection method for the plant pathogen Sclerotinia sclerotiorum based on loop-mediated isothermal amplification (LAMP). Australas Plant Pathol 43:61–66

    CAS  Google Scholar 

  • Eshraghi L, Aryamanesh N, Anderson JP, Shearer B, McComb JA, Hardy GESJ, O’Brien PA (2011) A quantitative PCR assay for accurate in planta quantification of the necrotrophic pathogen Phytophthora cinnamomi. Eur J Plant Pathol 131:419

    CAS  Google Scholar 

  • Fegan M, Prior P (2005) How complex is the Ralstonia Solanacearum species complex. Bact Wilt Dis Ralstonia Solanacearum Species Complex. APS, St. Paul, pp 449–462

  • Fernández-Álvarez C, González SF, Santos Y (2016) Development of a SYBR green I real-time PCR assay for specific identification of the fish pathogen Aeromonas salmonicida subspecies salmonicida. Appl Microbiol Biotechnol 100:10585–10595

    PubMed  Google Scholar 

  • Fukuta S, Takahashi R, Kuroyanagi S, Miyake N, Nagai H, Suzuki H, Hashizume F, Tsuji T, Taguchi H, Watanabe H, Kageyama K (2013) Detection of Pythium aphanidermatum in tomato using loop-mediated isothermal amplification (LAMP) with species-specific primers. Eur J Plant Pathol 136:689–701

    CAS  Google Scholar 

  • Goto M, Honda EA, Nomoto A, Hanaki KJB (2009) Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. Biotechniques 46:167–172

    CAS  PubMed  Google Scholar 

  • Hayward AC (1991) Biology and epidemiology of bacterial wilt caused by Pseudomonas Solanacearum. Annu Rev Phytopathol 29:65–87

    CAS  PubMed  Google Scholar 

  • He L, Sequerira L, Kelman A (1983) Characteristics of strains of Pseudomonas solanacearum from China. Plant Dis 67:1357–1361

    Google Scholar 

  • Huang L, Chen YX, Huang, HY (1956) A preliminary report on the control of sweet potato blast. Acta Phytopathologica Sinica 2:210–211 (in chinese)

    Google Scholar 

  • Huang LF, Fang BP, Luo ZX, Chen JY, Zhang XJ, Wang ZY (2010) First report of bacterial stem and root rot of sweetpotato caused by a Dickeya sp (Erwinia chrysanthemi) in China. Plant Dis 94:1503

    CAS  PubMed  Google Scholar 

  • Huang W, Zhang H, Xu J, Wang S, Kong X, Ding W, Xu J, Feng JJFPS (2017) Loop-mediated isothermal amplification method for the rapid detection of Ralstonia solanacearum phylotype I mulberry strains in China. Front Plant Sci 8:76

    PubMed  PubMed Central  Google Scholar 

  • Imai M, Ai N, Minekawa H, Notomi T, Ishizaki T, Tashiro M, Odagiri TJV (2006) Development of H5-RT-LAMP (loop-mediated isothermal amplification) system for rapid diagnosis of H5 avian influenza virus infection. Vaccine 24:6679–6682

    CAS  PubMed  Google Scholar 

  • Jiang G, Wei Z, Xu J, Chen H, Zhang Y, She X, Macho AP, Ding W, Liao B (2017) Bacterial wilt in China: history, current status, and future perspectives. Front Plant Sci 8:1–10

    Google Scholar 

  • Kubota R (2012) Engineering a simple, rapid, inexpensive, nucleic acid based system for strain selective detection of the bacterial wilt pathogen Ralstonia solanacearum. Ph.D. University of Hawaii at Manoa

  • Kubota R, Vine BG, Alvarez AM, Jenkins DMJP (2008) Detection of Ralstonia solanacearum by loop-mediated isothermal amplification. Phytopathology 98:1045

    CAS  PubMed  Google Scholar 

  • Le Floch G, Tambong J, Vallance J, Tirilly Y, Lévesque A, Rey P (2007) Rhizosphere persistence of three Pythium oligandrum strains in tomato soilless culture assessed by DNA macroarray and real-time PCR. FEMS Microbiol Ecol 61:317–326

    PubMed  Google Scholar 

  • Maeda H, Kokeguchi S, Fujimoto C, Tanimoto I, Yoshizumi W, Nishimura F, Takashiba S (2005) Detection of periodontal pathogen Porphyromonas gingivalis by loop-mediated isothermal amplification method. FEMS Immunol Med Microbiol 43:233–239

    CAS  PubMed  Google Scholar 

  • Nagamine K, Hase T, Notomi TJMCP (2002) Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes 16:223–229

    CAS  PubMed  Google Scholar 

  • Nair S, Manimekalai R, Ganga RP, Hegde VJWJOM (2016) Loop mediated isothermal amplification (LAMP) assay for detection of coconut root wilt disease and arecanut yellow leaf disease phytoplasma. World J Microbiol Biotechnol 32:108

    PubMed  Google Scholar 

  • Niu JH, Jian H, Guo QX, Chen CL, Guo YDJPP (2012) Evaluation of loop-mediated isothermal amplification (LAMP) assays based on 5S rDNA-GS2 regions for detecting Meloidogyne enterolobii. Plant Pathol 61:809–819

    CAS  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  • Pastrik KH, Elphinstone JG, Pukall R (2002) Sequence analysis and detection of Ralstonia solanacearum by multiplex PCR amplification of 16S–23S ribosomal intergenic spacer region with internal positive control. Eur J Plant Pathol 108:831–842

    CAS  Google Scholar 

  • Peng D, Xie J, Qiang W, Ling KS, Zhou T (2017) One-step reverse transcription loop-mediated isothermal amplification assay for detection of Apple chlorotic leaf spot virus. J Virol Methods 248:154–158

    CAS  PubMed  Google Scholar 

  • Sunani SK, Bashyal BM, Rawat K, Manjunatha C, Sharma S, Prakash G, Krishnan SG, Singh AK, Aggarwal R (2019) Development of PCR and loop mediated isothermal amplification assay for the detection of bakanae pathogen Fusarium fujikuroi. Eur J Plant Pathol 154:715–725

    CAS  Google Scholar 

  • Tan HM, Cao LX, He ZF, Su GJ, Lin B, Zhou SNJWJOM (2006) Isolation of endophytic actinomycetes from different cultivars of tomato and their activities against Ralstonia solanacearum in vitro. World J Microbiol Biotechnol 22:1275–1280

    CAS  Google Scholar 

  • Thangavelu R, Devi PGJB (2018) Rapid and sensitive detection of Pseudocercospora eumusae pathogen causing eumusae leaf spot disease of banana by loop-mediated isothermal amplification (LAMP) method. 3 Biotech 8:442

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tomlinson J, Boonham N, Hughes KJD, Griffen RL, Barker I (2005) On-site DNA extraction and real-time PCR for detection of Phytophthora ramorum in the field. Appl Environ Microbiol 71:6702–6710

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wang Z, Gu Q, Sun H, Li H, Sun B, Liang X, Yuan Y, Liu R, Shi YJJOVM (2014) One-step reverse transcription loop mediated isothermal amplification assay for sensitive and rapid detection of Cucurbit chlorotic yellows virus. J Virol Methods 195:63–66

    CAS  PubMed  Google Scholar 

  • Wei W, Miaohong R, Qiu YX, Wang WY, Ke YQ, Pan TG (2009) Phenylaprapanoid metabolism of sweet potato against Pseudomonas solanacearum. Chin J Eco-Agric 17:944–948

    Google Scholar 

Download references

Acknowledgements

This work was supported by the following grants: China Agriculture Research System (CARS-10-B14); Program of Seed Industry Innovation and Industrialization of Fujian Province (fjzycxny2017005); Program of Innovative Research Team of Fujian Academy of Agricultural Sciences (STIT2017-2-3).

Author information

Authors and Affiliations

  1. Institute of Crop Sciences, Fujian Academy of Agricultural Sciences, Fuzhou, 350013, Fujian, China

    Huawei Li, Hong Zhang, Zhonghua Liu, Zhijian Lin, Yongxiang Qiu, Hao Tang & Sixin Qiu

  2. Scientific Observing and Experimental Station of Tuber and Root Crops in South China, Ministry of Agriculture, Fuzhou, 350013, Fujian, China

    Huawei Li, Hong Zhang, Zhonghua Liu, Zhijian Lin, Yongxiang Qiu, Hao Tang & Sixin Qiu

Authors
  1. Huawei Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhonghua Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhijian Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yongxiang Qiu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hao Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sixin Qiu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sixin Qiu.

Ethics declarations

Conflict of interest

The authors declare no competing interests.

Additional information

Communicated by Erko Stackebrandt.

Publisher's Note

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

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, H., Zhang, H., Liu, Z. et al. Rapid diagnosis of Ralstonia solanacearum infection sweet potato in China by loop-mediated isothermal amplification. Arch Microbiol 203, 777–785 (2021). https://doi.org/10.1007/s00203-020-02059-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00203-020-02059-8

Keywords

Search

Navigation