Advertisement

Journal of Plant Pathology

, Volume 101, Issue 1, pp 51–57 | Cite as

Loop-mediated isothermal amplification (LAMP) detection of Phytophthora hibernalis, P. syringae and P. cambivora

  • Guan-Rong LiEmail author
  • Guo-Ming Huang
  • Lin-Hui Zhu
  • Dajin Lv
  • Baohong Cao
  • Fang Liao
  • Jia-Feng Luo
Original Article
  • 29 Downloads

Abstract

Phytophthora hibernalis, P. syringae and P. cambivora are devastating quarantine pathogens to many economically important plants worldwide. They often co-infect the same plant, causing rots and leading to great loss in yield and even to the death of the plant. Conventionally, the detection of the three pathogens has been based on time-consuming and laborious morphological methods and, although PCR-based detection is much better, it is still thermal cycler- and electrophoresis-dependent. A high throughput, rapid, convenient and cost-effective detection is required for quarantine detection at the port of entry. In this study, based on the novel LAMP technique, three sets of four LAMP primers, PH-F3/PH-B3/PH-FIP/PH-BIP, PS-F3/PS-B3/PS-FIP/PS-BIP and PC-F3/PC-B3/PC-FIP/PC-BIP, designed on the sequences of enolase (Enol), internal transcribed spacers (ITS) and ras-like protein Ypt1 genes of P. hibernalis, P. syringae and P. cambivora, were designed. Through the optimization of the reaction system and conditions, a highly specific and extremely sensitive LAMP detection system was developed for P. hibernalis, P. syringae and P. cambivora. The LAMP products showed the typical DNA ladders upon agarose gel electrophoresis, the visual turbidity of the reaction solution and the white precipitate of byproduct magnesium pyrophosphate upon centrifugation were observed; the LAMP solution turned light green from orange upon the addition of SYBR Green I. No cross reactions with the other closely related Phytophthora species were found, whereas specificity and sensitivity were very high, for the detection of 0.02 fg of genomic DNA was more than 105 times higher than the corresponding conventional PCR. To the best of our knowledge, this represents the first detection of the three quarantine Phytophthora pathogens by LAMP, a technique with a great potential for the identification of other Phytophthora pathogens.

Keywords

P. hibernalis P. syringae P. cambivora Loop-mediated isothermal amplification (LAMP) detection 

Notes

Acknowledgements

This work was funded by Rapid Identification of Quarantine Fungal Phytophthora Pathogens of Imported Fruits and Seedlings, a Program from The General Administration of Quality Supervision, Inspection and Quarantine of PRC (No: 2012IK286) and Plant Quarantine Pathogen Detection and Application by DNA Barcoding, National “12th Five-Year” Science and Technology Support Program (No:2012BAK 11- B02).

References

  1. Chen Q, Li B, Liu P, Lan C, Zhan Z, Weng Q (2013) Development and evaluation of specific PCR and LAMP assays for the rapid detection of Phytophthora melonis. Eur J Plant Pathol 137:597–607CrossRefGoogle Scholar
  2. Dai TT, Lu CC, Lu J, Dong S, Ye W, Wang Y, Zheng X (2012) Development of a loop-mediated isothermal amplification assay for detection of Phytophthora sojae. FEMS Microbiol Lett 334:27–34CrossRefGoogle Scholar
  3. Du HZ, Wu PS, Yan J, Zhang QW (2013) Detection of Phytophthora hibernalis by real-time fluorescent PCR. Plant Quarantine 27:36–39 (in Chinese)Google Scholar
  4. Félix-Gastélum R, Mircetich SM (2005) Influence of flooding duration on the development root and crown rot of Lovell peach [Prunus persica (L.) Bstsch] caused by three different Phytophthora species. Revista Mexicana de Fitopatología 23:33–41Google Scholar
  5. Feng LP, Liu HM, Wang YC, Li Y, Wu XH, Ji Y, Wu PS (2011) SN/T 2759-2011. The entry-exit inspection and quarantine industry standards of the People's Republic of China-quarantine and identification of Phytophthora cambivora (petri) Buisman. China Standards Press, Beijing, China (in Chinese)Google Scholar
  6. Fukuta S, Kato S, Yoshida K, Mizukami Y, Ishida AM, Ueda J, Kanbe M, Ishimoto Y (2003) Detection of tomato yellow leaf curl virus by loop-mediated isothermal amplification reaction. J Virol Methods 112:35–40CrossRefGoogle Scholar
  7. Fukuta S, Ohishi K, Yoshida K, Mizukami Y, Ishida A, Kanbe M (2004) Development of immunocapture reverse transcription loop-mediated isothermal amplification for the detection of tomato spotted wilt virus from chrysanthemum. J Virol Methods 121:49–55CrossRefGoogle Scholar
  8. Gao R, Zhang G (2013) Potential of DNA barcoding for detecting quarantine fungi. Phytopathology 103:1103–1107CrossRefGoogle Scholar
  9. Grote D, Olmos A, Kofoet A, Tuset JJ, Bertolini E, Cambra M (2002) Specific and sensitive detection of Phytophthora nicotianae by simple and nested-PCR. Eur J Plant Pathol 108:197–207CrossRefGoogle Scholar
  10. Hirayama H, Kageyama S, Moriyasu S, Sawai K, Minamihashi A (2013) Embryo sexing and sex chromosomal chimerism analysis by loop-mediated isothermal amplification in cattle and water buffaloes, 2013. J Reprod Dev 59:321–326CrossRefPubMedCentralGoogle Scholar
  11. Ippolito A, Schena L, Nigro F (2004) Real-time detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soil. Eur J Plant Pathol 110:833–843CrossRefGoogle Scholar
  12. Judelson HS, Tooley PW (2000) Enhanced polymerase chain reaction methods for detecting and quantifying Phytophthora infestans in plants. Phytopathology 90:1112–1119CrossRefGoogle Scholar
  13. Kroon LP, Brouwer H, de Cock AW, Govers F (2012) The genus Phytophthora anno 2012. Phytopathology 102:348–364CrossRefGoogle Scholar
  14. Li B, Liu P, Xie S, Yin R, Weng Q, Chen Q (2015) Specific and sensitive detection of Phytophthora nicotianae by nested PCR and loop-mediated isothermal amplification assays. J Phytopathol 163:185–193CrossRefGoogle Scholar
  15. Liu YT, Zhu LH, Li PJ, Liao F, Ren XY, Li GR (2015) Triplex-PCR molecular detection of two quarantine fungal diseases of Prunus-Phytophthora syringae and P. cambivora. Acta Phys Sin 42:571–577 (in Chinese)Google Scholar
  16. Niessen L, Vogel RF (2010) Detection of Fusarium graminearum DNA using a loop-mediated isothermal amplification (LAMP) assay. Int J Food Microbiol 140:183–191CrossRefGoogle Scholar
  17. 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:E63CrossRefPubMedCentralGoogle Scholar
  18. Okuda M, Matsumoto M, Tanaka Y, Subandiyah S, Iwanami T (2005) Characterization of the tufB- secE-nusG-rplKAJL-rpoB gene cluster of the citrus greening organism and detection by loop-mediated isothermal amplification. Plant Dis 89:705–711CrossRefGoogle Scholar
  19. Parida M, Horioke K, Ishida H, Dash PK, Saxena P, Jana AM, Islam MA, Inoue S, Hosaka N, Morita K (2005) Rapid detection and differentiation of dengue virus serotype by a real time reverse transcription-loop mediated isothermal amplification assay. J Clin Microbiol 43:2895–2903.  https://doi.org/10.1128/JCM.43.6.2895-2903.2005 CrossRefPubMedCentralGoogle Scholar
  20. Ristaino JB, Madritch M, Trout CL (1998) PCR amplification of ribosomal DNA for species identification in the plant pathogen genus Phytophthora. Appl Environ Microbiol 64:948–954PubMedCentralGoogle Scholar
  21. Schena L, Hughes KJD, David E (2006) Detection and quantification of Phytophthora ramorum, P. kernoviae, P. citricola and P. quercina in symptomatic leaves by multiplex real-time PCR. Mol Plant Pathol 7:365–379CrossRefGoogle Scholar
  22. Wang YC, Li Y, Wu PS, Shao XL, Zhang JX, Yu DD, Gan QH, Song T (2011) SN/T 2756—2011. The entry-exit inspection and quarantine industry standards of the People's Republic of China - inspection and identification of Phytophthora syringe (Berk.) Kleb. China standards press, Beijing, China (in Chinese)Google Scholar
  23. Zhang ZG, Li YQ, Fan H, Wang YC, Zheng XB (2006) Molecular detection of Phytophthora capsici in infected plant tissues, soil and water. Plant Pathol 55:770–775CrossRefGoogle Scholar
  24. Zhang HF, Ren Z, Liu X, Zhang ZG, Wang YC, Wu XH, Zheng XB (2008) Rapid molecular detection of Phytophthora hibernalis by PCR. Acta Phys Sin 38:231–237 (in Chinese)Google Scholar
  25. Zhang YJ, Liu YT, Liao F, Luo JF, Huang GM (2011) Research on molecular detection of Phytophthora medicaginis on LAMP. China Plant Protection 31:6–9 (in Chinese)Google Scholar
  26. Zhu LH, Guo JZ, Liao F, Luo JF, Huang GM, Ren XY, Li GR (2015) Simultaneous triplex PCR detection of two quarantine fungal pathogens of citrus, Phytophthora hiberalis and Phytophthora syringae. J Southwest Univ 37:1–8 (in Chinese)Google Scholar

Copyright information

© Società Italiana di Patologia Vegetale (S.I.Pa.V.) 2018

Authors and Affiliations

  • Guan-Rong Li
    • 1
    Email author
  • Guo-Ming Huang
    • 2
  • Lin-Hui Zhu
    • 1
  • Dajin Lv
    • 1
  • Baohong Cao
    • 1
  • Fang Liao
    • 2
  • Jia-Feng Luo
    • 2
  1. 1.College of Agronomy and BiotechnologySouthwest UniversityChongqingChina
  2. 2.Tianjin Entry-exit Inspection and Quarantine BureauTianjinChina

Personalised recommendations