European Journal of Plant Pathology

, Volume 155, Issue 1, pp 173–179 | Cite as

Development of a loop-mediated isothermal amplification for rapid diagnosis of Aphelenchoides ritzemabosi

  • Dong-Wei Wang
  • Chun-Ling Xu
  • Zong-Shi Bai
  • Jun-Yi Li
  • Yu-Chun Han
  • Li-Rong Zhao
  • Hui XieEmail author


To establish the amplification and detection system for the chrysanthemum foliar nematode (CFN), Aphelenchoides ritzemabosi, the loop-mediated isothermal amplification (LAMP), which includes two external primers (F3/B3), two inner primers (FIP/BIP) and one loop primer (LF), was designed based on the CFN 18S ribosomal RNA gene. The DNA samples were amplified by LAMP primers using isothermal amplification (65 °C). The amplified products were detected by electrophoresis and visual inspection of fluorescent dyes. Detection of CFN in a sample was confirmed when the electrophoresis results show a DNA ladder or the mixtures in the tubes showed green fluorescence. The results showed that the LAMP method could not only detect and identify a single female, male, juvenile and egg of CFN but also directly detect CFN in mixed nematode species samples and plant tissue samples. The sensitivity of the LAMP assay was 100-fold dilution of single nematode DNA.


Aphelenchoides ritzemabosi Loop-mediated isothermal amplification 18S ribosomal RNA Single nematode Mixed samples 



This work was supported by National Natural Science Foundation of China (No. 31871939), Key science and technology project of Hainan Province (No. 080151) and Special research and development of Applied Technology in Hainan (No. ZDXM2014020).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This study does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

Informed consent was obtained from all individual participants included in the study.


  1. Bai, Z., Qin, M., Zhao, L., et al. (2017). Loop-mediated isothermal amplification assay for rapid diagnosis of Aphelenchoides besseyi. Chinese Journal of Rice Science, 31, 432–440.Google Scholar
  2. Chen, C., Xie, H., Wei, Y., et al. (2006). Studies on cultural technique and reproduction fitness of Radopholus similis. Journal of South China Agricultural University, 26, 61–64.Google Scholar
  3. Crozzoli, R., Hurtado, T., Perichi, G., et al. (2008). Characterization of a Venezuelan population of Aphelenchoides ritzemabosi on chrysanthemum. Nematologia Mediterranea, 36, 79–83.Google Scholar
  4. Cui, R. Q., Zhao, L. R., & Zhong, G. Q. (2010). A rapid method to detect Aphlenchoides ritzemabosi by PCR. Acta Agriculturae Universitatis Jiangxiensis, 32, 714–717.Google Scholar
  5. Dai, S., Wang, W., & Huang, J. (2002). Advances of researches on phylogeny of Dendranthema and origin of chrysanthemum. Journal of Beijing Forestry University, 24, 230–234.Google Scholar
  6. Evans, K., Trudgill, D. L., & Webster, J. M. (1993). Plant parasitic nematodes in temperate agriculture (p. 648). Wallingford: CBA International.Google Scholar
  7. He, X. F., Peng, H., Ding, Z., et al. (2013). Loop-mediated isothermal amplification assay for rapid diagnosis of Meloidogyne enterolobii directly from infected plants. Scientia Agricultura Sinica, 46, 534–544.Google Scholar
  8. Hooper, D. J., & Cowland, J. A. (1986). Fungal hosts for the chrysanthemum nematode, Aphelenchoides ritzemabosi. Plant Pathology, 35, 128–129.CrossRefGoogle Scholar
  9. Hunt, D. J. (1993). Aphelenchida, Longidoridae and Trichodoridae, their systematic and blonomics (p. 352). Wallingford: CBA International.Google Scholar
  10. Imai, M., Ninomiya, A., Minekawa, H., Notomi, T., Ishizaki, T., Tashiro, M., & Odagiri, T. (2006). Development of H5-RT LAMP (loop-mediated isothermal amplification) system for rapid diagnosis of H5 avian influenza virus infection. Vaccine, 24, 6679–6682.CrossRefGoogle Scholar
  11. Kikuchi, T., Aikawa, T., Oeda, Y., Karim, N., & Kanzaki, N. (2009). A rapid and precise diagnostic method for detecting the pinewood nematode Bursaphelenchus xylophilus by loop-mediated isothermal amplification. Phytopathology, 99, 1365–1369.CrossRefGoogle Scholar
  12. Leal, I., Foord, B., Allen, E., et al. (2013). Development of a reverse transcription loop mediated isothermal amplification (RTLAMP) method to detect living pinewood nematode, Bursaphelenchus xylophilus, in wood. Forest Pathology, 43, 101–114.CrossRefGoogle Scholar
  13. Niu, J. H., Guo, Q. X., Jian, H., Chen, C. L., Yang, D., Liu, Q., & Guo, Y. D. (2011). Rapid detection of Meloidogyne spp. by LAMP assay in soil and roots. Crop Protection, 30, 1063–1069.CrossRefGoogle Scholar
  14. Niu, J. H., Jian, H., Guo, Q. X., Chen, C. L., Wang, X. Y., Liu, Q., & Guo, Y. D. (2012). Evaluation of loop-mediated isothermal amplification (LAMP) assays based on 5S r DNA-IGS2 regions for detecting Meloidogyne enterolobii. Plant Pathology, 61, 809–819.CrossRefGoogle Scholar
  15. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., & Hase, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 28, E63.CrossRefGoogle Scholar
  16. Peng, H., Peng, D. L., Hu, X. Q., et al. (2012). Loop-mediated isothermal amplification for rapid and precise detection of the burrowing nematode, Radopholus similis, directly from diseased plant tissues. Nematology, 14, 977–986.CrossRefGoogle Scholar
  17. Peng, D. L., Xu, X. Q., Peng, H., et al. (2013). Loop-mediated isothermal amplification assay for rapid diagnosis of Heterodera avenae. China, 2013100203834.4.Google Scholar
  18. Peng, X. F., Chen, D. Q., Wang, D. W., et al. (2015). Culture conditions and reproductive characteristics of Aphelenchoides ritzemabosi on callus. Journal of Huazhong Agricultural University, 34, 48–51.Google Scholar
  19. Reise, R. W., Huettel, R. N., & Sayre, R. M. (1987). Carrot callus tissue for culture of endoparasitic nematodes. Journal of Nematology, 19, 387–389.PubMedPubMedCentralGoogle Scholar
  20. Rybarczyk-Mydłowska, K., Mooyman, P., van Megen, H., van den Elsen, S., Vervoort, M., Veenhuizen, P., van Doorn, J., Dees, R., Karssen, G., Bakker, J., & Helder, J. (2012). Small subunit ribosomal DNA-based phylogenetic analysis of foliar nematodes (Aphelenchoides spp.) and their quantitative detection in complex DNA backgrounds. Phytopathology, 102, 1153–1160.CrossRefGoogle Scholar
  21. Samaliev, H. Y., & Mohamedova, M. (2011). Plant-parasitic nematodes associated with strawberry (Fragaria aiianassa Duch.) in Bulgaria. Bulgarian Journal of Agricultural Science, 17, 730–735.Google Scholar
  22. Siddiqi, M. R. (1974). Aphelenchoides ritzemabosi. In: CIH descriptions of plant prarasitic nematodes, Set 3, N0.32, England: Commonwealth Institute of Helminthology, 4pp.Google Scholar
  23. Vovlas, N., Minuto, A., Gar-ibaldi, A., et al. (2005). Identification and histopathology of the foliar nematode Aphelenchoides ritzemabosi (Nematoda: Aphelenchoididae) on basil in Italy. Nematology, 7, 301–308.CrossRefGoogle Scholar
  24. Wang, J. L., Zhang, J. C., & Gu, J. F. (2011). Method of extract DNA from a single nematode. Plant Quarantine, 25, 32–35.Google Scholar
  25. Wang, X., Li, H. M., Gu, J. F., et al. (2014). Loop-mediated isothermal amplification assay for rapid diagnosis of Aphlenchoides besseyi. China, CN201410081273.3.Google Scholar
  26. Wei, H. Y., Wang, X., Li, H. M., et al. (2016). Loop-mediated isothermal amplification assay for rapid diagnosis of Meloidogyne mali. Journal of Plant Protection, 43, 260–266.Google Scholar
  27. Xie, H. (2005). Taxonomy of plant nematodes (2nd ed.p. 435). Beijing: China Higher Education Press.Google Scholar
  28. Xie, H. (2007). The epidemic and detection methods of Aphelenchoides ritzemabosi. Plant Quarantine, 21, 190–192.Google Scholar
  29. Xiong, Y. F., Wang, X., Ge, J. J., et al. (2014). Loop-mediated isothermal amplification for detecting of Burrowing nematode (Radopholus similis). Plant Quarantine, 28, 48–51.Google Scholar
  30. Zhang, Y. W., Li, X. N., Guo, P. P., et al. (2010). Fluorescent reagent in detection of porcine reproductive and respiratory syndrome virus by loop-mediated isothermal amplification (LAMP). Journal of Agricultural Biotechnology, 18, 508–513.Google Scholar
  31. Zhu, L. F. (2014). Detection of Meloidogyne hapla and glyphosate-resistant soybean by LAMP [M. D]. BeiJing: Chinese Academy of Agricultural Sciences.Google Scholar

Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2019

Authors and Affiliations

  • Dong-Wei Wang
    • 1
  • Chun-Ling Xu
    • 1
  • Zong-Shi Bai
    • 1
  • Jun-Yi Li
    • 1
  • Yu-Chun Han
    • 2
  • Li-Rong Zhao
    • 3
  • Hui Xie
    • 1
    Email author
  1. 1.Laboratory of Plant Nematology/Research Center of Nematodes of Plant Quarantine, Department of Plant Pathology/Guangdong Province Key Laboratory of Microbial Signals and Disease Control, College of AgricultureSouth China Agricultural UniversityGuangzhouPeople’s Republic of China
  2. 2.Postentry Quarantine Station for Tropical Plant, Hainan Entry-Exit Inspection and Quarantine BureauHaikouPeople’s Republic of China
  3. 3.Inspection & Quarantine Technology, Guangdong Entry-Exit Inspection and Quarantine BureauGuangzhouPeople’s Republic of China

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