The Plant Pathology Journal

The Korean Society of Plant Pathology (한국식물병리학회)
권호 표지
DOI QR코드

DOI QR Code

Rapid and Efficient Detection of 16SrI Group Areca Palm Yellow Leaf Phytoplasma in China by Loop-Mediated Isothermal Amplification

  • Yu, Shao-shuai (Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences) ;
  • Che, Hai-yan (Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences) ;
  • Wang, Sheng-jie (Research Institute of Tropical Forestry, Chinese Academy of Forestry) ;
  • Lin, Cai-li (Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry) ;
  • Lin, Ming-xing (Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences) ;
  • Song, Wei-wei (Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences) ;
  • Tang, Qing-hua (Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences) ;
  • Yan, Wei (Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences) ;
  • Qin, Wei-quan (Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences)
  • Received : 2020.06.04
  • Accepted : 2020.08.20
  • Published : 2020.10.01
Download PDF
⟨ Previous Next ⟩

Abstract

Areca palm yellow leaf (AYL) disease caused by the 16SrI group phytoplasma is a serious threat to the development of the Areca palm industry in China. The 16S rRNA gene sequence was utilized to establish a rapid and efficient detection system efficient for the 16SrI-B subgroup AYL phytoplasma in China by loop-mediated isothermal amplification (LAMP). The results showed that two sets of LAMP detection primers, 16SrDNA-2 and 16SrDNA-3, were efficient for 16SrI-B subgroup AYL phytoplasma in China, with positive results appearing under reaction conditions of 64℃ for 40 min. The lowest detection limit for the two LAMP detection assays was the same at 200 ag/μl, namely approximately 53 copies/μl of the target fragments. Phytoplasma was detected in all AYL disease samples from Baoting, Tunchang, and Wanning counties in Hainan province using the two sets of LAMP primers 16SrDNA-2 and 16SrDNA-3, whereas no phytoplasma was detected in the negative control. The LAMP method established in this study with comparatively high sensitivity and stability, provides reliable results that could be visually detected, making it suitable for application and research in rapid diagnosis of AYL disease, detection of seedlings with the pathogen and breeding of disease-resistant Areca palm varieties.

Keywords

References

  1. Che, H. and Luo, D. 2011. A method for the detection of phytoplasma of Areca palm yellow leaf disease and its special kit. China Patent No. ZL200910077044.3.
  2. Che, H., Wu, C., Fu, R., Wen, Y. and Luo, D. 2010. Molecular identification of pathogens from arecanut yellow leaf disease in Hainan. Chinese J. Trop. Crops 31:83-87. https://doi.org/10.3969/j.issn.1000-2561.2010.01.016
  3. Deng, S. and Hiruki, C. 1991. Amplification of 16S rRNA genes from culturable and nonculturable Mollicutes. J. Microbiol. Methods 14:53-61. https://doi.org/10.1016/0167-7012(91)90007-D
  4. Dickinson, M. and Hodgetts, J. 2013. Phytoplasma: methods and protocols. Humana Press, Totowa, NJ, USA. 421 pp.
  5. Doi, Y., Teranaka, M., Yora, K. and Asuyama, H. 1967. Mycoplasma or PLT group-like microorganisms found in the phloem elements of plants infected with mulberry dwarf, potato witches' broom, aster yellows or paulownia witches' broom. Jpn. J. Phytopathol. 33:259-266. https://doi.org/10.3186/jjphytopath.33.259
  6. Lee, I. M., Hammond, R. W., Davis, R. E. and Gundersen, D. E. 1993. Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasmalike organisms. Phytopathology 83:834-842. https://doi.org/10.1094/Phyto-83-834
  7. Muddumadiah, C., Madhupriya, Kumar, S., Manimekalai, R. and Rao, G. P. 2014. Detection and characterization of 16SrI-B phytoplasmas associated with yellow leaf disease of arecanut palm in India. Phytopathogenic Mollicutes 4:77-82. https://doi.org/10.5958/2249-4677.2014.00585.4
  8. Nagamine, K., Hase, T. and Notomi, T. 2002. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol. Cell. Probes 16:223-229. https://doi.org/10.1006/mcpr.2002.0415
  9. Nair, S., Manimekalai, R., Ganga Raj, P. and Hegde, V. 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. https://doi.org/10.1007/s11274-016-2078-4
  10. Nair, S., Roshna, O. M., Soumya, V. P., Hegde, V., Manimekalai, R. and Thomas, G. V. 2014. Real-time PCR technique for detection of arecanut yellow leaf disease phytoplasma. Australasian Plant Pathol. 43:527-529. https://doi.org/10.1007/s13313-014-0278-7
  11. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N. and Hase, T. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28:e63. https://doi.org/10.1093/nar/28.12.e63
  12. Obura, E., Masiga, D., Wachira, F., Gurja, B. and Khan, Z. R. 2011. Detection of phytoplasma by loop-mediated isother mal amplification of DNA (LAMP). J. Microbiol. Methods 84:312-316. https://doi.org/10.1016/j.mimet.2010.12.011
  13. Ramaswamy, M., Nair, S., Soumya, V. P. and Thomas, G. V. 2013. Phylogenetic analysis identifies a 'Candidatus Phytoplasma oryzae'-related strain associated with yellow leaf disease of areca palm (Areca catechu L.) in India. Int. J. Syst. Evol. Microbiol. 63:1376-1382. https://doi.org/10.1099/ijs.0.043315-0
  14. Schneider, B., Seemueller, E., Smart, C. D. and Kirkpatrick, B. C. 1995. Phylogenetic classification of plant pathogenic mycoplasma-like organisms or phytoplasmas. In: Molecular and diagnostic procedures in mycoplasmology, eds. by S. Razin and J. G. Tully, pp. 369-380. Academic Press, San Diego, CA, USA.
  15. Silva, C. K., Damayanthi, M., de Silva, R., Dickinson, M., de Silva, N. and Udagama, P. 2015. Molecular and scanning electron microscopic proof of phytoplasma associated with areca palm yellow leaf disease in Sri Lanka. Plant Dis. 99:1641.
  16. Sugawara, K., Himeno, M., Keima, T., Kitazawa, Y., Maejima, K., Oshima, K. and Namba, S. 2012. Rapid and reliable detection of phytoplasma by loop-mediated isothermal amplification targeting a housekeeping gene. J. Gen. Plant Pathol. 78:389-397. https://doi.org/10.1007/s10327-012-0403-9
  17. Wang, S., Wang, S., Lin, C., Yu, S., Wang, L., Piao, C., Guo, M. and Tian, G. 2017. Loop-mediated isothermal amplification assay for detection of five phytoplasmas belonging to 16SrI group based on target tuf gene. Sci. Silvae Sin. 53:54-63.
  18. Yu, S., Li, Y., Ren, Z., Song, C., Lin, C., Piao, C. and Tian, G. 2017. Multilocus sequence analysis for revealing finer genetic variation and phylogenetic interrelatedness of phytoplasma strains in 16SrI group in China. Sci. Silvae Sin. 53:105-118.
  19. Zhou, Y., Gan, B., Zhang, Z., Sui, C., Wei, J., Yang, Y. and Yang, X. 2010. Detection of the phytoplasmas associated with yellow leaf disease of Areca catechu L. in Hainan province of China by nested PCR. Chinese Agric. Sci. Bull. 26:381-384.