Skip to main content
SpringerLink
Log in

Development of a Loop-Mediated Isothermal Amplification Assay for Rapid and Specific Identification of ACT Producing Alternaria alternata, the Agent of Brown Spot Disease in Tangerine

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Rapid, accurate, and easy identification of pathogenic agents has always been important in medicine, veterinary, and agriculture. The brown spot infection is among the most common diseases in tangerine caused by Alternaria alternata. Due to the existence of seven various pathotypes of A. alternata species, it is challenging and time consuming to detect a pathotype responsible for citrus brown spot. In this study, we were seeking a rapid and specific approach to identify the tangerine pathotype within the A. alternata-pathogenic species, using the loop-mediated isothermal amplification (LAMP) method and actts2 gene as a marker molecule. Nine pathogenic samples were obtained from the region of Ramsar, Iran, and certified as A. alternata-pathogenic isolates. Specific primers were designed for regions coding for Alternaria citri toxin (ACT), and the PCR and LAMP reactions were performed. Our data showed that the primers designed for the tangerine pathotype of A. alternata were specific, and in both reactions, positive results were only observed in desired pathotypes. In the other pathotypes of this species as well as other standard fungal samples as negative controls, no positive result was observed. Therefore, our results suggest the possibility to detect the tangerine-specific A. alternata pathotype from other related species with a high accuracy and in early stages of the disease.

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

Similar content being viewed by others

References

  1. Pimentel, D. (2009). Pesticides and pest control. In Integrated pest management: innovation development process (pp. 83–87). Netherlands: Springer.

    Chapter  Google Scholar 

  2. Henson, J. M., & French, R. (1993). The polymerase chain reaction and plant disease diagnosis. Annual Review of Phytopathology, 31, 81–109.

    Article  CAS  Google Scholar 

  3. Walton, J. D. (1996). Host-selective toxins: agents of compatibility. Plant Cell, 8, 1723–1733.

    Article  CAS  Google Scholar 

  4. Tsuge, T., Harimoto, Y., Akimitsu, K., Ohtani, K., Kodama, M., Akagi, Y., Egusa, M., Yamamoto, M., & Otani, H. (2013). Host‐selective toxins produced by the plant pathogenic fungus Alternaria alternata. FEMS Microbiology Reviews, 37(1), 44–66.

    Article  CAS  Google Scholar 

  5. Johnson, R. D., Johnson, L., Kohomoto, K., Otani, H., Lane, C. R., & Kodama, M. (2000). A polymerase chain reaction-based method to specifically detect Alternaria alternata apple pathotype (A. mali), the causal agent of Alternaria blotch of apple. Phytopathology, 90(9), 973–976.

    Article  CAS  Google Scholar 

  6. Kohmoto, K., Itoh, Y., Shimomura, N., Kondoh, Y., Otani, H., Kodama, M., Nishimura, S., & Nakatsuka, S. (1993). Isolation and biological activities of two host-specific toxins from the tangerine pathotype of Alternaria alternata. Phytopathology, 83(5), 495–502.

    Article  CAS  Google Scholar 

  7. Ajiro, N., Miyamoto, Y., Masunaka, A., et al. (2010). Role of the host-selective ACT-toxin synthesis gene ACTTS2 encoding an enoyl-reductase in pathogenicity of the tangerine pathotype of Alternaria alternata. Phytopathology, 100(2), 120–126.

    Article  CAS  Google Scholar 

  8. Miyamoto, Y., Masunaka, A., Tsuge, T., Yamamoto, M., Ohtani, K., Fukumoto, T., Gomi, K., Peever, T. L., Tada, Y., Ichimura, K., & Akimitsu, K. (2010). ACTTS3 encoding a polyketide synthase is essential for the biosynthesis of ACT-toxin and pathogenicity in the tangerine pathotype of Alternaria alternata. Molecular Plant-Microbe Interactions, 23(4), 406–414.

    Article  CAS  Google Scholar 

  9. Gill, P., & Ghaemi, A. (2008). Nucleic acid isothermal amplification technologies—a review. Nucleosides, Nucleotides and Nucleic Acids, 27(3), 224–243.

    Article  CAS  Google Scholar 

  10. 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(12), e63–e63.

    Article  CAS  Google Scholar 

  11. Hamedi, J., Papiran, R., & Moghimi, H. (2014). Isolation and screening of phytotoxin-producing actinomycetes for biological control of Cardaria draba. Progress in Biological Sciences, 4(1), 111–119.

    Google Scholar 

  12. Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning (A laboratory manual 2nd ed.). New York: Cold Spring Harbor Laboratory.

    Google Scholar 

  13. Borneman, J., & Hartin, R. J. (2000). PCR primers that amplify fungal rRNA genes from environmental samples. Applied and Environmental Microbiology, 66(10), 4356–4360.

    Article  CAS  Google Scholar 

  14. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic Local Alignment Search Tool. Journal of Molecular Biology, 215(3), 403–410.

    Article  CAS  Google Scholar 

  15. Kuninaga, S., & Yokosawa, R. (1987). Studies on the taxonomy of plant pathogenic fungi by a comparison of DNA homology. I. Genetic relatedness among species in the genus Alternaria. Annals of the Phytopathological Society of Japan, 53, 368–369 (in Japanese).

    Article  Google Scholar 

  16. Kusaba, M., & Tsuge, T. (1994). Nuclear ribosomal DNA variation and pathogenic specialization in Alternaria fungi known to produce host-specific toxins. Applied and Environmental Microbiology, 60(9), 3055–3062.

    CAS  Google Scholar 

  17. Kusaba, M., & Tsuge, T. (1995). Phylogeny of Alternaria fungi known to produce host-specific toxins on the basis of variation in internal transcribed spacers of ribosomal DNA. Current Genetics, 28(5), 491–498.

    Article  CAS  Google Scholar 

  18. Moradi, A., Nasiri, J., Abdollahi, H., & Almasi, M. (2012). Development and evaluation of a loop-mediated isothermal amplification assay for detection of Erwinia amylovora based on chromosomal DNA. European Journal of Plant Pathology, 133, 609–620.

    Article  CAS  Google Scholar 

  19. Tomlinson, J. A., Dickinson, M. J., & Boonham, N. (2010). Detection of Botrytis cinerea by loop-mediated isothermal amplification. Letters in Applied Microbiology, 51, 650–657.

    Article  CAS  Google Scholar 

  20. Huang, C., Sun, Z., Yan, J., Luo, Y., Wang, H., & Ma, Z. (2011). Rapid and precise detection of latent infections of wheat stripe rust in wheat leaves using loop-mediated isothermal amplification. Journal of Phytopathology, 159, 582–584.

    Article  Google Scholar 

  21. Li, B., Du, J., Lan, C., Liu, P., Weng, Q., & Chen, Q. (2013). Development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of Fusarium oxysporum f. sp. cubense race 4. European Journal of Plant Pathology, 135, 903–911.

    Article  CAS  Google Scholar 

  22. Grote, D., Olmos, A., Kofoet, A., Tuset, J. J., Bertolini, E., & Cambra, M. (2002). Specific and sensitive detection of Phytophthora nicotianae by simple and nested-PCR. European Journal of Plant Pathology, 108(3), 197–207.

    Article  CAS  Google Scholar 

  23. Sawamura, K. (1962). Studies on spotted disease of apples. I. Causal agent of Alternaria blotch. Bull Tohoku Natl Agric Exp Stn Ser, 23, 163–175.

    Google Scholar 

  24. Okuno, T., Ishita, Y., Sawai, K., & Matsumoto, T. (1974). Characterization of alternariolide, a host-specific toxin produced by Alternaria mali Roberts. Chemistry Letters, 1974, 635–638.

    Article  Google Scholar 

  25. Maekawa, N., Yamamoto, M., Nishimura, S., Kohmoto, K., Kuwada, K., & Watanabe, Y. (1984). Studies on host-specific AF-toxins produced by Alternaria alternata strawberry pathotype causing Alternaria black spot of strawberry. Production of host-specific toxins and their biological activities. Annals of the Phytopathological Society of Japan, 50, 600–609.

    Article  CAS  Google Scholar 

  26. Nakatsuka, S., Ueda, K., Goto, T., Yamamoto, M., Nishimura, S., & Kohmoto, K. (1986). Structure of AF-toxin II, one of the host-specific toxins produced by Alternaria alternata strawberry pathotype. Tetrahedron Letters, 27, 2753–2756.

    Article  CAS  Google Scholar 

  27. Tanaka, S. (1933). Studies on black spot disease of the Japanese pear (Pyrus serotina Rehd.). Mem Coll Agric Kyoto Imp Univ, 28, 1–31.

    Google Scholar 

  28. Pegg, K. G. (1966). Studies of a strain of Alternaria citri Pierce, the causal organism of brown spot of emperor mandarin (Citrus reticulata). Queensl J Agric Anim Sci, 23, 15–28.

    Google Scholar 

  29. Timmer, L. W., Solel, Z., & Orozco-Santos, M. (2000). Alternaria brown spot of mandarins. Compendium of citrus diseases (pp. 19–21). St. Paul: The American Phytopathological Society Press.

    Google Scholar 

  30. Doidge, E. M. (1929). A study of some Alternarias infecting citrus in South Africa. Union South Africa Dept Agric Sci Bull, 69, 1–29.

    Google Scholar 

  31. Gardner, J. M., Kono, Y., Tatum, J. H., Suzuki, Y., & Takeuchi, S. (1985). Structure of major component of ACRL toxins, host-specific phytotoxic compound produced by Alternaria citri. Agricultural and Biological Chemistry, 49, 1235–1238.

    Article  CAS  Google Scholar 

  32. Grogan, R. G., Kimble, K. A., & Misagi, I. (1975). A stem canker disease of tomato caused by Alternaria alternata f. sp. lycopersici. Phytopathology, 65, 880–886.

    Article  Google Scholar 

  33. Lucas, G. B. (1975). Disease of tobacco. Raleigh: Biological Consulting Association.

    Google Scholar 

  34. Kodama, M., Suzuki, T., Otani, H., Kohmoto, K., & Nishimura, S. (1990). Purification and bioassay of host-selective AT-toxin from Alternaria alternata causing brown spot of tobacco. Annals of the Phytopathological Society of Japan, 56, 628–636.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Mr. Hossein Taheri from the Iran Citrus Research Institute in Ramsar for his valuable support.

Author information

Authors and Affiliations

  1. Department of Microbial Biotechnology, School of Biology and Center of Excellence in Phylogeny of Living Organisms, College of Science, University of Tehran, Enghelab Ave., Tehran, 141556455, Iran

    Hamid Moghimi, Amir Moradi, Javad Hamedi & Mina Basiri

  2. Microbial Technology and Products Research Center, University of Tehran, Ghods St., Enghelab Ave., Tehran, 1417864411, Iran

    Hamid Moghimi, Amir Moradi, Javad Hamedi & Mina Basiri

Authors
  1. Hamid Moghimi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Amir Moradi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Javad Hamedi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mina Basiri
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hamid Moghimi.

Ethics declarations

All the experiments undertaken in this study comply with the current laws of the country where they were performed.

Conflict of Interest

The authors declare that they have no competing interests.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Moghimi, H., Moradi, A., Hamedi, J. et al. Development of a Loop-Mediated Isothermal Amplification Assay for Rapid and Specific Identification of ACT Producing Alternaria alternata, the Agent of Brown Spot Disease in Tangerine. Appl Biochem Biotechnol 178, 1207–1219 (2016). https://doi.org/10.1007/s12010-015-1939-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-015-1939-x

Keywords

Search

Navigation