Skip to main content

Establishment and evaluation of a TEF1-α based loop-mediated isothermal amplification assay for detection of Phomopsis longicolla

Abstract

Phomopsis longicolla is an important seed-borne pathogen affecting soybean. In this research, transcription elongation factor 1-α (TEF1-α) was identified as a suitable target for detecting P. longicolla using Loop-mediated isothermal amplification (LAMP) method. The specificity of the method was tested against 54 P. longicolla isolates, 13 oomycetes isolates and 28 other fungal isolates. With the addition of hydroxynaphthol blue (HNB) prior to amplification, a sky-blue color was only observed in the presence of P. longicolla, whereas other isolates showed no color change. The method was sensitive enough to detect as little as 100 pg/uL fungal DNA. In addition, the assay also detected P. longicolla from diseased soybean tissues and residues from different origins. The results demonstrated that the LAMP assay provides a rapid and sensitive tool for detecting P. longicolla from plant tissues.

This is a preview of subscription content, access via your institution.

Fig. 1

References

  • Baird RE, Abney TS, Mullinix BG (2001) Fungi associated with pods and seeds during the R6 and R8 stages of four soybean cultivars in southwestern Indiana. Phytoprotection 82:1–11

    Article  Google Scholar 

  • Cui YL, Duan CX, Wang XM, Li HJ, Zhu ZD (2009) First report of Phomopsis longicolla causing soybean stem blight in China. Plant Pathol 58:799–799

    Article  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Harrington T, Steimel J, Workneh F, Yang X (2000) Molecular identification of fungi associated with vascular discoloration of soybean in the north central United States. Plant Dis 84:83–89

    Article  CAS  Google Scholar 

  • Hepperly P, Sinclair J (1978) Quality losses in Phomopsis-infected soybean seeds. Phytopathology 68:1684–1687

    Article  Google Scholar 

  • Hobbs TW, Schmitthenner A, Kuter GA (1985) A new Phomopsis species from soybean. Mycologia 77:535–544

    Article  Google Scholar 

  • Morgan-Jones G (1989) The Diaporthe/Phomopsis complex: taxonomic considerations Proceedings of the World Soybean Research Conference IV. A. Pascale, ed. Orientación Gráfica, Buenos Aires, Argentin 161:1699–1706

  • Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:63–64

    Article  Google Scholar 

  • Shan Z, Li S, Liu Y, Yang Z, Yang C, Sha A, Chen H, Chen S, Zhou XA (2012) First report of phomopsis seed decay of soybean caused by Phomopsis longicolla in South China. Plant Dis 96:1693–1693

    Article  Google Scholar 

  • Sinclair J (1993) Phomopsis seed decay of soybeans: a prototype for studying seed disease. Plant Dis 77:329–334

    Article  Google Scholar 

  • Sinclair J (1995) Reevaluation of grading standards and discounts for fungus-damaged soybean seeds. J Am Oil Chem Soc 72:1415–1419

    Article  CAS  Google Scholar 

  • Wang H, Qi M, Cutler AJ (1993) A simple method of preparing plant samples for PCR. Nucleic Acids Res 21:4153--4154

  • Zhang Y, Shan X, Shi L, Lu X, Tang S, Wang Y, Li Y, Alam M, Yan H (2012) Development of a fimY-based loop-mediated isothermal amplification assay for detection of Salmonella in food. Food Res Int 45:1011–1015

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by National Natural Science Foundation of China under Grant 31500526, the National ‘863’ Program under Grant 2012AA101501, China Postdoctoral Science Foundation under Grant 2014 M561657, Jiangsu Postdoctoral Sustentation Fund under Grant 1402159c and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Ting-ting Dai or Xiao-Bo Zheng.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Table S1

(DOC 77 kb)

Table S2

(DOC 60 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Dai, Tt., Shen, H. & Zheng, XB. Establishment and evaluation of a TEF1-α based loop-mediated isothermal amplification assay for detection of Phomopsis longicolla . Australasian Plant Pathol. 45, 335–337 (2016). https://doi.org/10.1007/s13313-016-0415-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13313-016-0415-6

Keywords

  • TEF1-α
  • HNB
  • Loop-mediated isothermal amplification
  • Phomopsis longicolla