Australasian Plant Pathology

, Volume 48, Issue 6, pp 553–562 | Cite as

A loop-mediated isothermal amplification assay can rapidly diagnose soybean root-rot and damping-off diseases caused by Pythium spinosum

  • Hui Feng
  • Jiajia Chen
  • Ze Yu
  • Zhe Li
  • Wenwu Ye
  • Yuanchao Wang
  • Xiaobo ZhengEmail author
Original Paper


The oomycete pathogen causing root-rot and damping-off diseases in soybean (Glycine max) fields in the Huang-Huai region of China was identified as Pythium spinosum. To detect P. spinosum for disease diagnosis and control, we developed a loop-mediated isothermal amplification (LAMP) reaction with a primer set designed from the rDNA internal transcribed spacer 2 (ITS2) sequence of P. spinosum. The LAMP assay can efficiently amplify the target gene within 60 min at 63 °C. In specificity tests using 31 Pythium spp., 12 Phytophthora spp., 6 Phytopythium spp., and 9 other fungi strains, no cross-reactions were observed in the LAMP assay. The detection limit was 100 pg·μL−1 of genomic DNA per reaction. In cases of suspected disease, P. spinosum could be detected directly, using the LAMP assay, from soybean tissues and soil collected from fields in soybean production areas in the Huang-Huai region. This study provides a rapid method for diagnosing soybean root-rot and damping-off diseases caused by P. spinosum.


Pythium spinosum ITS2 LAMP assay Soybean root rot 



We thank Xiaoli Wang and Yue Yang for maintaining the strains used in this study. This work was supported by the China Agriculture Research System (CARS-004-PS14), the Fundamental Research Funds for the Central Universities (KJQN201738), the National Natural Science Foundation of China (31601618) and the Special Fund for Agro-scientific Research in the Public Interest of China (201303018).

Author contributions

Conceived and designed the experiments: XBZ and YCW.

Performed the experiments: HF, JJC, ZY, and ZL.

Analyzed the data: HF, JJC, WWY, and XBZ.

Contributed reagents/materials/analysis tools: HF, JJC, ZY, ZL, WWY, and YCW.

Wrote the paper: HF, JJC, XBZ, and WWY.

Supplementary material

13313_2019_659_Fig6_ESM.png (2 mb)
Figure S1

Asexual and sexual reproductive structures of P. spinosum. (A) Mycelia. (B, C) Hyphal swellings. (D) A terminal oogonium. (E) An intercalary oogonium. (F) Catenulate oogonia. (PNG 2048 kb)

13313_2019_659_MOESM1_ESM.tif (8.5 mb)
High resolution image (TIF 8713 kb)


  1. Benard D, Punja ZK (1995) Role of Pythium species in cavity spot development on carrots in British Columbia. Can J Plant Pathol 17:31–45CrossRefGoogle Scholar
  2. Benson D (1997) Phytophthora diseases worldwide. 1st ed. Crop Protection 16(4): 399Google Scholar
  3. Botha WJ (1993) Zoospore production in Pythium spinosum. Mycol Res 97:1495–1498CrossRefGoogle Scholar
  4. Botton SA, Pereira DI, Costa MM, Azevedo MI, Argenta JS, Jesus FP, Alves SH, Santurio JM (2011) Identification of Pythium insidiosum by nested PCR in cutaneous lesions of Brazilian horses and rabbits. Curr Microbiol 62(4):1225–1229CrossRefGoogle Scholar
  5. Cao YY, Li YQ, Li JJ, Wang LF, Cheng ZQ, Wang H et al (2016) Rapid and quantitative detection of Pythium inflatum by real-time fluorescence loop-mediated isothermal amplification assay. Eur J Plant Pathol 144:83–95CrossRefGoogle Scholar
  6. Chen JJ, LÜ L, Ye WW, Wang YC, Zheng XB (2017) Pythium cedri sp. nov. (Pythiaceae, Pythiales) from southern China based on morphological and molecular characters. Phytotaxa 309(2):135CrossRefGoogle Scholar
  7. Duan YB, Zhang XK, Ge CY, Wang Y, Cao JH, Jia XJ, Wang JX, Zhou MG (2014) Development and application of loop-mediated isothermal amplification for detection of the F167Y mutation of carbendazim-resistant isolates in Fusarium graminearum. Sci Report 4:7094CrossRefGoogle Scholar
  8. Feng W, Ishiguro Y, Hotta K, Watanabe H, Suga H, Kageyama K (2015) Simple detection of Pythium irregulare using loop-mediated isothermal amplification assay. FEMS Microbiol Lett 362(21)Google Scholar
  9. Feng W, Hieno A, Kusunoki M, Suga H, Kageyama K (2019) LAMP detection of four plant-pathogenic oomycetes and its application in lettuce fields. Plant Dis 103(2):298–307CrossRefGoogle Scholar
  10. Fukuta S, Takahashi R, Kuroyanagi S, Miyake N, Nagai H, Suzuki H (2013) Detection of Pythium aphanidermatum in tomato using loop-mediated isothermal amplification (lamp) with species-specific primers. Eur J Plant Pathol 136:689–701CrossRefGoogle Scholar
  11. Fukuta S, Takahashi R, Kuroyanagi S, Ishiguro Y, Miyake N, Nagai H, Suzuki H et al (2014) Development of loop-mediated isothermal amplification assay for the detection of Pythium myriotylum. Lett Appl Microbiol 59(1):49–57CrossRefGoogle Scholar
  12. Ghosh R, Nagavardhini A, Sengupta A, Sharma M (2015) Development of Loop-Mediated Isothermal Amplification (LAMP) assay for rapid detection of Fusarium oxysporum f. sp. ciceris - wilt pathogen of chickpea BMC Research Notes (2015) 8:40Google Scholar
  13. Goto M, Honda E, Ogura A, Nomoto A, Hanaki K (2009) Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. Biotechniques 46(3):167–172CrossRefGoogle Scholar
  14. Hall TA (1999) A user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  15. Hendricks KE, Roberts PD (2015) First report of Pythium spinosum as a pathogen of watermelon and in association with a dieback of watermelon in Southwest Florida. Plant Health ProgressGoogle Scholar
  16. Hendrix FF, Campbell WA (1973) Pythiums as plant pathogens. Annu Rev Phytopathol 11:77–98CrossRefGoogle Scholar
  17. Ivimey CW, Collins WB (1937) A pythium wilt of primula caused by Pythium spinosum, sawada. Trans Br Mycol Soc 21:29–33CrossRefGoogle Scholar
  18. Le Floch G, Tambong J, Vallance J, Tirilly Y, Levesque A, Rey P (2007) Rhizosphere persistence of three Pythium oligandrum strains in tomato soilless culture assessed by DNA macroarray and real-time PCR. FEMS Microbiol Ecol 61(2):317–326CrossRefGoogle Scholar
  19. Lu CC, Zhang HF, Wang YC, Zheng XB (2015) Rapid diagnosis of Fusarium root rot in soybean caused by Fusarium equiseti or Fusarium graminearum using loop-mediated isothermal amplification (LAMP) assays. Australas Plant Pathol 44:437–443CrossRefGoogle Scholar
  20. Martin FN, Loper JE (2010) Soilborne plant diseases caused by Pythium spp.: ecology, epidemiology, and prospects for biological control. Crit Rev Plant Sci 18(2):111–181CrossRefGoogle Scholar
  21. Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325CrossRefGoogle Scholar
  22. Nawaz K, Shahid AA, Subhani MN, Anwar W (2016) First report of Pythium spinosum causing root rot of chili (Capsicum annuum) in Pakistan. Plant Dis 100:526CrossRefGoogle Scholar
  23. 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(12):e63–e663Google Scholar
  24. Nzungize J, Gepts P, Buruchara R, Buah S, Ragama P, Busogoro JP, Baudoin JP (2011) Pathogenic and molecular characterization of Pythium species inducing root rot symptoms of common bean in Rwanda. Afr J Microbiol Res 5:1169–1181CrossRefGoogle Scholar
  25. Park MJ, Back CG, Han KS, Park JH (2016) Occurrence of damping-off caused by Pythium spinosum on Cucumis melo in Korea. Res Plant Dis 22(3):190–193CrossRefGoogle Scholar
  26. Pettitt TR, Wakeham AJ, Wainwright MF, White JG (2002) Comparison of serological, culture, and bait methods for detection of Pythium and Phytophthora zoospores in water. Plant Pathol 51:720–727CrossRefGoogle Scholar
  27. Plaats-Niterink AJVD (1981) Monograph of the genus Pythium. Stud Mycol 21Google Scholar
  28. Robideau GP, De Cock AW, Coffey MD, Voglmayr H, Brouwer H, Bala K et al (2011) DNA barcoding of oomycetes with cytochrome coxidase subunit I and internal transcribed spacer. Mol Ecol Resour 11(6):1002–1011CrossRefGoogle Scholar
  29. Shen DY, Li QL, Yu J, Zhao YY, Zhu Y, Xu H, Dou DL (2017) Development of a loop-mediated isothermal amplification method for the rapid detection of Pythium ultimum. Australas Plant Pathol 46(6):571–576CrossRefGoogle Scholar
  30. Takeuchi J, Horie H, Nishimura S (2002) First report of Pythium rot of Sansevieria trifasciata caused by Pythium spinosum in Japan. Annu Rep Kanto-Tosan Soc Plant Prot 49:89–91Google Scholar
  31. Toda T, Iwasa A, Fuji S, Furuya H (2015) Widespread occurrence of Pythium arrhenomanes pathogenic to Rice seedlings around Japanese Rice fields. Plant Dis 99(12):1823–1831CrossRefGoogle Scholar
  32. Villa NO, Kageyama K, Asano T, Suga H (2006) Phylogenetic relationships of Pythium and Phytophthora species based on ITS rDNA, cytochrome oxidase II and β-tubulin gene sequences. Mycologia 98(3):410–422PubMedGoogle Scholar
  33. Wang PH, Chung CY, Lin YS, Yeh Y (2003) Use of polymerase chain reaction to detect the soft rot pathogen, Pythium myriotylum, in infected ginger rhizomes. Lett Appl Microbiol 36:116–120CrossRefGoogle Scholar
  34. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols:315–322Google Scholar
  35. Wrather JA, Koenning SR (2006) Estimates of disease effects on soybean yields in the United States 2003 to 2005. J Nematol 38:173–180PubMedPubMedCentralGoogle Scholar
  36. Zeng DD, Ye WW, Xu M, Lu CC, Tian Q, Zheng XB (2017) Rapid diagnosis of soya bean root rot caused by Fusarium culmorum using a loop-mediated isothermal amplification assay. J Phytopathol 165(4):249–256CrossRefGoogle Scholar
  37. Zhang BD, Yang XB (2000) Pathogenicity of Pythium populations from corn–soybean rotation fields. Plant Dis 84:94–99CrossRefGoogle Scholar
  38. Zheng XB (1997) Methods in Phytophthora. China, Chinese Agriculture Press, BeijingGoogle Scholar

Copyright information

© Australasian Plant Pathology Society Inc. 2019

Authors and Affiliations

  1. 1.Department of Plant Pathology, College of Plant ProtectionNanjing Agricultural UniversityNanjingChina
  2. 2.Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of EducationNanjingChina

Personalised recommendations