Skip to main content
SpringerLink
Log in

Rapid diagnosis of Fusarium root rot in soybean caused by Fusarium equiseti or Fusarium graminearum using loop-mediated isothermal amplification (LAMP) assays

  • Original Paper
  • Published:
Australasian Plant Pathology Aims and scope Submit manuscript

Abstract

Rapid diagnostic assays for Fusarium root rot in soybean, caused by Fusarium equiseti or Fusarium graminearum were developed using the target gene CYP51C. Both assays amplified the target gene in 60 min at 62 °C, after which they were assessed for specificity and sensitivity. Specificity was evaluated against other Fusarium spp., other fungal species, and oomycetes. A positive yellow-green color (by the naked eye) or intense green fluorescence (under ultraviolet light) was observed only in the presence of F. equiseti or F. graminearum after adding SYBR Green I, whereas other strains showed either no color change or weak fluorescence. The detection limit of the CYP51C-Fe-LAMP assay was 10 pg. μL−1 genomic DNA per reaction, and as few as four conidia per gram of soil could be detected; and the detection limit of the CYP51C-Fg-LAMP assay was 100 pg. μL−1 genomic DNA per reaction, and 40 conidia per gram of soil could be identified. The assays also detected F. equiseti or F. graminearum from inoculated soybean tissues and diseased plants in the field. These results suggest that CYP51C-Fe-LAMP assay and CYP51C-Fg-LAMP assay are effective in rapidly diagnosing soybean root rot caused by F. equiseti and F. graminearum.

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

Similar content being viewed by others

References

  • Abd-Elsalam KA, Aly IN, Abdel-Satar MA, Khalil MS, Verreet JA (2004) PCR identification of Fusarium genus based on nuclear ribosomal-DNA sequence data. Afr J Biotechnol 2:82–85

    Google Scholar 

  • Almasi MA, Moradi A, Ojaghkandi MA, Aghaei S (2013) Development and application of loop-mediated isothermal amplification assay for rapid detection of Fusarium oxysporum f. sp. Lycopersici. J Plant Pathol Microbiol 4:2

    Article  Google Scholar 

  • Caipang CMA, Haraguchi I, Ohira T, Hirono I, Aoki T (2004) Rapid detection of a fish iridovirus using loop-mediated isothermal amplification (LAMP). J Virol Methods 121:155–161

    Article  CAS  PubMed  Google Scholar 

  • Dai TT, Lu CC, Lu J, Dong S, Ye W, Wang Y, Zheng X (2012) Development of a loop‐mediated isothermal amplification assay for detection of Phytophthora sojae. FEMS Microbiol Lett 334:27–34

    Article  CAS  PubMed  Google Scholar 

  • Deng J (2007) Structural, functional and evolutionary analyses of the rice blast fungal genome. North Carolina State University, North Carolina

    Google Scholar 

  • Diaz-Guerra T, Mellado E, Cuenca-Estrella M, Rodriguez-Tudela J (2003) A point mutation in the 14α-sterol demethylase gene cyp51A contributes to itraconazole resistance in Aspergillus fumigatus. Antimicrob Agents Chemother 47:1120–1124

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • El-Kazzaz M, El-Fadly G, Hassan M, El-Kot G (2008) Identification of some Fusarium spp. Using molecular biology techniques. Egypt J Phytopathol 36:57–69

    Google Scholar 

  • Fang Z (1998) Research methods of plant disease. China Agricultural Publishing House, Beijing

    Google Scholar 

  • Fernández-Ortuño D, Loza-Reyes E, Atkins SL, Fraaije BA (2010) The CYP51C gene, a reliable marker to resolve interspecific phylogenetic relationships within the Fusarium species complex and a novel target for species-specific PCR. Int J Food Microbiol 144:301–309

    Article  PubMed  Google Scholar 

  • Hartman GL, Sinclair JB, Rupe JC (1999) Compendium of soybean diseases. American Phytopathological Society (APS Press), St. Paul

    Google Scholar 

  • Iwamoto T, Sonobe T, Hayashi K (2003) Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples. J Clin Microbiol 41:2616–2622

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Jurado M, Vázquez C, Patiño B, Teresa González-Jaén M (2005) PCR detection assays for the trichothecene-producing species Fusarium graminearum, Fusarium culmorum, Fusarium poae, Fusarium equiseti and Fusarium sporotrichioides. Syst Appl Microbiol 28:562–568

    Article  CAS  PubMed  Google Scholar 

  • Karlsen F, Steen HB, Nesland JM (1995) SYBR green I DNA staining increases the detection sensitivity of viruses by polymerase chain reaction. J Virol Methods 55:153–156

    Article  CAS  PubMed  Google Scholar 

  • Lévesque CA, Rahe JE, Eaves DM (1987) Effects of glyphosate on Fusarium spp.: its influence on root colonization of weeds, propagule density in the soil, and crop emergence. Can J Microbiol 33:354–360

    Article  Google Scholar 

  • 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. Eur J Plant Pathol 135:903–911

    Article  CAS  Google Scholar 

  • Lu C, Dai T, Zhang H, Wang Y, Zheng X (2015) Development of a loop-mediated isothermal amplification assay to detect Fusarium oxysporum. J Phytopathol 163(1):63–66

    Article  CAS  Google Scholar 

  • Maciá - Vicente JG, Jansson HB, Talbot NJ, Lopez - Llorca LV (2009) Real-time PCR quantification and live-cell imaging of endophytic colonization of barley (Hordeum vulgare) roots by Fusarium equiseti and Pochonia chlamydosporia. New Phytol 182:213–228

    Article  PubMed  Google Scholar 

  • Marasas WFO, Nelson PE, Toussoun TA (1984) Toxigenic Fusarium species. Identity and mycotoxicology. The Pennsylvania State University Press, University Park, PA

    Google Scholar 

  • Martinelli JA, Bocchese CA, Xie W, O’Donnell K, Kistler HC (2004) Soybean pod blight and root rot caused by lineages of the Fusarium graminearum and the production of mycotoxins. Fitopatol Bras 29:492–498

    Article  Google Scholar 

  • Martinez-Culebras P, Querol A, Suarez-Fernandez M, Garcia-Lopez M, Barrio E (2003) Phylogenetic relationships among Colletotrichum pathogens of strawberry and design of PCR primers for their identification. J Phytopathol 151:135–143

    Article  CAS  Google Scholar 

  • McGee DC (1992) Soybean diseases: a reference source for seed technologists. American Phytopathological Society, St. Paul

    Google Scholar 

  • Mori Y, Hirano T, Notomi T (2006) Sequence specific visual detection of LAMP reactions by addition of cationic polymers. BMC Biotechnol 6:3

    Article  PubMed Central  PubMed  Google Scholar 

  • Nagamine K, Hase T, Notomi T (2002) Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes 16:223–229

    Article  CAS  PubMed  Google Scholar 

  • Naito S, Mohamad D, Nasution A & Purwanti H (1993) Soil-borne diseases and ecology of pathogens on soybean roots in Indonesia. Jpn Agric Res Q 26: 247–247.

  • Nelson BD, Hansen JM, Windels CE, Helms TC (1997) Reaction of soybean cultivars to isolates of Fusarium solani from the Red River Valley. Plant Dis 81:664–668

    Article  Google Scholar 

  • Nelson PE, Toussoun TA, Marasas W (1983) Fusarium species: an illustrated manual for identification. Pennsylvania State University Press, Universtity Park, Penn

    Google Scholar 

  • Nelson PE, Desjardins AE, Plattner RD (1993) Fumonisins, mycotoxins produced by Fusarium species: biology, chemistry, and significance. Annu Rev Phytopathol 31:233–252

    Article  CAS  PubMed  Google Scholar 

  • Nelson PE, Dignani MC, Anaissie EJ (1994) Taxonomy, biology, and clinical aspects of Fusarium species. Clin Microbiol Rev 7:479

    CAS  PubMed Central  PubMed  Google Scholar 

  • Niessen L, Vogel RF (2010) Detection of Fusarium graminearum DNA using a loop-mediated isothermal amplification (LAMP) assay. Int J Food Microbiol 140:183–191

    Article  CAS  PubMed  Google Scholar 

  • Njiru ZK, Mikosza ASJ, Armstrong T, Enyaru JC, Ndung’u JM, Thompson ARC (2008) Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense. PLoS Negl Trop Dis 2:e147

    Article  PubMed Central  PubMed  Google Scholar 

  • 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:e63–e63

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Pan W, Wang JY, Shen HY, Zhao MQ, Ju CM, Dong XY, Yi L, Chen JD (2011) Development and application of the novel visual loop-mediated isothermal amplification of Omp25 sequence for rapid detection of Brucella sp. J Anim Vet Adv 10:2120–2126

    Article  CAS  Google Scholar 

  • Parida M, Horioke K, Ishida H, Dash PK, Saxena P, Jana AM, Islam MA, Inoue S, Hosaka N, Morita K (2005) Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay. J Clin Microbiol 43:2895–2903

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Peng J, Zhan Y, Zeng F, Long H, Pei Y, Guo J (2013) Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. niveum in soil. FEMS Microbiol Lett 349:127–134

    Article  CAS  PubMed  Google Scholar 

  • Soliman H, El-Matbouli M (2006) Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of viral hemorrhagic septicaemia virus (VHS). Vet Microbiol 114:205–213

    Article  CAS  PubMed  Google Scholar 

  • Tang J, Wang W, Wang Y (2005) Molecular detection of Colletotrichum orbiculare. Zhongguo Nongye Kexue 39:2028–2035

    Google Scholar 

  • Tomita N, Mori Y, Kanda H, Notomi T (2008) Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc 3:877–882

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wrather J, Koenning S (2009) Effects of diseases on soybean yields in the United States 1996 to 2007. Plant Health Prog. doi:10.1094/PHP-2009-0401-01-RS

    Google Scholar 

  • Wrather J, Stienstra W, Koenning S (2001) Soybean disease loss estimates for the United States from 1996 to 1998. Can J Plant Pathol 23:122–131

    Article  Google Scholar 

  • Xing L, Westphal A (2006) Interaction of Fusarium solani f. sp. glycines and Heterodera glycines in sudden death syndrome of soybean. Phytopathology 96:763–770

    Article  PubMed  Google Scholar 

  • Yang X, Lundeen P (1997) Occurrence and distribution of soybean sudden death syndrome in Iowa. Plant Dis 81:719–722

    Article  Google Scholar 

  • YongYang G, Nan W, GuanPeng G, Wei W (2010) Triplex PCR detection of Cladosporium cucumerinum, Fusarium oxysporum f. sp. niveum and Mycosphaerella melonis in infected plant tissues. Acta Phytopathol Sin 40:343–350

    Google Scholar 

  • Zhang Z, Zhang J, Wang Y, Zheng X (2005) Molecular detection of Fusarium oxysporum f. sp. niveum and Mycosphaerella melonis in infected plant tissues and soil. FEMS Microbiol Lett 249:39–47

    Article  CAS  PubMed  Google Scholar 

  • Zheng X (1995) Methods in Phytophthora. Chinese Agriculture Press, Beijing

    Google Scholar 

Download references

Acknowledgments

This research was supported by the National High-Tech R&D Program (863 Program) (2012AA101501), China Agriculture Research System (CARS-004-PS14), Chinese National Science Foundation Committee (project 31225022), public sector research funding (201303018), and Genetically Modified Organisms Breeding Major Projects of China (grant no. 2014ZX08011-003).

Author information

Authors and Affiliations

  1. Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, 210095, China

    C. Lu, H. Zhang, Y. Wang & X. Zheng

Authors
  1. C. Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. H. Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Y. Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. X. Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to X. Zheng.

Electronic supplementary material

Table S1

GenBank accession numbers of the Fusarium spacies used for alignment (DOC 39 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lu, C., Zhang, H., Wang, Y. et al. Rapid diagnosis of Fusarium root rot in soybean caused by Fusarium equiseti or Fusarium graminearum using loop-mediated isothermal amplification (LAMP) assays. Australasian Plant Pathol. 44, 437–443 (2015). https://doi.org/10.1007/s13313-015-0361-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13313-015-0361-8

Keywords

Search

Navigation