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A novel LAMP assay using hot water in vacuum insulated bottle for rapid detection of the soybean red crown rot pathogen Calonectria ilicicola

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Abstract

Red crown rot is an important disease of soybean crops worldwide. It is caused by the fungal pathogen Calonectria ilicicola. Infection by C. ilicicola results in a significant reduction in soybean yield and quality. Therefore, accurate pathogen detection in the early stages of the disease is paramount to implementing disease management promptly and minimizing losses. In this study, we developed a sensitive and specific loop-mediated isothermal amplification (LAMP) assay (TUB-Ci-LAMP) targeting the β-tubulin gene of C. ilicicola. Furthermore, an in-field TUB-Ci-LAMP assay was developed using Buffer C to perform on-site crude DNA extraction and a vacuum insulated bottle as a temperature-stable, portable container for isothermal amplification under the field environment. This assay only had positive reaction to C. ilicicola, but not to any of 22 other tested fungal or oomycete species. Under both laboratory and field conditions, genomic DNA of C. ilicicola at 0.01 ng per µL or higher was consistently detected using this assay. Additionally, the in-field assay detected C. ilicicola in artificially inoculated and naturally infected soybean plants. These findings indicate that TUB-Ci-LAMP is useful in aiding researchers, diagnosticians, and first responders on rapid and accurate detection of soybean red crown rot under both laboratory and field environments.

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References

  • Bell DK, Sobers EK (1966) A peg, pod, and root necrosis of peanuts caused by a species of Calonectria. Phytopathology 56:1361–1364

    Google Scholar 

  • Crous PW, Wingfield MJ, Alfenas AC (1993) Cylindrocladium parasiticum sp. nov., a new name for C. crotalariae. Mycol Res 97:889–896

    Article  Google Scholar 

  • Crous PW, Groenewald JZ, Risede J, Hyweljones N (2004) Calonectria species and their cylindrocladium anamorphs: species with sphaeropedunculate vesicles. Stud Mycol 50:415–430

    Google Scholar 

  • Dai TT, Lu CC, Lu J, Dong SM, Ye WW, Wang YC et al (2012) Development of a loop-mediated isothermal amplification assay for detection of Phytophthora sojae. FEMS Microbiol Lett 334(1):27–34

    Article  CAS  Google Scholar 

  • Dann EK, Cooke AW, Forsberg LI, Pegg KG, Tan YP, Shivas RG (2012) Pathogenicity studies in avocado with three nectriaceous fungi, Calonectria ilicicola, Gliocladiopsis sp. and Ilyonectria liriodendri. Plant Pathol 61:896–902

    Article  Google Scholar 

  • Fei NY, Qi YB, Meng TT, Fu JF, Yan XR (2018) First report of root rot caused by Calonectria ilicicola on blueberry in Yunnan Province. China Plant Disease 102:1036

    Article  Google Scholar 

  • Gai J, Lin M (1992) A Report on Black Root Rot of Soybeans in Jiangsu, CHINA. Soybean Science 81(1):183–187

  • Gao X, Chen X, Pan R, Yan X, Liao H (2012) Preliminary investigation of diseases on new extensive soybean varieties in Guangdong Province. Plant Prot 38(2):147–151

    Google Scholar 

  • Gao X, Wu M, Xu R, Wang X, Pan R, Kim HJ, Liao H (2014) Root interactions in a maize/soybean intercropping system control soybean soil-borne disease, red crown rot. PLoS One 9(5):e95031

  • Guan M, Pan R, Gao X, Xu D, Deng Q, Deng M (2010) First Report of Red Crown Rot Caused by Cylindrocladium parasiticum on Soybean in Guangdong. Southern China Plant Disease 94(4):485–485

    Article  CAS  Google Scholar 

  • Hartman GL, Rupe JC, Sikora EJ, Domier LL, Davis JA, Steffey KL (2015) Compendium of Soybean Diseases and Pests. American Phytopathological Society, Saint Paul

    Google Scholar 

  • Huang S, D Jaephil, Madhumita M, Fan A, Lei Z, Lisa J et al (2013) Low cost extraction and isothermal amplification of dna for infectious diarrhea diagnosis. Plos One 8(3):e60059

  • Jayanath NY, Nguyen LT, Vu TT, Tran LD (2018) Development of a portable electrochemical loop mediated isothermal amplification (LAMP) device for detection of hepatitis B virus. RSC Adv 8:34954–34959

    Article  CAS  Google Scholar 

  • Jiang S, Li YR (2020) Principle and application of isothermal amplification technology. Chinese Journal of Laboratory Medicine 43(05):591–596

    Google Scholar 

  • Kuruppu PU, Schneider RW, Russin JS (2004) Effects of soil temperature on microsclerotia of Calonectria ilicicola and soybean root colonization by this fungus. Plant Dis 88(6):620–624

    Article  CAS  Google Scholar 

  • Labarre P, Hawkins KR, Gerlach J, Wilmoth J, Beddoe A, Singleton J et al (2011) A simple, inexpensive device for nucleic acid amplification without electricity—toward instrument-free molecular diagnostics in low-resource settings. PLoS One 6(5):e19738

  • Liu HH, Wang J, Wu PH, Lu M, Chang HX (2021) The whole genome sequence resource of Calonectria ilicicola, the casual pathogen of soybean red crown rot. Molecular Plantmicrobe Interactions 34(7):848–851

    Article  Google Scholar 

  • Lu CC, Dai TT, Zhang HF, Wang YC, Zheng XB (2014) Development of a Loop-Mediated Isothermal Amplification Assay to Detect Fusarium oxysporum. J Phytopathol 163(1):63–66

    Article  Google Scholar 

  • Lu CC, Song B, Zhang HF, Wang YC, Zheng XB (2015) Rapid diagnosis of soybean seedling blight caused by Rhizoctonia solani and soybean charcoal rot caused by Macrophomina phaseolina using lamp assays. Phytopathology 105(12):1612–1617

    Article  CAS  Google Scholar 

  • Ma ZH, Zhang ZD, Wang YX, Yang XB (2004) Cylindrocladium Crotalariae Causing Red Crown Rot of Soybean in China. Plant Pathol 53(4):537–537

    Article  Google Scholar 

  • Malapi-Wight M, Demers JE, Veltri D, Marra RE, Crouch JA (2016) Lamp detection assays for boxwood blight pathogens: a comparative genomics approach. Sci Rep 6:26140

    Article  CAS  Google Scholar 

  • Martzy R, Kolm C, Brunner K, Mach RL, Krska R, Sinkovec H, Sommer R, Farnleitner AH, Reischer GH (2017) A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Enterococcus spp. in water. Water Res 122:62–69

    Article  CAS  Google Scholar 

  • Nagamine K, Hase T, Notomi T (2002) Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes 16(3):223–229

    Article  CAS  Google Scholar 

  • Niessen L, Niessen L (2013) Loop-Mediated Isothermal Amplification-Based Detection of Fusarium graminearum. Methods Mol Biol 968:177–193

    Article  CAS  Google Scholar 

  • Nishi K (1989) Present situation on Calonectria root rot of soybean (in Japanese). Journal of Agricultural Science (japan) 44:70–75

    Google Scholar 

  • Nkouawa A, Sako Y, Li T, Chen X, Nakao M, Yanagida T et al (2012) A loop-mediated isothermal amplification method for a differential identification of taenia tapeworms from human: application to a field survey. Parasitol Int 61(4):723–725

    Article  CAS  Google Scholar 

  • Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N et al (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28(12):E63–E63

    Article  CAS  Google Scholar 

  • Parida M, Sannarangaiah S, Dash PK, Rao PVL, Morita K (2008) Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases. Rev Med Virol 18(6):407–421

    Article  CAS  Google Scholar 

  • Qiao TM, Zhang J, Li SJ, Han S, Zhu TH (2016) Development of nested pcr, multiplex pcr, and loop-mediated isothermal amplification assays for rapid detection of Cylindrocladium scoparium on eucalyptus. The Plant Pathology Journal 32(5):414–422

    Article  CAS  Google Scholar 

  • Rowe RC, Beute MK, Wells JC (1974) 1973 Cylindrocladium black root rot of peanuts in North Carolina – 1972. Plant Disease Reporter 58(4):348–352

    CAS  Google Scholar 

  • Sabalza M, Yasmin R, Barber CA, Castro T, Malamud D, Kim BJ, Zhu H, Montagna RA, Abrams WR (2018) Detection of Zika virus using reverse-transcription LAMP coupled with reverse dot blot analysis in saliva. PLoS One 13:e0192398

  • Sema M, Alemu A, Bayih A, Getie S, Getnet G, Guelig D et al (2015) Evaluation of non-instrumented nucleic acid amplification by loop-mediated isothermal amplification (nina-lamp) for the diagnosis of malaria in northwest ethiopia. Malar J 14(1):44

    Article  Google Scholar 

  • Sung JM (1980) An Investigation of Undescribed Black Root Rot Disease of Soybean Caused by Cylindrocladium(Calonectria) crotalariae in Korea. Korean Journal of Mycology 8(1):53–57

    Google Scholar 

  • Velders AH, Schoen C, Saggiomo V (2018) Loop-mediated isothermal amplification (LAMP) shield for Arduino DNA detection. BMC Res Notes 11(1):93

    Article  Google Scholar 

  • Wang SS, Ye WW, Tian Q, Dong SM, Zheng XB (2017) Rapid detection of colletotrichum gloeosporioides using a loop-mediated isothermal amplification assay. Australas Plant Pathol 46(5):493–498

    Article  Google Scholar 

  • Wilisiani F, Tomiyama A, Katoh H, Hartono S, Neriya Y, Nishigawa H, Natsuaki T (2019) Development of a LAMP assay with a portable device for real-time detection of begomoviruses under field conditions. J Virol Methods 265:71–76

    Article  CAS  Google Scholar 

  • Yuan J, Liu W, Huang LY (2013) Method for preventing nucleic acid contamination and indicating reaction results in nucleic acid constant temperature amplification reaction. CN201210371448.5

  • Zhao W, Wang T, Qi R (2015) Ypt1gene-based detection of phytophthora sojae in a loop-mediated isothermal amplification assay. J Plant Dis Prot 122(2):66–73

    Article  Google Scholar 

  • Zheng XB (1997) Methods in phytophthora. Chinese Agriculture Press, Beijing

    Google Scholar 

  • Zhuo Y (2016) Some problems and relevant solutions on lamp research. Modern Journal of Animal Husbandry and Veterinary Medicine 7:53–57

    Google Scholar 

  • Zhang JH, Chen XF, Sun H (2014) Research on detection of Tobacco ringspot virus by RT-LAMP. Acta Phytopathologica Sinica 44(6):629-633

Download references

Acknowledgements

This research was supported by the National High-Tech Research and Development Program (863 Program) (2012AA101501) of China, the National Department Public Benefit Research Foundation (No. 200903004), Chinese National Science Foundation Committee (project 31225022), public sector research funding (201303018), and Science and technology project of Jiangsu entry-exit inspection and Quarantine Bureau (2016KJ62). We thank two anonymous reviewers for comments that improved this manuscript.

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Authors and Affiliations

  1. Lianyungang Customs (Formerly Lianyungang Entry-Exit Inspection and Quarantine Bureau), Lianyungang, 222000, China

    ChenChen Lu & WanFeng Yang

  2. Co-Innovation Center for the Sustainable Forestry in Southern China, Jiangsu Key Laboratory for Prevention and Control of Invasive Species, College of Forestry, Nanjing Forestry University, Nanjing, 210037, Jiangsu, China

    TingTing Dai & YuanChao Wang

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

    ChenChen Lu, HaiFeng Zhang, DanDan Zeng & XiaoBo Zheng

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  1. ChenChen Lu
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  2. TingTing Dai
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  3. HaiFeng Zhang
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  4. DanDan Zeng
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  5. YuanChao Wang
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Correspondence to XiaoBo Zheng.

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Lu, C., Dai, T., Zhang, H. et al. A novel LAMP assay using hot water in vacuum insulated bottle for rapid detection of the soybean red crown rot pathogen Calonectria ilicicola. Australasian Plant Pathol. 51, 251–259 (2022). https://doi.org/10.1007/s13313-022-00855-y

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