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Ypt1 gene-based recombinase polymerase amplification assay for Phytophthora capsici and P. tropicalis detection in black pepper

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Abstract

Foot rot caused by Phytophthora is one of the major diseases of black pepper (Piper nigrum L.). Accurate and timely diagnosis of the disease is crucial for its successful management. Although PCR and qPCR assays are used for detection, the cost and time required to perform these assays are high. Recombinase polymerase amplification (RPA) assay has the advantage of minimal assay time and it is performed under isothermal conditions. Hence, RPA assay was developed for the detection of P. capsici and P. tropicalis and compared with newly developed end-point PCR test. Out of three sets of primers analyzed, a primer set based on the Ypt1 gene successfully amplified a 230/231 bp product. Optimum amplification of RPA products were observed when the assay was performed at 37 °C with 14 mM magnesium acetate for 40 min. Sensitivity analysis using serial dilutions indicated that RPA is 10 times more sensitive than end-point PCR. During specificity analysis, non-specific bands were observed with other Phytophthora species, and hence the assay was further refined with betaine wherein addition of 1.0 M betaine avoided amplification of non-specific bands. The optimized RPA assay could detect Phytophthora from infected black pepper leaf, stem and root using both purified DNA and crude extracts. The end-point PCR test successfully differentiated the two species of Phytophthora in a validation test. These results indicate the robustness of the developed end-point PCR and RPA assays and its potential application in detection and differentiation of P. capsici and P. tropicalis infecting black pepper.

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References

  • Ahmed, F. A., Larrea-Sarmiento, A., Alvarez, A. M., & Arif, M. (2018). Genome informed diagnostics for specific and rapid detection of Pectobacterium species using recombinase polymerase amplification coupled with a lateral flow device. Scientific Reports, 8, 15972.

    Article  Google Scholar 

  • Anandaraj, M., & Sarma, Y. R. (1990). A simple baiting technique to detect and isolate Phytophthora capsici (‘P. palmivora’ MF4) from soil. Mycological Research, 94, 1003–1004.

    Article  Google Scholar 

  • Babu, B., Ochoa-Coronac, F. M., & Pareta, M. L. (2018). Recombinase polymerase amplification applied to plant virus detection and potential implications. Analytical Biochemistry, 546, 72–77.

    Article  CAS  Google Scholar 

  • Blair, J. E., Coffey, M. D., & Martin, F. N. (2012). Species tree estimation for the late blight pathogen, Phytophthora infestans and close relatives. PLoS One, 7, e37003.

    Article  CAS  Google Scholar 

  • Craw, P., & Balachandran, W. (2012). Isothermal nucleic acid amplification technologies for point-of-care diagnostics: A critical review. Lab on a Chip, 12, 2469–2486.

    Article  CAS  Google Scholar 

  • Dai, T., Yang, X., Hu, T., Jiao, B., Xu, Y., Zheng, X., & Shen, D. (2019). Comparative evaluation of a novel recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) assay, LAMP, conventional PCR and leaf-disc baiting methods for detection of Phytophthora sojae. Frontiers in Microbiology, 10, 1884.

    Article  Google Scholar 

  • Erwin, D. C., & Ribeiro, O. K. (1996). Phytophthora diseases worldwide. St. Paul: American Phytopathological Society Press.

    Google Scholar 

  • Henke, W., Herdel, K., Jung, K., Schnorr, D., & Loening, S. A. (1997). Betaine improves the PCR amplification of GC-rich DNA sequences. Nucleic Acids Research, 25, 3957–3958.

    Article  CAS  Google Scholar 

  • Kapoor, R., Srivastava, N., Kumar, S., Saritha, R. K., Sharma, S. K., Jain, R. K., & Baranwal, V. K. (2017). Development of a recombinase polymerase amplification assay for the diagnosis of banana bunchy top virus in different banana cultivars. Archives of Virology, 162, 2791–2796.

    Article  CAS  Google Scholar 

  • Kox, L. F. F., Brouwershaven, I. V., Vossenberg, B. V. D., Beld, H. V. D., Bonants, P. J. M., & Gruyter, J. D. (2007). Diagnostic values and utility of immunological, morphological, and molecular methods for in planta detection of Phytophthora ramorum. Phytopathology, 97, 1119–1129.

    Article  CAS  Google Scholar 

  • Li, J., Macdonald, J., & Stetten, F. (2019). Review: A comprehensive summary of a decade development of the recombinase polymerase amplification. Analyst, 144, 31–67.

    Article  CAS  Google Scholar 

  • Londoño, M. A., Harmon, C. L., & Polston, J. E. (2016). Evaluation of recombinase polymerase amplification for detection of begomoviruses by plant diagnostic clinics. Virology Journal, 13, 48.

    Article  Google Scholar 

  • Luo, G.-C., Yi, T.-T., Jiang, B., Guo, X.-l., & Zhang, G.-Y. (2019). Betaine-assisted recombinase polymerase assay with enhanced specificity. Analytical Biochemistry, 575, 36–39.

    Article  CAS  Google Scholar 

  • Martin, F. N., Abad, Z. G., Balci, Y., & Ivors, K. (2012). Identification and detection of Phytophthora: Reviewing our progress, identifying our needs. Plant Disease, 96, 1080–1103.

    Article  Google Scholar 

  • Miles, T. D., Martin, F. N., & Coffey, M. D. (2015). Development of rapid isothermal amplification assays for detection of Phytophthora spp. in plant tissue. Phytopathology, 105, 265–278.

    Article  CAS  Google Scholar 

  • Mok, E., Wee, E., Wang, Y., & Trau, M. (2016). Comprehensive evaluation of molecular enhancers of the isothermal exponential amplification reaction. Scientific Reports, 6, 37837.

    Article  CAS  Google Scholar 

  • Munawar, M., Toljamo, A., Martin, F., & Kokko, H. (2019). Recombinase polymerase amplification assay for fast, sensitive and on-site detection of Phytophthora cactorum without DNA extraction. European Journal of Horticultural Science, 84, 14–19.

    Article  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 Research, 28, E63.

    Article  CAS  Google Scholar 

  • Pandian, R. T. P., Bhat, A. I., Biju, C. N., & Sasi, S. (2018). Development of diagnostic assays for rapid and sensitive detection of Phytophthora infecting major spices and plantation crops. Journal of Spices and Aromatic Crops, 27, 119–130.

    Google Scholar 

  • Rojas, J. A., Miles, T. D., Coffey, M. D., Martin, F. N., & Chilvers, M. I. (2017). Development and application of qPCR and RPA genus- and species-specific detection of Phytophthora sojae and P. sansomeana root rot pathogens of soybean. Plant Disease, 101, 1171.

    Article  CAS  Google Scholar 

  • Sarma, Y. R., Anandaraj, M., & Venugopal, M. N. (1997). Phytophthora foot rot of black pepper. In: Agnihotri, V.P., Sarbhoy, A.K., Singh, D.V. (Eds.), Management of Threatening Diseases of National Importance. Malhotra Publishing House, New Delhi, pp. 237–248.

  • Schrader, C., Schielke, A., Ellerbroek, L., & Johne, R. (2012). PCR inhibitors–occurrence, properties and removal. Journal of Applied Microbiology, 113, 1014–1026.

    Article  CAS  Google Scholar 

  • Sheji, C., Renu, S. G., Balaji, S., & Anandaraj, M. (2009). Ribosomal DNA analysis of three Phytophthora species occurring in India. Indian Phytopathology, 62, 155–162.

    CAS  Google Scholar 

  • Si Ammour, M., Bilodeau, G. J., Tremblay, D. M., Heyden, H. V., Yaseen, T., Varvaro, L., & Carisse, O. (2017). Development of real-time isothermal amplification assays for on-site detection of Phytophthora infestans in potato leaves. Plant Disease, 101, 1269–1277.

    Article  Google Scholar 

  • Silvar, C., Duncan, J. M., Cooke, D. E. L., Williams, N. A., Díaz, J., & Merino, F. (2005). Development of specific PCR primers for identification and detection of Phytophthora capsici Leon. European Journal of Plant Pathology, 112, 43–52.

    Article  CAS  Google Scholar 

  • Tooley, P. W., Bunyard, B. A., Carras, M. M., & Hatziloukas, E. (1997). Development of PCR primers from internal transcribed spacer region 2 for detection of Phytophthora species infecting potatoes. Applied and Environmental Microbiology, 63, 1467–1475.

    Article  CAS  Google Scholar 

  • Yu, J., Shen, D., Dai, T., Lu, X., Xu, H., & Dou, D. (2019). Rapid and equipment free detection of Phytophthora capsici using lateral flow strip-based recombinase polymerase amplification assay. Letters in Applied Microbiology, 69, 64–70.

    CAS  PubMed  Google Scholar 

  • Zhang, Z. G., Li, Y. Q., Fan, H., Wang, Y. C., & Zheng, X. B. (2006). Molecular detection of Phytophthora capsici in infected plant tissues, soil and water. Plant Pathology, 55, 770–775.

    Article  CAS  Google Scholar 

  • Zhao, C., Sun, F., Li, X., Lan, Y., Du, L., Zhou, T., & Zhou, Y. (2019). Reverse transcription-recombinase polymerase amplification combined with lateral flow strip for detection of rice black-streaked dwarf virus in plants. Journal of Virological Methods, 263, 96–100.

    Article  CAS  Google Scholar 

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Acknowledgments

The authors are thankful to The Director, ICAR-Indian Institute of Spices Research, Kozhikode, Kerala for providing facilities and Indian Council of Agricultural Research, New Delhi for funding.

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Authors and Affiliations

  1. Division of Crop Protection, ICAR-Indian Institute of Spices Research, Kozhikode, Kerala, 673 012, India

    A. Jeevalatha, C. N. Biju & R. Suseela Bhai

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  1. A. Jeevalatha
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  2. C. N. Biju
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  3. R. Suseela Bhai
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Correspondence to A. Jeevalatha.

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Jeevalatha, A., Biju, C.N. & Bhai, R.S. Ypt1 gene-based recombinase polymerase amplification assay for Phytophthora capsici and P. tropicalis detection in black pepper. Eur J Plant Pathol 159, 863–875 (2021). https://doi.org/10.1007/s10658-021-02211-0

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