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Rapid genotyping of bacterial leaf blight resistant genes of rice using loop-mediated isothermal amplification assay

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Abstract

The use of resistant (R) genes is the most effective strategy to manage bacterial leaf blight (BLB) disease of rice. Several attempts were made to incorporate R genes into susceptible rice cultivars using marker-assisted backcross breeding (MABB). However, MABB relies exclusively on PCR for foreground selection of R genes, which requires expensive equipment for thermo-cycling and visualization of results; hence, it is limited to sophisticated research facilities. Isothermal nucleic acid amplification techniques such as loop-mediated isothermal amplification (LAMP) assay do not require thermo-cycling during the assay. Therefore, it will be the best alternative to PCR-based genotyping. In this study, we have developed a LAMP assay for the specific and sensitive genotyping of seven BLB resistance (R) genes viz., Xa1, Xa3, Xa4, Xa7, Xa10, Xa11, and Xa21 in rice. Gene-specific primers were designed for the LAMP assay. The LAMP assay was optimized for time, temperature, and template DNA concentration. For effective detection, incubation at 60 °C for 30 min was optimum for all seven R genes. A DNA intercalating dye ethidium bromide and a calorimetric dye hydroxynaphthol blue was used for result visualization. Further, sensitivity assay revealed that the LAMP assay could detect R genes at 100 fg of template DNA compared to 1 ng and 10 pg, respectively, in conventional PCR and q-PCR assays. The LAMP assay developed in this study provides a simple, specific, sensitive, robust, and cost-effective method for foreground selection of R genes in the resistance breeding programs of resource-poor laboratory.

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References

  1. Srinivasan B, Gnanamanickam SS (2005) Identification of a new source of resistance in wild rice, Oryza rufipogon to bacterial blight of rice caused by Indian strains of Xanthomonas oryzae pv. Oryzae. Curr Sci 88:1229–1231

    CAS  Google Scholar 

  2. Pramesh D, Maruti SA et al (2017) Bronopol (2-Bromo-2-Nitropropane-1, 3-diol), a chlorine based chemical compound for the management of bacterial leaf blight of rice. Int J Plant Soil Sci 15:1–7. https://doi.org/10.9734/ijpss/2017/32535

    Article  Google Scholar 

  3. Yasmin S, Hafeez FY, Mirza MS et al (2017) Biocontrol of bacterial leaf blight of rice and profiling of secondary metabolites produced by rhizospheric Pseudomonas aeruginosa BRp3. Front Microbiol 8. https://doi.org/10.3389/fmicb.2017.01895

  4. Fatima P, Mishra A, Khan NA, Singh KN (2018) Towards developing bacterial leaf blight ( BB ) resistance Rice varieties of eastern Uttar Pradesh by using marker assisted selection. 1090–1097

  5. Hari Y, Srinivasarao K, Viraktamath BC et al (2013) Marker-assisted introgression of bacterial blight and blast resistance into IR 58025B, an elite maintainer line of rice. Plant Breed 132:586–594. https://doi.org/10.1111/pbr.12056

    Article  CAS  Google Scholar 

  6. Shanti ML, Shenoy VV, Devi GL et al (2010) Marker-assisted breeding for resistance to bacterial leaf blight in popular cultivar and parental lines of hybrid rice. J Plant Pathol 92:495–501. https://doi.org/10.4454/jpp.v92i2.194

    Article  CAS  Google Scholar 

  7. Tiwari AK (2016) Current trends in plant disease diagnostics and management practices:207–219. https://doi.org/10.1007/978-3-319-27312-9

  8. Balachiranjeevi CH, Bhaskar NS, Abhilash V et al (2015) Marker-assisted introgression of bacterial blight and blast resistance into DRR17B, an elite, fine-grain type maintainer line of rice. Mol Breed 35. https://doi.org/10.1007/s11032-015-0348-8

  9. Shamim M, Singh K (2017) Biotic Stress management in rice: molecular approaches

  10. Arunakumari K, Durgarani CV, Satturu V et al (2016) Marker-assisted pyramiding of genes conferring resistance against bacterial blight and blast diseases into Indian Rice variety MTU1010. Rice Sci 23:306–316. https://doi.org/10.1016/j.rsci.2016.04.005

    Article  Google Scholar 

  11. Rumsby G (2006) An introduction to PCR techniques. Horm Assays Biol Fluids:75–89. https://doi.org/10.1385/1-59259-986-9:75

  12. Esteves LM, Bulhões SM, Branco CC et al (2018) Diagnosis of human leptospirosis in a clinical setting: real-time PCR high resolution melting analysis for detection of Leptospira at the onset of disease. Sci Rep 8:1–10. https://doi.org/10.1038/s41598-018-27555-2

    Article  CAS  Google Scholar 

  13. Fellahi S, El Harrak M, Kuhn JH et al (2016) Comparison of SYBR green i real-time RT-PCR with conventional agarose gel-based RT-PCR for the diagnosis of infectious bronchitis virus infection in chickens in Morocco. BMC Res Notes 9:1–9. https://doi.org/10.1186/s13104-016-2037-z

    Article  CAS  Google Scholar 

  14. Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE (2000) Multiplex PCR: optimization and application in diagnostic virology. Clin Microbiol Rev 13:559–570. https://doi.org/10.1128/CMR.13.4.559-570.2000

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Vliet M, Schukkink AF, EN B, Klatser R (2018) Nucleic acid sequence-based amplification ( NASBA ) for the identification of mycobacteria. J General Microbiol 130:2423–2429

    Google Scholar 

  16. LaBarre P, Gerlach J, Wilmoth J et al (2010) Non-instrumented nucleic acid amplification (NINA): instrument-free molecular malaria diagnostics for low-resource settings. 2010 Annu Int Conf IEEE Eng Med Biol Soc EMBC’10:1097–1099. https://doi.org/10.1109/IEMBS.2010.5627346

  17. Sood S, Verma R, Mir SS et al (2014) Nucleic acid amplification tests (NAATs) for gonorrhoea diagnosis in women: experience of a tertiary care hospital in North India. Indian J Med Res 140:649–652

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Florkowski C, Don-Wauchope A, Gimenez N et al (2017) Point-of-care testing (POCT) and evidence-based laboratory medicine (EBLM)–does it leverage any advantage in clinical decision making? Crit Rev Clin Lab Sci 54:471–494. https://doi.org/10.1080/10408363.2017.1399336

    Article  CAS  PubMed  Google Scholar 

  19. Notomi T, Okayama H, Masubuchi N et al (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:E63. https://doi.org/10.1093/nar/28.12.e63

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ma M, Ma C, Li M et al (2011) Loop-mediated isothermal amplification for rapid detection of Chinese sacbrood virus. J Virol Methods 176:115–119. https://doi.org/10.1016/j.jviromet.2011.05.028

    Article  CAS  PubMed  Google Scholar 

  21. Aviel-Ronen S, Zhu CQ, Coe BP et al (2006) Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues. BMC Genomics 7:1–10. https://doi.org/10.1186/1471-2164-7-312

    Article  CAS  Google Scholar 

  22. Manosas M, Spiering MM, Ding F et al (2012) Mechanism of strand displacement synthesis by DNA replicative polymerases. Nucleic Acids Res 40:6174–6186. https://doi.org/10.1093/nar/gks253

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Mori Y, Nagamine K, Tomita N, Notomi T (2001) Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun 289:150–154. https://doi.org/10.1006/bbrc.2001.5921

    Article  CAS  PubMed  Google Scholar 

  24. Poole CB, Li Z, Alhassan A et al (2017) Colorimetric tests for diagnosis of filarial infection and vector surveillance using noninstrumented nucleic acid loop-mediated isothermal amplification (NINA-LAMP). PLoS One 12. https://doi.org/10.1371/journal.pone.0169011

  25. Tanner NA, Zhang Y, Evans TC (2015) Visual detection of isothermal nucleic acid amplification using pH-sensitive dyes. Biotechniques 58:59–68. https://doi.org/10.2144/000114253

    Article  CAS  PubMed  Google Scholar 

  26. Lucchi NW (2018) Use of malachite green-loop mediated isothermal Ampli cation for detection of Plasmodium spp. Parasites 6–11

  27. Yuan D, Kong J, Li X et al (2018) Colorimetric LAMP microfluidic chip for detecting three allergens : peanut , sesame and soybean. Sci Rep:1–8. https://doi.org/10.1038/s41598-018-26982-5

  28. Britton S, Cheng Q, Sutherland CJ, McCarthy JS (2015) A simple, high-throughput, colourimetric, field applicable loop-mediated isothermal amplification (HtLAMP) assay for malaria elimination. Malar J 14:1–12. https://doi.org/10.1186/s12936-015-0848-3

    Article  CAS  Google Scholar 

  29. Lopez-Jimena B, Wehner S, Harold G et al (2018) Development of a single-tube one-step RT-LAMP assay to detect the chikungunya virus genome. PLoS Negl Trop Dis 12:1–14. https://doi.org/10.1371/journal.pntd.0006448

    Article  CAS  Google Scholar 

  30. Yan M, Li W, Zhou Z et al (2017) Direct detection of various pathogens by loop-mediated isothermal amplification assays on bacterial culture and bacterial colony. Microb Pathog 102:1–7. https://doi.org/10.1016/j.micpath.2016.10.025

    Article  CAS  PubMed  Google Scholar 

  31. Prasannakumar MK, Buela Parivallal P, Manjunatha C et al (2020) Loop-mediated isothermal amplification assay for pre-symptomatic stage detection of Xanthomonas axonopodis pv. punicae infection in pomegranate. Australas Plant Pathol:10–11. https://doi.org/10.1007/s13313-020-00720-w

  32. King KM, Krivova V, Canning GGM et al (2018) Loop-mediated isothermal amplification (LAMP) assays for rapid detection of Pyrenopeziza brassicae (light leaf spot of brassicas). Plant Pathol 67:167–174. https://doi.org/10.1111/ppa.12717

    Article  CAS  Google Scholar 

  33. Manjunatha C, Sharma S, Kulshreshtha D, Gupta S (2018) Rapid detection of Puccinia triticina causing leaf rust of wheat by PCR and loop mediated isothermal amplification. 1–14

  34. Kiddle G, Hardinge P, Buttigieg N et al (2012) GMO detection using a bioluminescent real time reporter (BART) of loop mediated isothermal amplification (LAMP) suitable for field use. BMC Biotechnol 12. https://doi.org/10.1186/1472-6750-12-15

  35. Ming R, Yu Q, Moore PH (2007) Sex determination in papaya. Semin Cell Dev Biol 18:401–408. https://doi.org/10.1016/j.semcdb.2006.11.013

    Article  CAS  PubMed  Google Scholar 

  36. Keb-Llanes M, González G, Chi-Manzanero B, Infante D (2002) A rapid and simple method for small-scale DNA extraction in Agavaceae and other tropical plants. Plant Mol Biol Report 20. https://doi.org/10.1007/BF02782465

  37. Hasan MM, Rafii MY, Ismail MR et al (2015) Marker-assisted backcrossing: a useful method for rice improvement. Biotechnol Biotechnol Equip 29:237–254. https://doi.org/10.1080/13102818.2014.995920

    Article  PubMed  PubMed Central  Google Scholar 

  38. Visscher PM, Haley CS, Thompson R (1996) Marker-assisted introgression in backcross breeding programs. Genetics 144:1923–1932

    Article  CAS  Google Scholar 

  39. Tewari S, Sharma S (2019) Molecular techniques for diagnosis of bacterial plant pathogens. Elsevier Inc.

Download references

Acknowledgements

We are thankful to the Director of Research, UAS Bengaluru, Karnataka, India, and Director of Research, UAS Raichur, Karnataka, India, for providing research facilities. The authors are thankful to Rashtriya Krishi Vikasa Yojana (RKVY), Govt of Karnataka, for providing financial support. The authors have no conflict of interest to declare.

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Authors and Affiliations

  1. Department of Plant Pathology, University of Agricultural Sciences, Bangalore, India

    M. K. Prasannakumar, Buela P. Parivallal, Sahana N. Banakar & K. T. Rangaswamy

  2. ICAR-Indian Agricultural Research Institute, Regional Station Wellington, Nilgiris, Tamil Nadu, India

    Chennappa Manjunatha

  3. Rice Pathology Laboratory, All India Coordinated Rice Improvement Programme, Gangavathi, University of Agricultural Sciences, Raichur, Karnataka, India

    Devanna Pramesh

  4. Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai, India

    Karthik S. Narayan & Gopal Venkatesh

  5. Department of Genetics and Plant Breeding, University of Agricultural Sciences, Bangalore, Karnataka, India

    H. B. Mahesh

  6. Department of Biotechnology, Regional Centre for Biotechnology, Gurgaon, Haryana, India

    Ramu S. Vemanna

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  1. M. K. Prasannakumar
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Contributions

MKP, CM, DP, HBM, and KTR conceived the idea. BPP, KSN, GV, SNB, RSV conducted the experiments. BPP, CM, DP, and MKP analyzed the data and wrote the manuscript. All authors read and approved the manuscript.

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Correspondence to M. K. Prasannakumar or Devanna Pramesh.

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Prasannakumar, M.K., Parivallal, B.P., Manjunatha, C. et al. Rapid genotyping of bacterial leaf blight resistant genes of rice using loop-mediated isothermal amplification assay. Mol Biol Rep 48, 467–474 (2021). https://doi.org/10.1007/s11033-020-06077-z

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