Abstract
Bacterial wilt caused by Ralstonia solanacearum, is one of the most notorious plant diseases of potato and causes severe yield losses globally. R. solanacearum produces fluidal white coloured colonies with a light pink centre on casamino acid-peptone-glucose medium. Bacterial wilt pathogen is soil as well as tuber borne, so early detection of bacterial wilt pathogen is necessary to take up timely management practices. Traditional methods used for detection of R. solanacearum are labour-intensive, expensive, time-consuming and can be carried out only in well-equipped laboratories. Hence, the present study was aimed to develop a loop-mediated isothermal amplification (LAMP) assay for R. solanacearum, as an alternative approach for quick and efficient detection of the bacterial wilt. LAMP reaction mix was optimized by adjusting the concentrations of MgSO4, dNTPs, betaine, and time, temperature etc. The optimum temperature and time for the detection of R. solanacearum were 65 °C and 50 min, respectively. The positive samples showed colour change from violet and were reconfirmed in 2% agarose gel electrophoresis which produced ladder like bands. The LAMP assay developed was highly specific to detect R. solanacearum from other bacteria and sensitive with a lowest detection limit of 10 pg/µl of template DNA. The developed LAMP assay was validated with R. solanacearum isolates, infected stems, tubers and soil, and also, it was capable of detecting latent infection of R. solanacearum in seed tubers. Hence, the LAMP assay protocol provides a rapid, specific and sensitive tool for the latent detection of R. solanacearum in seed potato tubers.
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Abbreviations
- LAMP:
-
Loop mediated isothermal amplification
- MgSO4 :
-
Magnesium sulphate
- dNTP:
-
Deoxynucleoside triphosphate
- HNB:
-
Hydroxy naphthol blue
- Bst DNA polymerase:
-
Bacillus stearothermophilus DNA polymerase
- PCR:
-
Polymerase chain reaction
- RT PCR:
-
Reverse transcription-polymerase chain reaction
- DNA:
-
Deoxyribonucleic acid
- CPG:
-
Casamino acid peptone glucose
- TZC:
-
Triphenyl tetrazolium chloride
- SDS:
-
Sodium dodecyl sulphate
- Nacl:
-
Sodium chloride
- CTAB:
-
Cetyl trimethyl ammonium bromide
- egl :
-
endoglucanase
- NCBI:
-
National Center for Biotechnology Information
- IpxC :
-
UDP-(3-O-acyl)-N-acetylglucosamine deacetylase gene
- F3:
-
Forward outer primer
- B3:
-
Backward outer primer
- FIP:
-
Forward inner primer
- BIP:
-
Backward inner primer
- ng:
-
Nanogram
- pg:
-
Picogram
- fg:
-
Femtogram
- PEG:
-
Polyethylene glycol
- dpi:
-
Day post inoculation
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The authors gratefully acknowledge the Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, for extending the necessary facilities to carry out the experiments.
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Archana, T., Kamalakannan, A., Gopalakrishnan, C. et al. Detection of Indian Isolates of Soil and Tuber Borne Ralstonia solanacearum (Smith) Infecting Potato (Solanum tuberosum L.) Through a Colorimetric LAMP Assay. Potato Res. (2024). https://doi.org/10.1007/s11540-024-09699-z
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DOI: https://doi.org/10.1007/s11540-024-09699-z