Development and validation of a LAMP-based method for rapid and reliable detection of Xanthomonas albilineans, the causal agent of sugarcane leaf scald
IntroductionXanthomonas albilineans (Xalb)-induced leaf scald (LS) is a significant bacterial disease affecting sugarcane and posing a global threat to the sugarcane industry. The presence of irregular symptoms makes traditional phenotypic detection difficult, and molecular methods necessitate costl...
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Frontiers Media S.A.
2025-01-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1537812/full |
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| author | Moutoshi Chakraborty Moutoshi Chakraborty Shamsul Arafin Bhuiyan Shamsul Arafin Bhuiyan Simon Strachan Simon Strachan Muhammad J. A. Shiddiky Nam-Trung Nguyen Rebecca Ford Rebecca Ford |
| author_facet | Moutoshi Chakraborty Moutoshi Chakraborty Shamsul Arafin Bhuiyan Shamsul Arafin Bhuiyan Simon Strachan Simon Strachan Muhammad J. A. Shiddiky Nam-Trung Nguyen Rebecca Ford Rebecca Ford |
| author_sort | Moutoshi Chakraborty |
| collection | DOAJ |
| description | IntroductionXanthomonas albilineans (Xalb)-induced leaf scald (LS) is a significant bacterial disease affecting sugarcane and posing a global threat to the sugarcane industry. The presence of irregular symptoms makes traditional phenotypic detection difficult, and molecular methods necessitate costly equipment, labor, and extended sample-to-answer processing times.MethodsThis study introduces an innovative rapid DNA isolation method requiring no reagents, combined with an isothermal amplification-based assay for efficient detection of Xalb DNA in sugarcane xylem sap, leaf tissue, and meristematic tissue samples. Sugarcane samples from infected plants were subjected to heat lysis, followed by loop-mediated isothermal amplification (LAMP)-based fluorescence and colorimetric quantification within a single microcentrifuge tube.ResultsThe method exhibited exceptional detection sensitivity (detecting as low as 1 cell/μL), reproducibility [with a standard deviation (SD) of <5% for n = 3], and a broad linear dynamic range (10 pM to 1 aM or 107–100 copies/μL, r = 0.99). Quantification of Xalb was accurately correlated with sugarcane cultivar disease ratings. Validation using qPCR showed 91–98% agreement. This assay also effectively determined optimal sampling times and plant parts by monitoring the progression of the disease over time.DiscussionThis diagnostic assay holds significant potential as a commercial opportunity for a kit-based DNA extraction/purification-free molecular detection alternative. It can be adapted into a handheld device, enabling on-farm detection and quantification of the pathogen responsible for LS disease. |
| format | Article |
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| institution | Kabale University |
| issn | 1664-302X |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Microbiology |
| spelling | doaj-art-4bbddefd9d8742f0a692c272c36fe90d2025-01-30T06:23:05ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2025-01-011610.3389/fmicb.2025.15378121537812Development and validation of a LAMP-based method for rapid and reliable detection of Xanthomonas albilineans, the causal agent of sugarcane leaf scaldMoutoshi Chakraborty0Moutoshi Chakraborty1Shamsul Arafin Bhuiyan2Shamsul Arafin Bhuiyan3Simon Strachan4Simon Strachan5Muhammad J. A. Shiddiky6Nam-Trung Nguyen7Rebecca Ford8Rebecca Ford9Centre for Planetary Health and Food Security (CPHFS), Griffith University (GU), Brisbane, QLD, AustraliaSchool of Environment and Science (ESC), Griffith University (GU), Brisbane, QLD, AustraliaSugar Research Australia (SRA), Brisbane, QLD, AustraliaQueensland Micro- and Nanotechnology Centre (QMNC), Griffith University (GU), Brisbane, QLD, AustraliaSchool of Environment and Science (ESC), Griffith University (GU), Brisbane, QLD, AustraliaQueensland Micro- and Nanotechnology Centre (QMNC), Griffith University (GU), Brisbane, QLD, AustraliaRural Health Research Institute (RHRI), Charles Sturt University, Bathurst, NSW, AustraliaQueensland Micro- and Nanotechnology Centre (QMNC), Griffith University (GU), Brisbane, QLD, AustraliaCentre for Planetary Health and Food Security (CPHFS), Griffith University (GU), Brisbane, QLD, AustraliaSchool of Environment and Science (ESC), Griffith University (GU), Brisbane, QLD, AustraliaIntroductionXanthomonas albilineans (Xalb)-induced leaf scald (LS) is a significant bacterial disease affecting sugarcane and posing a global threat to the sugarcane industry. The presence of irregular symptoms makes traditional phenotypic detection difficult, and molecular methods necessitate costly equipment, labor, and extended sample-to-answer processing times.MethodsThis study introduces an innovative rapid DNA isolation method requiring no reagents, combined with an isothermal amplification-based assay for efficient detection of Xalb DNA in sugarcane xylem sap, leaf tissue, and meristematic tissue samples. Sugarcane samples from infected plants were subjected to heat lysis, followed by loop-mediated isothermal amplification (LAMP)-based fluorescence and colorimetric quantification within a single microcentrifuge tube.ResultsThe method exhibited exceptional detection sensitivity (detecting as low as 1 cell/μL), reproducibility [with a standard deviation (SD) of <5% for n = 3], and a broad linear dynamic range (10 pM to 1 aM or 107–100 copies/μL, r = 0.99). Quantification of Xalb was accurately correlated with sugarcane cultivar disease ratings. Validation using qPCR showed 91–98% agreement. This assay also effectively determined optimal sampling times and plant parts by monitoring the progression of the disease over time.DiscussionThis diagnostic assay holds significant potential as a commercial opportunity for a kit-based DNA extraction/purification-free molecular detection alternative. It can be adapted into a handheld device, enabling on-farm detection and quantification of the pathogen responsible for LS disease.https://www.frontiersin.org/articles/10.3389/fmicb.2025.1537812/fullnucleic acid isolationplant pathogen diagnosticLS diseaseXanthomonas albilineansrapid detection |
| spellingShingle | Moutoshi Chakraborty Moutoshi Chakraborty Shamsul Arafin Bhuiyan Shamsul Arafin Bhuiyan Simon Strachan Simon Strachan Muhammad J. A. Shiddiky Nam-Trung Nguyen Rebecca Ford Rebecca Ford Development and validation of a LAMP-based method for rapid and reliable detection of Xanthomonas albilineans, the causal agent of sugarcane leaf scald Frontiers in Microbiology nucleic acid isolation plant pathogen diagnostic LS disease Xanthomonas albilineans rapid detection |
| title | Development and validation of a LAMP-based method for rapid and reliable detection of Xanthomonas albilineans, the causal agent of sugarcane leaf scald |
| title_full | Development and validation of a LAMP-based method for rapid and reliable detection of Xanthomonas albilineans, the causal agent of sugarcane leaf scald |
| title_fullStr | Development and validation of a LAMP-based method for rapid and reliable detection of Xanthomonas albilineans, the causal agent of sugarcane leaf scald |
| title_full_unstemmed | Development and validation of a LAMP-based method for rapid and reliable detection of Xanthomonas albilineans, the causal agent of sugarcane leaf scald |
| title_short | Development and validation of a LAMP-based method for rapid and reliable detection of Xanthomonas albilineans, the causal agent of sugarcane leaf scald |
| title_sort | development and validation of a lamp based method for rapid and reliable detection of xanthomonas albilineans the causal agent of sugarcane leaf scald |
| topic | nucleic acid isolation plant pathogen diagnostic LS disease Xanthomonas albilineans rapid detection |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1537812/full |
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