Development and validation of genome-informed and multigene-based qPCR and LAMP assays for accurate detection of Dickeya solani: a critical quarantine pathogen threatening the potato industry
ABSTRACT Dickeya solani one of the most aggressive pectinolytic phytopathogens, causes blackleg disease in potatoes, resulting in significant economic losses and adversely impacting one of the world’s most important food crops. The diagnostics methods are critical in monitoring the latent infection...
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| Language: | English |
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American Society for Microbiology
2025-01-01
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| Series: | Microbiology Spectrum |
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| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.00784-24 |
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| author | Shefali Dobhal Gem Santillana Michael J. Stulberg Dario Arizala Anne M. Alvarez Mohammad Arif |
| author_facet | Shefali Dobhal Gem Santillana Michael J. Stulberg Dario Arizala Anne M. Alvarez Mohammad Arif |
| author_sort | Shefali Dobhal |
| collection | DOAJ |
| description | ABSTRACT Dickeya solani one of the most aggressive pectinolytic phytopathogens, causes blackleg disease in potatoes, resulting in significant economic losses and adversely impacting one of the world’s most important food crops. The diagnostics methods are critical in monitoring the latent infection for international trade of potato seed tubers and in implementation of control strategies. Our study employed a whole-genome comparative approach, identifying unique target gene loci (LysR and TetR family of transcriptional regulators gene regions) and designing loop-mediated isothermal amplification (LAMP) and a multi-gene-based multiplex TaqMan qPCR assays for specific detection and differentiation of D. solani. Both methods underwent meticulous validation with extensive inclusivity and exclusivity panels, exhibiting 100% accuracy and no false positives or negatives. The LAMP method demonstrated the detection limit of 100 fg and 1 CFU per reaction using pure genomic DNA and crude bacterial cell lysate, respectively. The qPCR detection limit was 1 pg, 100 fg and 10 fg with quadplex, triplex, and singleplex, respectively. None of the assays were impacted by any inhibitory or competitive effects after adding host DNA (in qPCR) or crude lysate (in LAMP). The assays proved robust and reproducible in detecting the target pathogen in infected samples, with the LAMP assay being field-deployable due to its simplicity and rapid results acquisition within approximately 9 min. The reproducibility was confirmed by performing the assay in two independent laboratories. These assays offer a robust, rapid, and reliable solution for routine testing, with applications in phytosanitary inspection, disease diagnosis, and epidemiological studies.IMPORTANCEDickeya solani, one of the most aggressive soft rot causing bacteria and a quarantine pathogen, poses a severe threat to food security by causing substantial economic losses to the potato industry. Accurate and timely detection of this bacterium is vital for monitoring latent infections, particularly for international trade of potato seed tubers, and for implementing effective control strategies. In this research, we have developed LAMP and multi-gene-based multiplex TaqMan qPCR assays for specific detection of D. solani. These assays, characterized by their precision, rapidity, and robustness, are crucial for distinguishing D. solani from related species. Offering unparalleled sensitivity and specificity, these assays are indispensable for phytosanitary inspection and epidemiological monitoring, providing a powerful tool enabling management of this threatening pathogen. |
| format | Article |
| id | doaj-art-dfa9c6acbdf246e599012b3344d9dd87 |
| institution | Kabale University |
| issn | 2165-0497 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | American Society for Microbiology |
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| series | Microbiology Spectrum |
| spelling | doaj-art-dfa9c6acbdf246e599012b3344d9dd872025-01-07T14:04:05ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972025-01-0113110.1128/spectrum.00784-24Development and validation of genome-informed and multigene-based qPCR and LAMP assays for accurate detection of Dickeya solani: a critical quarantine pathogen threatening the potato industryShefali Dobhal0Gem Santillana1Michael J. Stulberg2Dario Arizala3Anne M. Alvarez4Mohammad Arif5Department of Plant and Environmental Protection Sciences, University of Hawaii at Manoa, Honolulu, Hawaii, USAPlant Pathogen Confirmatory Diagnostics Laboratory (PPCDL), APHIS PPQ, Science and Technology, United States Department of Agriculture, Beltsville, Maryland, USAPlant Pathogen Confirmatory Diagnostics Laboratory (PPCDL), APHIS PPQ, Science and Technology, United States Department of Agriculture, Beltsville, Maryland, USADepartment of Plant and Environmental Protection Sciences, University of Hawaii at Manoa, Honolulu, Hawaii, USADepartment of Plant and Environmental Protection Sciences, University of Hawaii at Manoa, Honolulu, Hawaii, USADepartment of Plant and Environmental Protection Sciences, University of Hawaii at Manoa, Honolulu, Hawaii, USAABSTRACT Dickeya solani one of the most aggressive pectinolytic phytopathogens, causes blackleg disease in potatoes, resulting in significant economic losses and adversely impacting one of the world’s most important food crops. The diagnostics methods are critical in monitoring the latent infection for international trade of potato seed tubers and in implementation of control strategies. Our study employed a whole-genome comparative approach, identifying unique target gene loci (LysR and TetR family of transcriptional regulators gene regions) and designing loop-mediated isothermal amplification (LAMP) and a multi-gene-based multiplex TaqMan qPCR assays for specific detection and differentiation of D. solani. Both methods underwent meticulous validation with extensive inclusivity and exclusivity panels, exhibiting 100% accuracy and no false positives or negatives. The LAMP method demonstrated the detection limit of 100 fg and 1 CFU per reaction using pure genomic DNA and crude bacterial cell lysate, respectively. The qPCR detection limit was 1 pg, 100 fg and 10 fg with quadplex, triplex, and singleplex, respectively. None of the assays were impacted by any inhibitory or competitive effects after adding host DNA (in qPCR) or crude lysate (in LAMP). The assays proved robust and reproducible in detecting the target pathogen in infected samples, with the LAMP assay being field-deployable due to its simplicity and rapid results acquisition within approximately 9 min. The reproducibility was confirmed by performing the assay in two independent laboratories. These assays offer a robust, rapid, and reliable solution for routine testing, with applications in phytosanitary inspection, disease diagnosis, and epidemiological studies.IMPORTANCEDickeya solani, one of the most aggressive soft rot causing bacteria and a quarantine pathogen, poses a severe threat to food security by causing substantial economic losses to the potato industry. Accurate and timely detection of this bacterium is vital for monitoring latent infections, particularly for international trade of potato seed tubers, and for implementing effective control strategies. In this research, we have developed LAMP and multi-gene-based multiplex TaqMan qPCR assays for specific detection of D. solani. These assays, characterized by their precision, rapidity, and robustness, are crucial for distinguishing D. solani from related species. Offering unparalleled sensitivity and specificity, these assays are indispensable for phytosanitary inspection and epidemiological monitoring, providing a powerful tool enabling management of this threatening pathogen.https://journals.asm.org/doi/10.1128/spectrum.00784-24isothermal amplificationquarantine plant pathogensdetectionpotatoblacklegdiagnostics |
| spellingShingle | Shefali Dobhal Gem Santillana Michael J. Stulberg Dario Arizala Anne M. Alvarez Mohammad Arif Development and validation of genome-informed and multigene-based qPCR and LAMP assays for accurate detection of Dickeya solani: a critical quarantine pathogen threatening the potato industry Microbiology Spectrum isothermal amplification quarantine plant pathogens detection potato blackleg diagnostics |
| title | Development and validation of genome-informed and multigene-based qPCR and LAMP assays for accurate detection of Dickeya solani: a critical quarantine pathogen threatening the potato industry |
| title_full | Development and validation of genome-informed and multigene-based qPCR and LAMP assays for accurate detection of Dickeya solani: a critical quarantine pathogen threatening the potato industry |
| title_fullStr | Development and validation of genome-informed and multigene-based qPCR and LAMP assays for accurate detection of Dickeya solani: a critical quarantine pathogen threatening the potato industry |
| title_full_unstemmed | Development and validation of genome-informed and multigene-based qPCR and LAMP assays for accurate detection of Dickeya solani: a critical quarantine pathogen threatening the potato industry |
| title_short | Development and validation of genome-informed and multigene-based qPCR and LAMP assays for accurate detection of Dickeya solani: a critical quarantine pathogen threatening the potato industry |
| title_sort | development and validation of genome informed and multigene based qpcr and lamp assays for accurate detection of dickeya solani a critical quarantine pathogen threatening the potato industry |
| topic | isothermal amplification quarantine plant pathogens detection potato blackleg diagnostics |
| url | https://journals.asm.org/doi/10.1128/spectrum.00784-24 |
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