A new and accurate qPCR protocol to detect plant pathogenic bacteria of the genus ‘Candidatus Liberibacter’ in plants and insects
Abstract Four pathogenic bacterial species of the genus ‘Candidatus Liberibacter’, transmitted by psyllid vectors, have been associated with serious diseases affecting economically important crops of Rutaceae, Apiaceae and Solanaceae families. The most severe disease of citrus plants, huanglongbing...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2023-02-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-30345-0 |
| _version_ | 1797864905609576448 |
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| author | María Quintana-González de Chaves Félix Morán Silvia Barbé Edson Bertolini Felipe Siverio de la Rosa Ester Marco-Noales |
| author_facet | María Quintana-González de Chaves Félix Morán Silvia Barbé Edson Bertolini Felipe Siverio de la Rosa Ester Marco-Noales |
| author_sort | María Quintana-González de Chaves |
| collection | DOAJ |
| description | Abstract Four pathogenic bacterial species of the genus ‘Candidatus Liberibacter’, transmitted by psyllid vectors, have been associated with serious diseases affecting economically important crops of Rutaceae, Apiaceae and Solanaceae families. The most severe disease of citrus plants, huanglongbing (HLB), is associated with ‘Ca. Liberibacter asiaticus’ (CaLas), ‘Ca. Liberibacter americanus’ (CaLam) and ‘Ca. Liberibacter africanus’ (CaLaf), while ‘Ca. Liberibacter solanacearum’ (CaLsol) is associated with zebra chip disease in potatoes and vegetative disorders in apiaceous plants. Since these bacteria remain non-culturable and their symptoms are non-specific, their detection and identification are done by molecular methods, mainly based on PCR protocols. In this study, a new quantitative real-time PCR protocol based on TaqMan probe, which can also be performed in a conventional PCR version, has been developed to detect the four known phytopathogenic species of the genus Liberibacter. The new protocol has been validated according to European Plant Protection Organization (EPPO) guidelines and is able to detect CaLas, CaLam, CaLaf and CaLsol in both plants and vectors, not only using purified DNA but also using crude extracts of potato and citrus or psyllids. A comparative analysis with other previously described qPCR protocols revealed that this new one developed in this study is more specific and equally or more sensitive. Thus, other genus-specific qPCR protocols have important drawbacks regarding the lack of specificity, while with the new protocol there was no cross-reactions in 250 samples from 24 different plant and insect species from eight different geographical origins. Therefore, it can be used as a rapid and time-saving screening test, as it allows simultaneous detection of all plant pathogenic species of ‘Ca. Liberibacter’ in a one-step assay. |
| first_indexed | 2024-04-09T22:59:27Z |
| format | Article |
| id | doaj.art-5fff1538d90d4263bd1af86ad2397f66 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-09T22:59:27Z |
| publishDate | 2023-02-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-5fff1538d90d4263bd1af86ad2397f662023-03-22T11:02:13ZengNature PortfolioScientific Reports2045-23222023-02-0113111210.1038/s41598-023-30345-0A new and accurate qPCR protocol to detect plant pathogenic bacteria of the genus ‘Candidatus Liberibacter’ in plants and insectsMaría Quintana-González de Chaves0Félix Morán1Silvia Barbé2Edson Bertolini3Felipe Siverio de la Rosa4Ester Marco-Noales5Unidad de Protección Vegetal, Instituto Canario de Investigaciones Agrarias (ICIA)Unidad de Bacteriología, Centro de Protección Vegetal y Biotecnología. Instituto Vaslenciano de Investigaciones Agrarias (IVIA)Unidad de Bacteriología, Centro de Protección Vegetal y Biotecnología. Instituto Vaslenciano de Investigaciones Agrarias (IVIA)Department of Plant Health, Faculty of Agronomys, Federal University of Rio Grande do Sul (UFRGS)Unidad de Protección Vegetal, Instituto Canario de Investigaciones Agrarias (ICIA)Unidad de Bacteriología, Centro de Protección Vegetal y Biotecnología. Instituto Vaslenciano de Investigaciones Agrarias (IVIA)Abstract Four pathogenic bacterial species of the genus ‘Candidatus Liberibacter’, transmitted by psyllid vectors, have been associated with serious diseases affecting economically important crops of Rutaceae, Apiaceae and Solanaceae families. The most severe disease of citrus plants, huanglongbing (HLB), is associated with ‘Ca. Liberibacter asiaticus’ (CaLas), ‘Ca. Liberibacter americanus’ (CaLam) and ‘Ca. Liberibacter africanus’ (CaLaf), while ‘Ca. Liberibacter solanacearum’ (CaLsol) is associated with zebra chip disease in potatoes and vegetative disorders in apiaceous plants. Since these bacteria remain non-culturable and their symptoms are non-specific, their detection and identification are done by molecular methods, mainly based on PCR protocols. In this study, a new quantitative real-time PCR protocol based on TaqMan probe, which can also be performed in a conventional PCR version, has been developed to detect the four known phytopathogenic species of the genus Liberibacter. The new protocol has been validated according to European Plant Protection Organization (EPPO) guidelines and is able to detect CaLas, CaLam, CaLaf and CaLsol in both plants and vectors, not only using purified DNA but also using crude extracts of potato and citrus or psyllids. A comparative analysis with other previously described qPCR protocols revealed that this new one developed in this study is more specific and equally or more sensitive. Thus, other genus-specific qPCR protocols have important drawbacks regarding the lack of specificity, while with the new protocol there was no cross-reactions in 250 samples from 24 different plant and insect species from eight different geographical origins. Therefore, it can be used as a rapid and time-saving screening test, as it allows simultaneous detection of all plant pathogenic species of ‘Ca. Liberibacter’ in a one-step assay.https://doi.org/10.1038/s41598-023-30345-0 |
| spellingShingle | María Quintana-González de Chaves Félix Morán Silvia Barbé Edson Bertolini Felipe Siverio de la Rosa Ester Marco-Noales A new and accurate qPCR protocol to detect plant pathogenic bacteria of the genus ‘Candidatus Liberibacter’ in plants and insects Scientific Reports |
| title | A new and accurate qPCR protocol to detect plant pathogenic bacteria of the genus ‘Candidatus Liberibacter’ in plants and insects |
| title_full | A new and accurate qPCR protocol to detect plant pathogenic bacteria of the genus ‘Candidatus Liberibacter’ in plants and insects |
| title_fullStr | A new and accurate qPCR protocol to detect plant pathogenic bacteria of the genus ‘Candidatus Liberibacter’ in plants and insects |
| title_full_unstemmed | A new and accurate qPCR protocol to detect plant pathogenic bacteria of the genus ‘Candidatus Liberibacter’ in plants and insects |
| title_short | A new and accurate qPCR protocol to detect plant pathogenic bacteria of the genus ‘Candidatus Liberibacter’ in plants and insects |
| title_sort | new and accurate qpcr protocol to detect plant pathogenic bacteria of the genus candidatus liberibacter in plants and insects |
| url | https://doi.org/10.1038/s41598-023-30345-0 |
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