A new and accurate qPCR protocol to detect plant pathogenic bacteria of the genus ‘Candidatus Liberibacter’ in plants and insects
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...
| Autores principales: | , , , , , |
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| Formato: | article |
| Lenguaje: | Inglés |
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Springer Nature
2023
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| Acceso en línea: | https://hdl.handle.net/20.500.11939/8539 https://www.nature.com/articles/s41598-023-30345-0 |
| _version_ | 1855032809452732416 |
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| author | Quintana-González de Chaves, María Morán, Félix Barbé, Silvia Bertolini, Edson Siverio, Felipe Marco-Noales, Ester |
| author_browse | Barbé, Silvia Bertolini, Edson Marco-Noales, Ester Morán, Félix Quintana-González de Chaves, María Siverio, Felipe |
| author_facet | Quintana-González de Chaves, María Morán, Félix Barbé, Silvia Bertolini, Edson Siverio, Felipe Marco-Noales, Ester |
| author_sort | Quintana-González de Chaves, María |
| collection | ReDivia |
| description | 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. |
| format | article |
| id | ReDivia8539 |
| institution | Instituto Valenciano de Investigaciones Agrarias (IVIA) |
| language | Inglés |
| publishDate | 2023 |
| publishDateRange | 2023 |
| publishDateSort | 2023 |
| publisher | Springer Nature |
| publisherStr | Springer Nature |
| record_format | dspace |
| spelling | ReDivia85392025-04-25T14:49:03Z A new and accurate qPCR protocol to detect plant pathogenic bacteria of the genus ‘Candidatus Liberibacter’ in plants and insects Quintana-González de Chaves, María Morán, Félix Barbé, Silvia Bertolini, Edson Siverio, Felipe Marco-Noales, Ester PCR-based techniques Huanglongbing Candidatus Liberibacter asiaticus Candidatus Liberibacter africanus Candidatus Liberibacter americanus RT-PCR Detection H10 Pests of plants H20 Plant diseases U30 Research methods Pathogens PCR Psyllids Vectors Citrus protocols 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. 2023-03-03T08:45:15Z 2023-03-03T08:45:15Z 2023 article publishedVersion Quintana-González, M., Morán, F., Barbé, S., Bertolini, E., Siverio, F. & Marco-Noales, E. (2023). A new and accurate qPCR protocol to detect plant pathogenic bacteria of the genus ‘Candidatus Liberibacter’ in plants and insects. Scientific Reports, 13(1), 3338. 2045-2322 (online) https://hdl.handle.net/20.500.11939/8539 10.1038/s41598-023-30345-0 https://www.nature.com/articles/s41598-023-30345-0 en The authors would like to thank for the Grants IVIA/52202D–Sostenible–funded by Instituto Valenciano de Investigaciones Agrarias, Generalitat Valenciana, LIFE18 CCA/ES/001109 (Development of sustainable control strategies for citric under threat of climate change & preventing entry of HLB in EU -LIFE Vida for Citrus-) and PID2021-124145OR-C21 (Breeding, selection and evaluation of new citrus materials for more sustainable plantations in the face of emerging threats due to global change -FORTECITRUS-). This research was funded by INIA E-RTA2014-00008-C4-01 and INIA E-RTA2015-00005-C06-01 from the Spanish Government. MQGC is recipient of PhD grant 2017-2020 from Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria. Attribution-NonCommercial-NoDerivatives 4.0 Internacional http://creativecommons.org/licenses/by-nc-nd/4.0/ openAccess Springer Nature electronico |
| spellingShingle | PCR-based techniques Huanglongbing Candidatus Liberibacter asiaticus Candidatus Liberibacter africanus Candidatus Liberibacter americanus RT-PCR Detection H10 Pests of plants H20 Plant diseases U30 Research methods Pathogens PCR Psyllids Vectors Citrus protocols Quintana-González de Chaves, María Morán, Félix Barbé, Silvia Bertolini, Edson Siverio, Felipe Marco-Noales, Ester A new and accurate qPCR protocol to detect plant pathogenic bacteria of the genus ‘Candidatus Liberibacter’ in plants and insects |
| 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 |
| topic | PCR-based techniques Huanglongbing Candidatus Liberibacter asiaticus Candidatus Liberibacter africanus Candidatus Liberibacter americanus RT-PCR Detection H10 Pests of plants H20 Plant diseases U30 Research methods Pathogens PCR Psyllids Vectors Citrus protocols |
| url | https://hdl.handle.net/20.500.11939/8539 https://www.nature.com/articles/s41598-023-30345-0 |
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