The Challenge of Environmental Samples for PCR Detection of Phytopathogenic Bacteria: A Case Study of Citrus Huanglongbing Disease
Huanglongbing (HLB) is the most devastating citrus disease and is associated with three bacterial species of the genus ‘Candidatus Liberibacter’ transmitted by insect vectors. The early detection of HLB is based on PCR methods, and it is one of the cornerstones for preventing incursion into disease-...
| Main Authors: | , , , , , , , , , , |
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| Format: | publishedVersion |
| Language: | Inglés |
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MDPI
2020
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| Online Access: | http://hdl.handle.net/20.500.11939/6927 https://www.mdpi.com/2073-4395/11/1/10/htm |
| _version_ | 1855032516003495936 |
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| author | Morán, Félix Barbé, Silvia Bastin, Saskia Navarro-Herrero, Inmaculada Bertolini, Edson López, María M. Hernández-Suárez, Estrella Urbaneja, Alberto Tena, Alejandro Siverio, Felipe Marco-Noales, Ester |
| author_browse | Barbé, Silvia Bastin, Saskia Bertolini, Edson Hernández-Suárez, Estrella López, María M. Marco-Noales, Ester Morán, Félix Navarro-Herrero, Inmaculada Siverio, Felipe Tena, Alejandro Urbaneja, Alberto |
| author_facet | Morán, Félix Barbé, Silvia Bastin, Saskia Navarro-Herrero, Inmaculada Bertolini, Edson López, María M. Hernández-Suárez, Estrella Urbaneja, Alberto Tena, Alejandro Siverio, Felipe Marco-Noales, Ester |
| author_sort | Morán, Félix |
| collection | ReDivia |
| description | Huanglongbing (HLB) is the most devastating citrus disease and is associated with three bacterial species of the genus ‘Candidatus Liberibacter’ transmitted by insect vectors. The early detection of HLB is based on PCR methods, and it is one of the cornerstones for preventing incursion into disease-free countries. However, the detection of phytopathogenic bacteria with PCR-based methods is problematic in surveys that include a variety of samples of different origins. Here, we first report the proportion of amplifications obtained by two standardized real-time PCR methods for the diagnosis of HLB in various environmental samples that include plants, psyllid vectors, and parasitic wasps of the psyllids. The results of 4915 samples showed that 9.3% of them were amplified by the first rapid screening test and only 0.3% by the more specific tests. Most of the amplifications were associated with parasitic wasps. We designed the primers external to the target regions of both real-time PCR protocols to determine if amplifications belonged to one of three ‘Ca. Liberibacter’ species associated with HLB. The bioinformatic analysis of the sequences obtained with these primers revealed that all these amplifications came from the presence of other prokaryotic organisms in the samples. The primers developed in this study overcome the problem of undesired amplification in environmental samples. Thus, they could be used in future survey protocols to prevent the eradication of negative trees and the generation of unjustified alarms. |
| format | publishedVersion |
| id | ReDivia6927 |
| institution | Instituto Valenciano de Investigaciones Agrarias (IVIA) |
| language | Inglés |
| publishDate | 2020 |
| publishDateRange | 2020 |
| publishDateSort | 2020 |
| publisher | MDPI |
| publisherStr | MDPI |
| record_format | dspace |
| spelling | ReDivia69272025-04-25T14:47:56Z The Challenge of Environmental Samples for PCR Detection of Phytopathogenic Bacteria: A Case Study of Citrus Huanglongbing Disease Morán, Félix Barbé, Silvia Bastin, Saskia Navarro-Herrero, Inmaculada Bertolini, Edson López, María M. Hernández-Suárez, Estrella Urbaneja, Alberto Tena, Alejandro Siverio, Felipe Marco-Noales, Ester undesired amplification 16S real-time PCR sequence identity greening HLB Trioza erytreae Tamarixia dryi H20 Plant diseases Bacterial flora Candidatus Liberibacter HLB PCR Huanglongbing (HLB) is the most devastating citrus disease and is associated with three bacterial species of the genus ‘Candidatus Liberibacter’ transmitted by insect vectors. The early detection of HLB is based on PCR methods, and it is one of the cornerstones for preventing incursion into disease-free countries. However, the detection of phytopathogenic bacteria with PCR-based methods is problematic in surveys that include a variety of samples of different origins. Here, we first report the proportion of amplifications obtained by two standardized real-time PCR methods for the diagnosis of HLB in various environmental samples that include plants, psyllid vectors, and parasitic wasps of the psyllids. The results of 4915 samples showed that 9.3% of them were amplified by the first rapid screening test and only 0.3% by the more specific tests. Most of the amplifications were associated with parasitic wasps. We designed the primers external to the target regions of both real-time PCR protocols to determine if amplifications belonged to one of three ‘Ca. Liberibacter’ species associated with HLB. The bioinformatic analysis of the sequences obtained with these primers revealed that all these amplifications came from the presence of other prokaryotic organisms in the samples. The primers developed in this study overcome the problem of undesired amplification in environmental samples. Thus, they could be used in future survey protocols to prevent the eradication of negative trees and the generation of unjustified alarms. 2020-12-28T09:46:20Z 2020-12-28T09:46:20Z 2021 publishedVersion Morán, F.; Barbé, S.; Bastin, S.; Navarro, I.; Bertolini, E.; López, M. M. et al. (2020). The Challenge of Environmental Samples for PCR Detection of Phytopathogenic Bacteria: A Case Study of Citrus Huanglongbing Disease. Agronomy 2020, 11(1), 10. 2073-4395 http://hdl.handle.net/20.500.11939/6927 doi:10.3390/agronomy11010010 https://www.mdpi.com/2073-4395/11/1/10/htm en info:eu-repo/grantAgreement/EC/H2020/727459/EU/Insect-borne prokaryote-associated diseases in tropical and subtropical perennial crops/TropicSafe Project TROPICSAFE (Insect-borne prokaryote-associated diseases in tropical and subtropical perennial crops) (grant number 727459) E-RTA2015-00005-C06-01 (Métodos de control y contención de Trioza erytreae, vector del huanglongbing de los cítricos from Programa Estatal de I+D+I Orientada a los Retos de la Sociedad) Atribución-NoComercial-SinDerivadas 3.0 España http://creativecommons.org/licenses/by-nc-nd/3.0/es/ openAccess MDPI electronico |
| spellingShingle | undesired amplification 16S real-time PCR sequence identity greening HLB Trioza erytreae Tamarixia dryi H20 Plant diseases Bacterial flora Candidatus Liberibacter HLB PCR Morán, Félix Barbé, Silvia Bastin, Saskia Navarro-Herrero, Inmaculada Bertolini, Edson López, María M. Hernández-Suárez, Estrella Urbaneja, Alberto Tena, Alejandro Siverio, Felipe Marco-Noales, Ester The Challenge of Environmental Samples for PCR Detection of Phytopathogenic Bacteria: A Case Study of Citrus Huanglongbing Disease |
| title | The Challenge of Environmental Samples for PCR Detection of Phytopathogenic Bacteria: A Case Study of Citrus Huanglongbing Disease |
| title_full | The Challenge of Environmental Samples for PCR Detection of Phytopathogenic Bacteria: A Case Study of Citrus Huanglongbing Disease |
| title_fullStr | The Challenge of Environmental Samples for PCR Detection of Phytopathogenic Bacteria: A Case Study of Citrus Huanglongbing Disease |
| title_full_unstemmed | The Challenge of Environmental Samples for PCR Detection of Phytopathogenic Bacteria: A Case Study of Citrus Huanglongbing Disease |
| title_short | The Challenge of Environmental Samples for PCR Detection of Phytopathogenic Bacteria: A Case Study of Citrus Huanglongbing Disease |
| title_sort | challenge of environmental samples for pcr detection of phytopathogenic bacteria a case study of citrus huanglongbing disease |
| topic | undesired amplification 16S real-time PCR sequence identity greening HLB Trioza erytreae Tamarixia dryi H20 Plant diseases Bacterial flora Candidatus Liberibacter HLB PCR |
| url | http://hdl.handle.net/20.500.11939/6927 https://www.mdpi.com/2073-4395/11/1/10/htm |
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