High-Sensitivity ITS Real-Time PCR Assays for Detection of Ceratocystis lukuohia and Ceratocystis huliohia in Soil and Air Samples
Rapid Ohia Death (ROD) is caused by two pathogens, Ceratocystis lukuohia and Ceratocystis huliohia. The established species-specific real-time PCR assays targeting the single-copy cerato-platanin gene require DNA from between 2 and 16 spores per reaction for consistent detection, which is suitable f...
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| Format: | Article |
| Language: | English |
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The American Phytopathological Society
2023-06-01
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| Series: | PhytoFrontiers |
| Subjects: | |
| Online Access: | https://apsjournals.apsnet.org/doi/10.1094/PHYTOFR-09-22-0091-FI |
| _version_ | 1797326966615965696 |
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| author | Wade P. Heller Thomas C. Harrington Eva Brill Lisa M. Keith |
| author_facet | Wade P. Heller Thomas C. Harrington Eva Brill Lisa M. Keith |
| author_sort | Wade P. Heller |
| collection | DOAJ |
| description | Rapid Ohia Death (ROD) is caused by two pathogens, Ceratocystis lukuohia and Ceratocystis huliohia. The established species-specific real-time PCR assays targeting the single-copy cerato-platanin gene require DNA from between 2 and 16 spores per reaction for consistent detection, which is suitable for analysis of infected plant tissue but not sensitive enough to consistently detect low spore loads in environmental samples. Here, we present two redesigned qPCR assays targeting the first internal transcribed spacer (ITS) region of the multi-copy ribosomal DNA operon from the respective species, both capable of consistent detection of the pathogen at concentrations as low as 1 fg per reaction, less than the size of the haploid genome. Due to this increased sensitivity, these ITS qPCR assays are superior for analysis of DNA extracted from material collected in airborne particle samplers and from soil. Here, we demonstrate the utility of these qPCR assays for the characterization of windblown and soilborne dispersal of the pathogens, which is critical for the development of management practices that mitigate disease spread. [Graphic: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023. |
| first_indexed | 2024-03-08T06:31:43Z |
| format | Article |
| id | doaj.art-d74968a132f24ad3a0c1291d6c49819d |
| institution | Directory Open Access Journal |
| issn | 2690-5442 |
| language | English |
| last_indexed | 2024-03-08T06:31:43Z |
| publishDate | 2023-06-01 |
| publisher | The American Phytopathological Society |
| record_format | Article |
| series | PhytoFrontiers |
| spelling | doaj.art-d74968a132f24ad3a0c1291d6c49819d2024-02-03T11:32:54ZengThe American Phytopathological SocietyPhytoFrontiers2690-54422023-06-013114815510.1094/PHYTOFR-09-22-0091-FIHigh-Sensitivity ITS Real-Time PCR Assays for Detection of Ceratocystis lukuohia and Ceratocystis huliohia in Soil and Air SamplesWade P. Heller0Thomas C. Harrington1Eva Brill2Lisa M. Keith3Eastern Regional Research Center, USDA Agricultural Research Service, Wyndmoor, PADepartment of Plant Pathology and Microbiology, Iowa State University, Ames, IACollege of Tropical Agriculture and Human Resources, University of Hawaii, Hilo, HIDaniel K. Inouye U.S. Pacific Basin Agricultural Research Center, USDA Agricultural Research Service, Hilo, HIRapid Ohia Death (ROD) is caused by two pathogens, Ceratocystis lukuohia and Ceratocystis huliohia. The established species-specific real-time PCR assays targeting the single-copy cerato-platanin gene require DNA from between 2 and 16 spores per reaction for consistent detection, which is suitable for analysis of infected plant tissue but not sensitive enough to consistently detect low spore loads in environmental samples. Here, we present two redesigned qPCR assays targeting the first internal transcribed spacer (ITS) region of the multi-copy ribosomal DNA operon from the respective species, both capable of consistent detection of the pathogen at concentrations as low as 1 fg per reaction, less than the size of the haploid genome. Due to this increased sensitivity, these ITS qPCR assays are superior for analysis of DNA extracted from material collected in airborne particle samplers and from soil. Here, we demonstrate the utility of these qPCR assays for the characterization of windblown and soilborne dispersal of the pathogens, which is critical for the development of management practices that mitigate disease spread. [Graphic: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.https://apsjournals.apsnet.org/doi/10.1094/PHYTOFR-09-22-0091-FIdispersalfrasssoilspore |
| spellingShingle | Wade P. Heller Thomas C. Harrington Eva Brill Lisa M. Keith High-Sensitivity ITS Real-Time PCR Assays for Detection of Ceratocystis lukuohia and Ceratocystis huliohia in Soil and Air Samples PhytoFrontiers dispersal frass soil spore |
| title | High-Sensitivity ITS Real-Time PCR Assays for Detection of Ceratocystis lukuohia and Ceratocystis huliohia in Soil and Air Samples |
| title_full | High-Sensitivity ITS Real-Time PCR Assays for Detection of Ceratocystis lukuohia and Ceratocystis huliohia in Soil and Air Samples |
| title_fullStr | High-Sensitivity ITS Real-Time PCR Assays for Detection of Ceratocystis lukuohia and Ceratocystis huliohia in Soil and Air Samples |
| title_full_unstemmed | High-Sensitivity ITS Real-Time PCR Assays for Detection of Ceratocystis lukuohia and Ceratocystis huliohia in Soil and Air Samples |
| title_short | High-Sensitivity ITS Real-Time PCR Assays for Detection of Ceratocystis lukuohia and Ceratocystis huliohia in Soil and Air Samples |
| title_sort | high sensitivity its real time pcr assays for detection of ceratocystis lukuohia and ceratocystis huliohia in soil and air samples |
| topic | dispersal frass soil spore |
| url | https://apsjournals.apsnet.org/doi/10.1094/PHYTOFR-09-22-0091-FI |
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