High-Resolution Melting Analysis Enables Efficient Detection and Differentiation of Two Boxwood Blight Pathogens by qPCR Assays
Boxwood blight is a devastating disease caused by the fungal pathogens Calonectria henricotiae (Che) and C. pseudonaviculata (Cps). Identification and detection of these pathogens from infected plant material could play a significant role in breeding and selection of resistant cultivars and developm...
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| Format: | Article |
| Language: | English |
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The American Phytopathological Society
2022-09-01
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| Series: | PhytoFrontiers |
| Subjects: | |
| Online Access: | https://apsjournals.apsnet.org/doi/10.1094/PHYTOFR-09-21-0066-SC |
| _version_ | 1827095683718447104 |
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| author | Fred E. Gouker Yonghong Guo Margaret R. Pooler |
| author_facet | Fred E. Gouker Yonghong Guo Margaret R. Pooler |
| author_sort | Fred E. Gouker |
| collection | DOAJ |
| description | Boxwood blight is a devastating disease caused by the fungal pathogens Calonectria henricotiae (Che) and C. pseudonaviculata (Cps). Identification and detection of these pathogens from infected plant material could play a significant role in breeding and selection of resistant cultivars and development of disease management strategies in the ornamental nursery industry. We designed a simple, single-tube method for extraction of PCR-amplifiable DNA from boxwood leaves and cultures of the Calonectria pathogens. Previously developed fungal-specific primers based on histone and calmodulin regions were used to detect and distinguish between Che and Cps using real-time PCR and high-resolution melting (HRM) analysis, with discernable melting temperature differences of 0.5°C between amplified products. Here, we describe a single-tube acetone-based DNA extraction method and qPCR-HRM assay targeting single nucleotide polymorphisms within the calmodulin and histone H3 DNA regions as a fast and highly sensitive molecular method to detect and differentiate between Che and Cps species directly from plant tissue. [Figure: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license. |
| first_indexed | 2024-03-08T07:18:35Z |
| format | Article |
| id | doaj.art-85bbf92c77a74402be544be98bdb7cc4 |
| institution | Directory Open Access Journal |
| issn | 2690-5442 |
| language | English |
| last_indexed | 2025-03-20T07:03:47Z |
| publishDate | 2022-09-01 |
| publisher | The American Phytopathological Society |
| record_format | Article |
| series | PhytoFrontiers |
| spelling | doaj.art-85bbf92c77a74402be544be98bdb7cc42024-10-01T12:35:42ZengThe American Phytopathological SocietyPhytoFrontiers2690-54422022-09-012317618010.1094/PHYTOFR-09-21-0066-SCHigh-Resolution Melting Analysis Enables Efficient Detection and Differentiation of Two Boxwood Blight Pathogens by qPCR AssaysFred E. Gouker0Yonghong Guo1Margaret R. Pooler2U.S. Department of Agriculture, Agricultural Research Service, U.S. National Arboretum, Floral and Nursery Plants Research Unit, Beltsville, MD 20705U.S. Department of Agriculture, Agricultural Research Service, U.S. National Arboretum, Floral and Nursery Plants Research Unit, Beltsville, MD 20705U.S. Department of Agriculture, Agricultural Research Service, U.S. National Arboretum, Floral and Nursery Plants Research Unit, Beltsville, MD 20705Boxwood blight is a devastating disease caused by the fungal pathogens Calonectria henricotiae (Che) and C. pseudonaviculata (Cps). Identification and detection of these pathogens from infected plant material could play a significant role in breeding and selection of resistant cultivars and development of disease management strategies in the ornamental nursery industry. We designed a simple, single-tube method for extraction of PCR-amplifiable DNA from boxwood leaves and cultures of the Calonectria pathogens. Previously developed fungal-specific primers based on histone and calmodulin regions were used to detect and distinguish between Che and Cps using real-time PCR and high-resolution melting (HRM) analysis, with discernable melting temperature differences of 0.5°C between amplified products. Here, we describe a single-tube acetone-based DNA extraction method and qPCR-HRM assay targeting single nucleotide polymorphisms within the calmodulin and histone H3 DNA regions as a fast and highly sensitive molecular method to detect and differentiate between Che and Cps species directly from plant tissue. [Figure: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.https://apsjournals.apsnet.org/doi/10.1094/PHYTOFR-09-21-0066-SCboxwood blightBuxus spp.disease diagnosisdisease managementfungal pathogensHRM analysis |
| spellingShingle | Fred E. Gouker Yonghong Guo Margaret R. Pooler High-Resolution Melting Analysis Enables Efficient Detection and Differentiation of Two Boxwood Blight Pathogens by qPCR Assays PhytoFrontiers boxwood blight Buxus spp. disease diagnosis disease management fungal pathogens HRM analysis |
| title | High-Resolution Melting Analysis Enables Efficient Detection and Differentiation of Two Boxwood Blight Pathogens by qPCR Assays |
| title_full | High-Resolution Melting Analysis Enables Efficient Detection and Differentiation of Two Boxwood Blight Pathogens by qPCR Assays |
| title_fullStr | High-Resolution Melting Analysis Enables Efficient Detection and Differentiation of Two Boxwood Blight Pathogens by qPCR Assays |
| title_full_unstemmed | High-Resolution Melting Analysis Enables Efficient Detection and Differentiation of Two Boxwood Blight Pathogens by qPCR Assays |
| title_short | High-Resolution Melting Analysis Enables Efficient Detection and Differentiation of Two Boxwood Blight Pathogens by qPCR Assays |
| title_sort | high resolution melting analysis enables efficient detection and differentiation of two boxwood blight pathogens by qpcr assays |
| topic | boxwood blight Buxus spp. disease diagnosis disease management fungal pathogens HRM analysis |
| url | https://apsjournals.apsnet.org/doi/10.1094/PHYTOFR-09-21-0066-SC |
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