Development of a qPCR and a Lamp Assay for Verticillium longisporum Detection and a Triplex qPCR Assay for Simultaneous Detection of V. longisporum, Leptosphaeria biglobosa, and L. maculans from Canola Samples
Verticillium wilt, Verticillium stem striping, and Verticillium stripe are common disease names that all denote infection caused by Verticillium longisporum on canola or other brassica crops. In this study, a quantitative PCR (qPCR) and a loop-mediated isothermal amplification (LAMP) assay were deve...
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The American Phytopathological Society
2024-12-01
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| Series: | PhytoFrontiers |
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| Online Access: | https://apsjournals.apsnet.org/doi/10.1094/PHYTOFR-03-24-0031-R |
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| author | Heting Fu Yalong Yang Junye Jiang Greg C. Daniels Blake Hill Shiming Xue Kher Zahr L. Stellar Michael W. Harding David Feindel Carol Bvindi W. G. Dilantha Fernando Lipu Wang Jie Feng |
| author_facet | Heting Fu Yalong Yang Junye Jiang Greg C. Daniels Blake Hill Shiming Xue Kher Zahr L. Stellar Michael W. Harding David Feindel Carol Bvindi W. G. Dilantha Fernando Lipu Wang Jie Feng |
| author_sort | Heting Fu |
| collection | DOAJ |
| description | Verticillium wilt, Verticillium stem striping, and Verticillium stripe are common disease names that all denote infection caused by Verticillium longisporum on canola or other brassica crops. In this study, a quantitative PCR (qPCR) and a loop-mediated isothermal amplification (LAMP) assay were developed for the detection of V. longisporum from canola stem samples. Both assays are specific to V. longisporum at the species level and ubiquitous at the strain level across all whole-genome sequenced strains. The low limit for positive detection of the two assays is 1 pg fungal DNA in a 20-µl reaction or 700 fungal cells in 100-mg plant tissue. The qPCR assay was combined with the duplex qPCR assay for the two blackleg pathogens, Leptosphaeria biglobosa and L. maculans, to constitute a triplex qPCR system for simultaneous detection of all three pathogens. The usefulness of this triplex qPCR system was verified on canola samples collected from various locations in Alberta, Canada. Using this triplex qPCR system, V. longisporum was detected from one sample, while the two blackleg pathogens were detected at higher frequencies. Since it is sometimes difficult to differentiate Verticillium stripe and blackleg on Alberta canola samples based on visual symptoms, the triplex qPCR system is an important tool for the detection of V. longisporum, especially when its presence is masked or obscured by symptoms of blackleg. [Figure: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license. |
| format | Article |
| id | doaj-art-f9d9433d7c1a420db8973f369fecb767 |
| institution | Kabale University |
| issn | 2690-5442 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | The American Phytopathological Society |
| record_format | Article |
| series | PhytoFrontiers |
| spelling | doaj-art-f9d9433d7c1a420db8973f369fecb7672024-12-24T21:19:08ZengThe American Phytopathological SocietyPhytoFrontiers2690-54422024-12-014464365010.1094/PHYTOFR-03-24-0031-RDevelopment of a qPCR and a Lamp Assay for Verticillium longisporum Detection and a Triplex qPCR Assay for Simultaneous Detection of V. longisporum, Leptosphaeria biglobosa, and L. maculans from Canola SamplesHeting Fu0Yalong Yang1Junye Jiang2Greg C. Daniels3Blake Hill4Shiming Xue5Kher Zahr6L. Stellar7Michael W. Harding8David Feindel9Carol Bvindi10W. G. Dilantha Fernando11Lipu Wang12Jie Feng13Alberta Plant Health Lab, Alberta Agriculture and Irrigation (AGI), Edmonton, AB, T5Y 6H3, CanadaAlberta Plant Health Lab, Alberta Agriculture and Irrigation (AGI), Edmonton, AB, T5Y 6H3, CanadaAlberta Plant Health Lab, Alberta Agriculture and Irrigation (AGI), Edmonton, AB, T5Y 6H3, CanadaCrop Diversification Centre South, AGI, Brooks, AB, T1R 1E6, CanadaCrop Diversification Centre South, AGI, Brooks, AB, T1R 1E6, CanadaAlberta Plant Health Lab, Alberta Agriculture and Irrigation (AGI), Edmonton, AB, T5Y 6H3, CanadaAlberta Plant Health Lab, Alberta Agriculture and Irrigation (AGI), Edmonton, AB, T5Y 6H3, CanadaCrop Diversification Centre South, AGI, Brooks, AB, T1R 1E6, CanadaCrop Diversification Centre South, AGI, Brooks, AB, T1R 1E6, CanadaAlberta Plant Health Lab, Alberta Agriculture and Irrigation (AGI), Edmonton, AB, T5Y 6H3, CanadaDepartment of Plant Science, University of Manitoba, Winnipeg, MB, R3T 2N2, CanadaDepartment of Plant Science, University of Manitoba, Winnipeg, MB, R3T 2N2, CanadaDepartment of Plant Sciences, University of Saskatchewan, Saskatoon, SK, S7N 5A8, CanadaAlberta Plant Health Lab, Alberta Agriculture and Irrigation (AGI), Edmonton, AB, T5Y 6H3, CanadaVerticillium wilt, Verticillium stem striping, and Verticillium stripe are common disease names that all denote infection caused by Verticillium longisporum on canola or other brassica crops. In this study, a quantitative PCR (qPCR) and a loop-mediated isothermal amplification (LAMP) assay were developed for the detection of V. longisporum from canola stem samples. Both assays are specific to V. longisporum at the species level and ubiquitous at the strain level across all whole-genome sequenced strains. The low limit for positive detection of the two assays is 1 pg fungal DNA in a 20-µl reaction or 700 fungal cells in 100-mg plant tissue. The qPCR assay was combined with the duplex qPCR assay for the two blackleg pathogens, Leptosphaeria biglobosa and L. maculans, to constitute a triplex qPCR system for simultaneous detection of all three pathogens. The usefulness of this triplex qPCR system was verified on canola samples collected from various locations in Alberta, Canada. Using this triplex qPCR system, V. longisporum was detected from one sample, while the two blackleg pathogens were detected at higher frequencies. Since it is sometimes difficult to differentiate Verticillium stripe and blackleg on Alberta canola samples based on visual symptoms, the triplex qPCR system is an important tool for the detection of V. longisporum, especially when its presence is masked or obscured by symptoms of blackleg. [Figure: see text] Copyright © 2024 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-03-24-0031-Rblacklegcanolamultiplex qPCRstripeVerticillium stem stripingwilt |
| spellingShingle | Heting Fu Yalong Yang Junye Jiang Greg C. Daniels Blake Hill Shiming Xue Kher Zahr L. Stellar Michael W. Harding David Feindel Carol Bvindi W. G. Dilantha Fernando Lipu Wang Jie Feng Development of a qPCR and a Lamp Assay for Verticillium longisporum Detection and a Triplex qPCR Assay for Simultaneous Detection of V. longisporum, Leptosphaeria biglobosa, and L. maculans from Canola Samples PhytoFrontiers blackleg canola multiplex qPCR stripe Verticillium stem striping wilt |
| title | Development of a qPCR and a Lamp Assay for Verticillium longisporum Detection and a Triplex qPCR Assay for Simultaneous Detection of V. longisporum, Leptosphaeria biglobosa, and L. maculans from Canola Samples |
| title_full | Development of a qPCR and a Lamp Assay for Verticillium longisporum Detection and a Triplex qPCR Assay for Simultaneous Detection of V. longisporum, Leptosphaeria biglobosa, and L. maculans from Canola Samples |
| title_fullStr | Development of a qPCR and a Lamp Assay for Verticillium longisporum Detection and a Triplex qPCR Assay for Simultaneous Detection of V. longisporum, Leptosphaeria biglobosa, and L. maculans from Canola Samples |
| title_full_unstemmed | Development of a qPCR and a Lamp Assay for Verticillium longisporum Detection and a Triplex qPCR Assay for Simultaneous Detection of V. longisporum, Leptosphaeria biglobosa, and L. maculans from Canola Samples |
| title_short | Development of a qPCR and a Lamp Assay for Verticillium longisporum Detection and a Triplex qPCR Assay for Simultaneous Detection of V. longisporum, Leptosphaeria biglobosa, and L. maculans from Canola Samples |
| title_sort | development of a qpcr and a lamp assay for verticillium longisporum detection and a triplex qpcr assay for simultaneous detection of v longisporum leptosphaeria biglobosa and l maculans from canola samples |
| topic | blackleg canola multiplex qPCR stripe Verticillium stem striping wilt |
| url | https://apsjournals.apsnet.org/doi/10.1094/PHYTOFR-03-24-0031-R |
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