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Development of a PacBio Long-Read Sequencing Assay for High Throughput Detection of Fungicide Resistance in Zymoseptoria tritici

Fungicide resistance has become a challenging problem in management of Septoria tritici blotch (STB), caused by Zymoseptoria tritici, the most destructive disease of winter wheat throughout western and northern Europe. To ensure the continued effectiveness of those fungicides currently used, it is e...

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Autores principales: Samils, Berit, Andersson, Björn, Edin, Eva, Elfstrand, Malin, Rönneburg, Tilman, Bucur, Diana, Hutton, Fiona, Heick, Thies M., Hellin, Pierre, Kildea, Steven
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8256687/
https://www.ncbi.nlm.nih.gov/pubmed/34234765
http://dx.doi.org/10.3389/fmicb.2021.692845
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author Samils, Berit
Andersson, Björn
Edin, Eva
Elfstrand, Malin
Rönneburg, Tilman
Bucur, Diana
Hutton, Fiona
Heick, Thies M.
Hellin, Pierre
Kildea, Steven
author_facet Samils, Berit
Andersson, Björn
Edin, Eva
Elfstrand, Malin
Rönneburg, Tilman
Bucur, Diana
Hutton, Fiona
Heick, Thies M.
Hellin, Pierre
Kildea, Steven
author_sort Samils, Berit
collection PubMed
description Fungicide resistance has become a challenging problem in management of Septoria tritici blotch (STB), caused by Zymoseptoria tritici, the most destructive disease of winter wheat throughout western and northern Europe. To ensure the continued effectiveness of those fungicides currently used, it is essential to monitor the development and spread of such resistance in field populations of the pathogen. Since resistance to the key families of fungicides used for STB control (demethyalation inhibitors or azoles, succinate dehydrogenase inhibitors or SDHIs and Quinone outside Inhibitors or QoIs) is conferred through target-site mutations, the potential exists to monitor resistance through the molecular detection of alterations in the target site genes. As more efficient fungicides were developed and applied, the pathogen has continuously adapted through accumulating multiple target-site alterations. In order to accurately monitor these changes in field populations, it is therefore becoming increasingly important to completely sequence the targeted genes. Here we report the development of a PacBio assay that facilitates the multiplex amplification and long-read sequencing of the target gene(s) for the azole (CYP51), SDHI (Sdh B, C, and D), and QoI (cytochrome b) fungicides. The assay was developed and optimised using three Irish Z. tritici collections established in spring 2017, which capture the range of fungicide resistance present in modern European populations of Z. tritici. The sequences obtained through the PacBio assay were validated using traditional Sanger sequencing and in vitro sensitivity screenings. To further exploit the long-read and high throughput potential of PacBio sequencing, an additional nine housekeeping genes (act, BTUB, cal, cyp, EF1, GAPDH, hsp80-1, PKC, TFC1) were sequenced and used to provide comprehensive Z. tritici strain genotyping.
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spelling pubmed-82566872021-07-06 Development of a PacBio Long-Read Sequencing Assay for High Throughput Detection of Fungicide Resistance in Zymoseptoria tritici Samils, Berit Andersson, Björn Edin, Eva Elfstrand, Malin Rönneburg, Tilman Bucur, Diana Hutton, Fiona Heick, Thies M. Hellin, Pierre Kildea, Steven Front Microbiol Microbiology Fungicide resistance has become a challenging problem in management of Septoria tritici blotch (STB), caused by Zymoseptoria tritici, the most destructive disease of winter wheat throughout western and northern Europe. To ensure the continued effectiveness of those fungicides currently used, it is essential to monitor the development and spread of such resistance in field populations of the pathogen. Since resistance to the key families of fungicides used for STB control (demethyalation inhibitors or azoles, succinate dehydrogenase inhibitors or SDHIs and Quinone outside Inhibitors or QoIs) is conferred through target-site mutations, the potential exists to monitor resistance through the molecular detection of alterations in the target site genes. As more efficient fungicides were developed and applied, the pathogen has continuously adapted through accumulating multiple target-site alterations. In order to accurately monitor these changes in field populations, it is therefore becoming increasingly important to completely sequence the targeted genes. Here we report the development of a PacBio assay that facilitates the multiplex amplification and long-read sequencing of the target gene(s) for the azole (CYP51), SDHI (Sdh B, C, and D), and QoI (cytochrome b) fungicides. The assay was developed and optimised using three Irish Z. tritici collections established in spring 2017, which capture the range of fungicide resistance present in modern European populations of Z. tritici. The sequences obtained through the PacBio assay were validated using traditional Sanger sequencing and in vitro sensitivity screenings. To further exploit the long-read and high throughput potential of PacBio sequencing, an additional nine housekeeping genes (act, BTUB, cal, cyp, EF1, GAPDH, hsp80-1, PKC, TFC1) were sequenced and used to provide comprehensive Z. tritici strain genotyping. Frontiers Media S.A. 2021-06-18 /pmc/articles/PMC8256687/ /pubmed/34234765 http://dx.doi.org/10.3389/fmicb.2021.692845 Text en Copyright © 2021 Samils, Andersson, Edin, Elfstrand, Rönneburg, Bucur, Hutton, Heick, Hellin and Kildea. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Samils, Berit
Andersson, Björn
Edin, Eva
Elfstrand, Malin
Rönneburg, Tilman
Bucur, Diana
Hutton, Fiona
Heick, Thies M.
Hellin, Pierre
Kildea, Steven
Development of a PacBio Long-Read Sequencing Assay for High Throughput Detection of Fungicide Resistance in Zymoseptoria tritici
title Development of a PacBio Long-Read Sequencing Assay for High Throughput Detection of Fungicide Resistance in Zymoseptoria tritici
title_full Development of a PacBio Long-Read Sequencing Assay for High Throughput Detection of Fungicide Resistance in Zymoseptoria tritici
title_fullStr Development of a PacBio Long-Read Sequencing Assay for High Throughput Detection of Fungicide Resistance in Zymoseptoria tritici
title_full_unstemmed Development of a PacBio Long-Read Sequencing Assay for High Throughput Detection of Fungicide Resistance in Zymoseptoria tritici
title_short Development of a PacBio Long-Read Sequencing Assay for High Throughput Detection of Fungicide Resistance in Zymoseptoria tritici
title_sort development of a pacbio long-read sequencing assay for high throughput detection of fungicide resistance in zymoseptoria tritici
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8256687/
https://www.ncbi.nlm.nih.gov/pubmed/34234765
http://dx.doi.org/10.3389/fmicb.2021.692845
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