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

Samils, Berit and Andersson, Björn and Edin, Eva and Elfstrand, Malin and Rönneburg, Tilman and Bucur, Diana and Hutton, Fiona and Heick, Thies M. and Hellin, Pierre and Kildea, Steven (2021). Development of a PacBio Long-Read Sequencing Assay for High Throughput Detection of Fungicide Resistance in Zymoseptoria tritici. Frontiers in Microbiology. 12 , 692845
[Research article]

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Abstract

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.

Authors/Creators:Samils, Berit and Andersson, Björn and Edin, Eva and Elfstrand, Malin and Rönneburg, Tilman and Bucur, Diana and Hutton, Fiona and Heick, Thies M. and Hellin, Pierre and Kildea, Steven
Title:Development of a PacBio Long-Read Sequencing Assay for High Throughput Detection of Fungicide Resistance in Zymoseptoria tritici
Series Name/Journal:Frontiers in Microbiology
Year of publishing :2021
Volume:12
Article number:692845
Number of Pages:13
Publisher:FRONTIERS MEDIA SA
ISSN:1664-302X
Language:English
Publication Type:Research article
Article category:Scientific peer reviewed
Version:Published version
Copyright:Creative Commons: Attribution 4.0
Full Text Status:Public
Subjects:(A) Swedish standard research categories 2011 > 1 Natural sciences > 106 Biological Sciences (Medical to be 3 and Agricultural to be 4) > Microbiology (Microbiology in the medical area to be 30109)
Keywords:azole, SDHI, QoI, multiplex PCR, housekeeping genes, SNP markers, SSR markers
URN:NBN:urn:nbn:se:slu:epsilon-p-113115
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-p-113115
Additional ID:
Type of IDID
DOI10.3389/fmicb.2021.692845
Web of Science (WoS)000669587700001
ID Code:25069
Faculty:S - Faculty of Forest Sciences
NJ - Fakulteten för naturresurser och jordbruksvetenskap
Department:(NL, NJ) > Dept. of Forest Mycology and Plant Pathology
(S) > Dept. of Forest Mycology and Plant Pathology
Deposited By: SLUpub Connector
Deposited On:27 Aug 2021 08:25
Metadata Last Modified:27 Aug 2021 08:31

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