Molecular and experimental evidence of multi-resistance of Cercospora beticola field populations to MBC, DMI and QoI fungicides

Trkulja, Nenad; Milosavljević, Anja; Mitrović, Milana; Jović, Jelena; Toševski, Ivo; Khan, Mohamed F. R.; Secor, Gary A.

doi:10.1007/s10658-017-1239-0

Cited by 20 publications

(22 citation statements)

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“…Intriguingly, we identified a silent mutation (E170) associated with DMI resistance in our study. Obuya et al (2015) also associated this mutation with DMI resistance using RRV isolates, and it was also previously associated with resistance in C. beticola in isolates from Greece (Nikou et al 2009) and Serbia (Trkulja et al 2017). Obuya et al (2015) heterologously expressed a C. beticola CYP51 haplotype harboring E170 in S. cerevisiae strain R1 lacking multidrug resistance transporter Pdr5 (ΔPdr-5) and found no change in DMI sensitivity.…”

Section: Discussionmentioning

confidence: 66%

“…There was a stronger positive correlation between the allelic effects for tetraconazole sensitivity and radial growth under 1M NaCl salt stress (B) (coefficient = 0.3218). CbCYP51 induced overexpression gives rise to resistance (A) (Nikou et al 2009, Bolton et al 2012, as well as (B) amino acid substitutions in CbCYP51, leading to weakened binding and inhibition by DMIs (Trkulja et al 2017, Shrestha et al 2020. Multidrug transporters such as the ABC PDR transporter identified in this study may be pumping DMIs out across the membrane in a non-specific manner (C).…”

Section: Cas9 Rnp-mediated Gene Editingmentioning

confidence: 95%

“…However, Obuya et al (2015) could not demonstrate its involvement in DMI resistance through heterologous expression in yeast. Amino acid substitutions L144F, I387M and Y464S in CbCYP51 appeared to coincide with DMI resistance in C. beticola isolates from Serbia (Trkulja et al 2017) but overall, it has been difficult to clearly associate any CbCYP51 haplotype with resistance (Bolton et al 2012a; Trkulja et al 2017). Factors including small sample size may play a significant role in the interpretation of results in such studies.…”

Section: Introductionmentioning

confidence: 99%

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Genome-wide association studies reveal the complex genetic architecture of DMI fungicide resistance inCercospora beticola

Spanner

Taliadoros

Richards

et al. 2020

Preprint

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Cercospora leaf spot is the most important disease of sugar beet worldwide. The disease is caused by the fungus Cercospora beticola and is managed principally by timely application of fungicides including those of the sterol demethylation inhibitor (DMI) class. However, reliance on DMIs has caused an increase in resistance to this class of fungicides in multiple C. beticola populations. To better understand the genetic and evolutionary basis for resistance in C. beticola, a genome-wide association study (GWAS) and selective sweep analysis were conducted for the first time in this fungal plant pathogen. We performed whole genome resequencing of 190 C. beticola isolates predominantly from North Dakota and Minnesota that were phenotyped for sensitivity to tetraconazole, the most widely used DMI fungicide in this region. GWAS identified mutations in genes associated with DMI fungicide resistance including a Regulator of G-protein Signaling (RGS) protein, an ATP-binding cassette (ABC) pleiotropic drug resistance transporter, a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK), and a gene annotated as a hypothetical protein. A SNP upstream of CbCYP51, the gene encoding the target of DMI fungicides, was also identified via GWAS. Haplotype analysis of CbCYP51 identified a synonymous mutation (E170) in high linkage disequilibrium with the upstream SNP, and multiple non-synonymous mutations (L144F, I387M and Y464S) associated with DMI resistance. Additionally, a putative codon bias effect for the L144F substitution was identified that generated different resistance potentials. We also identified a CbCYP51 paralog in C. beticola, CbCYP51-like, with high protein homology to CYP51C found uniquely in Fusarium species but CbCYP51-like does not appear to influence DMI sensitivity. Genome-wide scans of selection showed that several of the GWAS mutations for fungicide resistance resided in regions that have recently undergone a selective sweep. Using radial plate growth on selected media as a fitness proxy, we did not find a trade-off associated with DMI fungicide resistance suggesting that resistance mutations can persist in C. beticola populations. Taken together, we show that population genomic data from a crop pathogen can allow the identification of mutations conferring fungicide resistance and inform about their origins in the pathogen population.

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Section: Discussionmentioning

confidence: 66%

Section: Cas9 Rnp-mediated Gene Editingmentioning

confidence: 95%

Section: Introductionmentioning

confidence: 99%

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Genome-wide association studies reveal the complex genetic architecture of DMI fungicide resistance inCercospora beticola

Spanner

Taliadoros

Richards

et al. 2020

Preprint

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“…Interestingly, a silent mutation at codon 170 has been identified to be present only in highly resistant C. beticola isolates (EC 50 values >50 ppm) from northern Greece (Nikou et al, 2009) and the RRV region of the United States (EC 50 values >20 ppm) (Obuya et al, 2015). Recently, nonsynonymous polymorphisms in CbCYP51 have also been discovered that appear to be linked to DMI resistance (Trkulja et al, 2017;Shrestha et al, 2020). The amino acid substitutions L144F, I309T, I387M, and Y464S in isolates from the RRV region of the United States (Spanner and Bolton, unpublished data) are all associated with DMI EC 50 values >1 ppm.…”

Section: Frac Groupmentioning

confidence: 99%

“…QoI-resistant isolates of C. beticola found to date have the substitution of glycine by alanine at codon 143 (designated G143A) (Birla et al, 2012;Bolton et al, 2013;Trkulja et al, 2017;Piszczek et al, 2018).…”

Section: Frac Group 11mentioning

confidence: 99%

Cercospora beticola: The intoxicating lifestyle of the leaf spot pathogen of sugar beet

Rangel

Spanner

Ebert

et al. 2020

Molecular Plant Pathology

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Evidence for the association of target‐site resistance in cyp51 with reduced DMI sensitivity in EuropeanCercospora beticola field isolates

Muellender¹,

Mahlein²,

Stammler³

et al. 2020

Pest Management Science

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BACKGROUND Cercospora leaf spot caused by Cercospora beticola is the most relevant foliar disease in sugar beet cultivation. In the last decade a decreasing sensitivity of C. beticola towards demethylation inhibitors (DMIs) occurred. Different mechanisms mediating a reduced sensitivity towards DMIs have been identified in different plant pathogens to date, such as target site mutations, over‐expression or active excretion of the fungicide. RESULTS A sequencing of the cytochrome P450‐dependent sterol 14α‐demethylase gene sequence (cyp51) of diverse C. beticola isolates collected in different European countries with reduced DMI sensitivity was performed in order to find a possible correlation of mutations with higher EC50 values. The amino acid alterations Y464S, L144F and I309T combined with L144F were found to be associated with a reduced sensitivity. Furthermore, mutations I387M, M145W and M145W with E460Q were found uniquely. Additionally, constitutive and fungicide triggered expression of cyp51 was assayed by means of RT‐qPCR. A very strong induction of cyp51 mRNA expression in sensitive isolates suggests that the fungal cells upregulate expression to maintain ergosterol biosynthesis in DMI presence. The less intensive cyp51 induction in isolates with higher EC50 values underlines the possible correlation of the found target‐site mutations with reduced sensitivity. CONCLUSION This study provides new results about possible alterations in the target gene mediating reduced sensitivity of C. beticola towards DMIs and hypothesized a fungicide induced over‐expression of the target enzyme CYP51 as natural reaction of the fungus to fungicide application. © 2020 Society of Chemical Industry

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Molecular and experimental evidence of multi-resistance of Cercospora beticola field populations to MBC, DMI and QoI fungicides

Cited by 20 publications

References 44 publications

Genome-wide association studies reveal the complex genetic architecture of DMI fungicide resistance inCercospora beticola

Genome-wide association studies reveal the complex genetic architecture of DMI fungicide resistance inCercospora beticola

Cercospora beticola: The intoxicating lifestyle of the leaf spot pathogen of sugar beet

Evidence for the association of target‐site resistance in cyp51 with reduced DMI sensitivity in EuropeanCercospora beticola field isolates

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