Use of PCR-RFLP Analysis to Monitor Fungicide Resistance in<i>Cercospora beticola</i>Populations from Sugarbeet (<i>Beta vulgaris</i>) in Michigan, United States

Rosenzweig, N.; Hanson, Linda E.; Clark, Gregory M.; Franc, G. D.; Stump, William L.; Jiang, Q. W.; Ja, Stewart; Kirk, W. W.

doi:10.1094/pdis-03-14-0241-re

Cited by 26 publications

(7 citation statements)

References 40 publications

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“…Sanger DNA sequencing can be used to detect single nucleotide polymorphisms (SNPs), however, it can be expensive for multiple samples and there is a time delay to receive results. Allele-specific PCR ( Aoki et al, 2011 ), PCR-cleaved amplified polymorphic sequences (CAPS; Lesemann et al, 2006 ), and PCR-restriction fragment length polymorphism (PCR-RFLP; Rosenzweig et al, 2014 ) have all been used for rapid detection of fungicide resistance but are not quantitative. Real-time PCR assays have also been used to quantify fungicide resistance in B. graminis f. sp.…”

Section: Introductionmentioning

confidence: 99%

Improved Detection and Monitoring of Fungicide Resistance in Blumeria graminis f. sp. hordei With High-Throughput Genotype Quantification by Digital PCR

et al. 2018

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The increased occurrence of triazole fungicide resistant strains of Blumeria graminis f. sp. hordei (Bgh) is an economic concern for the barley industry in Australia and elsewhere. High levels of resistance to triazoles in the field are caused by two separate point mutations in the Cyp51 gene, Y136F and S509T. Early detection of these mutations arising in pathogen field populations is important as this allows time for changes in fungicide practices to be adopted, thus mitigating potential yield losses due to fungicide failure and preventing the resistance from becoming dominant. A digital PCR (dPCR) assay has been developed for the detection and quantification of the Y136F and S509T mutations in the Bgh Cyp51 gene. Mutation levels were quantifiable as low as 0.2% in genomic DNA extractions and field samples. This assay was applied to the high throughput screening of Bgh field and bait trial samples from barley growing regions across Australia in the 2015 and 2016 growing seasons and identified the S509T mutation for the first time in the Eastern states of Australia. This is the first report on the use of digital PCR technology for fungicide resistance detection and monitoring in agriculture. Here we describe the potential application of dPCR for the screening of fungicide resistance mutations in a network of specifically designed bait trials. The combination of these two tools constitute an early warning system for the development of fungicide resistance that allows for the timely adjustment of management practices.

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Section: Introductionmentioning

confidence: 99%

Improved Detection and Monitoring of Fungicide Resistance in Blumeria graminis f. sp. hordei With High-Throughput Genotype Quantification by Digital PCR

et al. 2018

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“…Benzimidazole fungicides target and inhibit beta-tubulin in fungi, and the E198A target site mutation is known to confer high levels of resistance in C. beticola (Davidson et al 2006; Rosenzweig et al 2015; Shrestha et al 2020; Trkulja et al 2013). Therefore, we initially introduced the E198A beta-tubulin mutation into a benzimidazole-sensitive strain 16-1124 to establish a working gene editing method, which resulted in a benzimidazole-resistant phenotype (Fig.…”

Section: Resultsmentioning

confidence: 99%

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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“…In this research, we indicated that these techniques are highly specific and prompt for detecting fungicide responsiveness. Rosenzweig et al (2015) developed PCR-RFLP to detect the G143A and E198A point mutations of the cytochrome b and β-tubulin genes in Cercospora leaf spot (CLS) caused by Cercospora beticola in Michigan sugar beet (Beta vulgaris) fields. The results showed that the PCR-RFLP assay was effective in detecting the presence of the G143A and E198A mutations in C. beticola from field populations in Michigan.…”

Section: Discussionmentioning

confidence: 99%

Colletotrichum Species Related To “Nam Dork Mai See Tong” Mango In Central Thailand And Risk Detection of Carbendazim-Resistant Strains

Bincader

Pongpisutta

Rattanakreetakul

2021

Preprint

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Nam Dork Mai See Tong mango is a deluxe commercial fruit in Thailand, however anthracnose disease becoming a major problem affecting market-driven. Fungicide treatment is the main component in the mango anthracnose management, notwithstanding the appearance of fungal-resistance has been become a factor of fungicide limition and can affect to increasingly higher costs. Thirty-two isolates of the Colletotrichum species complex were obtained from anthracnose-diseased mango cv. Nam Dork Mai See Tong in 3 provinces of Thailand. Fungal identification based on morphological and molecular markers was investigated. All isolates were divided into 3 species: C. asianum , C. gloeosporioides and C. siamense . Pathogenicity tests revealed that all 3 species caused symptoms on artificially unwounded mango fruits and leaves. Only C. gloeosporioides have previously been reported on mango, while C. asianum and C. siamense were first reports associated with mango in Thailand. The responsiveness of Colletotrichum isolates to carbendazim was evaluated using an MIC assay. Nine isolates of C. asianum were highly resistant, while 5 and 8 isolates of C. gloeosporioides were resistant (R) and highly resistant (HR), respectively. Moreover, all isolates of C. siamense were HR. Mutations were detected by PCR of a partial sequence of the β-tubulin gene. The sequence of β-tubulin gene in HR strains showed a single nucleotide transversion of adenine to cytosine, resulting in a substitution at codon 198. However, R strains were found to have only an amino acid change at codon 200. Additionally, PCR-RFLP was applied as a rapid technique to detect carbendazim resistance. The results demonstrated that only the Bsh 1236I restriction enzyme generated two bands (200 and 300 bp) in the highly resistant strain, but these bands were absent in the sensitive (S) and R strains. Hence, PCR-RFLP technique showed the potential to specifically detect benzimidazole fungicide resistance in 3 species of Colletotrichum. Moreover, this technique is accessible and feasible for laboratory assessment

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Use of PCR-RFLP Analysis to Monitor Fungicide Resistance inCercospora beticolaPopulations from Sugarbeet (Beta vulgaris) in Michigan, United States

Cited by 26 publications

References 40 publications

Improved Detection and Monitoring of Fungicide Resistance in Blumeria graminis f. sp. hordei With High-Throughput Genotype Quantification by Digital PCR

Improved Detection and Monitoring of Fungicide Resistance in Blumeria graminis f. sp. hordei With High-Throughput Genotype Quantification by Digital PCR

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

Colletotrichum Species Related To “Nam Dork Mai See Tong” Mango In Central Thailand And Risk Detection of Carbendazim-Resistant Strains

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