Assessment of fungicide resistance and pathogen diversity in <i>Erysiphe necator</i> using quantitative real‐time PCR assays

Dufour, Marie‐Cécile; Fontaine, Séverine; Montarry, Josselin; Corio‐Costet, Marie‐France

doi:10.1002/ps.2032

Cited by 65 publications

(54 citation statements)

References 45 publications

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“…However, it has been suggested that such chemical fungicides represent a potential risk to the environment and to human health, with the additional problem of generating fungal resistance (Bardas et al. 2010; Dufour et al. 2011).…”

Section: Introductionmentioning

confidence: 99%

Changes in the Functionality of Plasma Membrane of Rhizopus stolonifer by Addition of Chitosan

Hernández-Lauzardo

Vega-Pérez

Valle

et al. 2011

Journal of Phytopathology

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The effect of chitosan (2 mg ⁄ ml) on the functionality of the plasma membrane of the Rhizopus stolonifer was studied. This study focuses on the changes in the integrity of the plasma membrane, external minimum medium pH, membrane potential, potassium efflux and determination of membrane phospholipids and proteins and of the H + -ATPase enzymatic kinetic activity. The results demonstrated that the membrane integrity diminishes gradually during 6 h of incubation, that there was no change in the external minimum medium pH and that the spores treated with chitosan showed the lowest membrane potential compared with the control. The results also revealed an increase of five times of potassium efflux by the addition of chitosan. There were no significant observed differences in the content of total phospholipids in both treatments. However, protein content was reduced approximately 40% and total H + -ATPase activity decreased 52% in the presence of chitosan. Chitosan treatments diminished the kinetic parameters (Vmax and Km) of the H + -ATPase activity. The damage to the plasma membrane of R. stolonifer by the presence of chitosan alters the H + -ATPase, affecting the physiological and metabolic functions of this phytopathogen fungus.

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Section: Introductionmentioning

confidence: 99%

Changes in the Functionality of Plasma Membrane of Rhizopus stolonifer by Addition of Chitosan

Hernández-Lauzardo

Vega-Pérez

Valle

et al. 2011

Journal of Phytopathology

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“…The resistance phenotype of these isolates was not determined, but in a separate study, 17 out of 18 isolates with Y136F had higher resistance factors (median RF = 27) to one or more DMIs than all isolates lacking the mutation (median RF = 1) (L. Rallos, un published data). The occurrence of double resistance to DMI and Qol in E. necator was earlier reported in Europe (14), and DMIresistant isolates of Monilinia fructicola have been found to be predisposed to accelerated development of Qol resistance (mech anism not investigated) (32). These phenomena can limit the use of DMIs and Qols as rotational or tank mix partners.…”

Section: Discussionmentioning

confidence: 93%

Fitness ofErysiphe necatorwith G143A-Based Resistance to Quinone Outside Inhibitors

et al. 2014

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Rallos, L. E. E., Johnson, N. G., Schmale, D. G., Ill, Prussin, A. J., II, and Baudoin, A. B. 2014. Fitness of Erysiphe necator with G143A-based resistance to quinone outside inhibitors. Plant Dis. 98:1494-1502.Management of grape powdery mildew (Erysiphe necator) using qui none outside inhibitors (Qols) has eroded in an increasing number of regions due to resistance development. To determine persistence of re sistance when Qols are withdrawn, competition assays were conducted on unsprayed grape plants (Vitis vinifera 'Chardonnay') by cycling mixtures of resistant and sensitive isolates characterized as genetically diverse based on microsatellite analyses. Under laboratory conditions, %G143A, quantified by quantitative polymerase chain reaction (qPCR), increased significantly, indicating competitiveness of the resistant fraction. To confirm competitiveness in the field, trials using potted plants were conducted. Percent G143A tended to decrease in one growing season, probably due to spore migration and mixing of populations with natural background inoculum. In a second season, Qol resistance persisted at high frequency for 4 weeks. Resistant popu lations were also found to persist in one vineyard without Qol applica tion for four consecutive years. The frequency was still about 25% in the fourth year, with higher frequency (36%) in a hotspot section. Qolresistant populations with >5% G143A also harbored Y136F in the cyp51 gene that confers some resistance to sterol demethylation inhibi tors, another fungicide class for powdery mildew control. Double re sistance could have been partly responsible for persistence of Qol resistance at this location.

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“…; Dufour et al . ) and (ii) the developed assay is based on genetic differences between alleles of the Avr3a gene, the proposed quantitative diagnostic tool can be efficiently used to screen virulent and avirulent natural P. infestans populations.…”

Section: Introductionmentioning

confidence: 99%

Specific detection and quantification of virulent/avirulent Phytophthora infestans isolates using a real-time PCR assay that targets polymorphisms of the Avr3a gene

Clément

Baldwin

Magalon

et al. 2013

Lett Appl Microbiol

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Significance and Impact of the Study: This tool clearly improves previously published methods of specific real-time quantitative PCR for Phytophthora infestans by allowing the intraspecific detection and quantification of Avr3a/avr3a genotypes. This reliable and sensitive assay has enabled to assess zoospore production as a fitness proxy and thus offers new opportunities for future researches on P. infestans/ host quantitative interactions. Abstract Molecular tools that allow intraspecific quantification and discrimination of pathogen isolates are useful to assess fitness of competitors during mixed infections. However, methods that were developed for quantifying Phytophthora infestans are only specific at the species level. Here, we reported a TaqMan-based real-time PCR assay allowing, according to the specificity of the used probes, an accurate quantification of different proportions of two genetically distinct clones of P. infestans in mixed fractions. Indeed, in addition to a primer specific to P. infestans, two primers and two TaqMan â probes that target single-nucleotide polymorphisms located in the Avr3a/avr3a virulence gene sequence were designed. The reliability of the method was tested on serially diluted fractions containing plasmid DNA with either the Avr3a or the avr3a sequences at concentrations ranging from 10 2 to 10 8 copies per ll. Based on its specificity, sensitivity and repeatability, the proposed assay allowed a quantification of the targeted DNA sequence in fractions with a Avr3a/avr3a ratio in the range 1/99 to 99/1. The reliability of the test was also checked for counting zoospores. Applications for future research in P. infestans/host quantitative interactions were also discussed.

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Assessment of fungicide resistance and pathogen diversity in Erysiphe necator using quantitative real‐time PCR assays

Abstract: The real-time PCR assay developed in this study provides a potentially useful tool for efficient quantification of different alleles of interest for fungicide monitoring and for population structure of E. necator.

Cited by 65 publications

References 45 publications

Changes in the Functionality of Plasma Membrane of Rhizopus stolonifer by Addition of Chitosan

Changes in the Functionality of Plasma Membrane of Rhizopus stolonifer by Addition of Chitosan

Fitness ofErysiphe necatorwith G143A-Based Resistance to Quinone Outside Inhibitors

Specific detection and quantification of virulent/avirulent Phytophthora infestans isolates using a real-time PCR assay that targets polymorphisms of the Avr3a gene

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