Expert Opinion: Powdery Mildew Qualitative vs Quantitative Resistance by Dr. Walt Wahaffe, USDA/ARS, Corvallis, OR
American Vineyard Foundation (AVF) funded research found that in Washington, Oregon and California, over 90% powdery mildew samples tested were found to be resistant to QoI (FRAC Class 11) and about 60% were resistant to both QOI and DMI (FRAC Class 3) (2017 data). The QoI resistance is attributed to a single point glycine to alanine mutation at position 143 of cytochrome b gene (G143A). Determining DMI resistance is more complex although a change from tyrosine to phenylalanine in the cytochrome 450 gene (Y136F) is associated with higher tolerance to DMI fungicides.
What is Qualitative Resistance? It is like a light switch on or off. For resistance to quinone outside inhibitor (QoI) fungicides (FRAC Group 11), the identification of the G143A mutation in Cytochrome b indicates that QoI resistance is present. This is a classic case of qualitative resistance where resistance comes from the modification of a single major gene (Cytochrome b), Erysiphe necator subpopulations are either sensitive or fully resistant to the fungicide. The FRAME Research team has only found the G143A in the CYTb gene from over 400 samples sequenced. Unlike Rallos et al (ref 1 below), we have not found any indication of heteroplasmy (two different mitochondria genomes) in a conidium or isolate.
For resistance to succinate dehydrogenase inhibitors (SDHI) fungicide (FRAC Group 7), like Cherrad et al (ref 2 below), the FRAME Research team has only found H242R mutation in the succinate dehydrogenase gene (SDH) A gene to be associated with Boscalid resistance. We have found I244V in SDH B gene to be associated with Boscalid and fluopyram resistance. We have also detected several other mutations in the population but have not gotten isolates with those mutations to confirm if they are associated with resistance. We know that G25R in the SDH C gene is not associated with resistance. Our SDHI work so far points to the possibility that SDHI resistance is not likely to have complete cross resistance within the FRAC class 7 and that it might be qualitative. I have to stress that we have not seen or heard of field control failure with SDHIs.
What is Quantitative Resistance? It is like getting a suntan. A slow progression in tolerating higher and higher dose or the gradual erosion of disease control over time. For resistance to DeMethylation Inhibitors (DMI) fungicide (FRAC group 3), like Rallos et al (ref 1), the FRAME Research team has found Y136F associated with most resistant isolates. There does seem to be a correlation of copy number of Y136F to DMI tolerance level. However, we have isolates that only have Y136F and were sensitive in our bioassay. They have a low copy number of the CYP51 gene. We also have isolates that do not have any known mutation and are highly tolerant. In this case, the isolates have higher copy number of CYP51. Combining our results with Rallos et al, it would appear that there are three mechanisms for DMI resistance. We have evidence of CYP51 mutation copy number being important and evidence that CYP51 copy number without mutation is also affecting tolerance. Our conclusion so far is that an isolate with high copy number of CYP51 with Y136F mutation is 99% likely to be highly tolerant. For practical purposes, Y136F is likely an indicator to be careful with DMIs. But the presence of the Y136F mutation alone does not necessarily not mean field failure is eminent . It is an indication that DMIs should not be the sole active ingredient used.
In summary, the identification of G143A mutation in CYTB is associated with qualitative resistance to QoI. Y136F mutation in CYP51 gene is likely an indicator for caution with DMIs but not the only indicator. We are still trying to figure out how to determine SDHI resistance.
1. Rallos LE, et al. Co-Occurrence of Two Allelic Variants of CYP51 in Erysiphe necator and Their Correlation with Over-Expression for DMI Resistance. PLoS One. 2016.
Abstract. Demethylation inhibitors (DMIs) have been an important tool in the management of grapevine powdery mildew caused by Erysiphe necator. Long-term, intensive use of DMIs has resulted in reduced sensitivity in field populations. To further characterize DMI resistance and understand resistance mechanisms in this pathogen, we investigated the cyp51 sequence of 24 single-spored isolates from Virginia and surrounding states and analyzed gene expression in isolates representing a wide range of sensitivity. Two cyp51 alleles were found with respect to the 136th codon of the predicted EnCYP51 sequence: the wild-type (TAT) and the mutant (TTT), which results in the known Y136F amino acid change. Some isolates possessed both alleles, demonstrating gene duplication or increased gene copy number and possibly a requirement for at least one mutant copy of CYP51 for resistance. Cyp51 was over-expressed 1.4- to 19-fold in Y136F-mutant isolates. However, the Y136F mutation was absent in one isolate with moderate to high resistance factor. Two additional synonymous mutations were detected as well, one of which, A1119C was present only in isolates with high cyp51 expression. Overall, our results indicate that at least two mechanisms, cyp51 over-expression and the known target-site mutation in CYP51, contribute to resistance in E. necator, and may be working in conjunction with each other
2. Cherrad S, Charnay A, Hernandez C, Steva H, Belbahri L, Vacher S. Emergence of boscalid-resistant strains of Erysiphe necator in French vineyards. Microbiol Res. 2018 Nov: 216:79-84.
Abstract. The grapevine powdery mildew Erysiphe necator (E. necator) is an obligate pathogen. Powdery mildew-diseased vines show an important reduction in plant size, winter hardiness and grape yield. Even a low-level infection with powdery mildew was shown to taint wine and ultimately reduce wine quality. For many years, succinate dehydrogenase inhibitor (SDHI) fungicides, mainly the new generation active ingredients (AIs) boscalid, penthiopyrad and fluopyram, have been widely used to control powdery mildew in grapevines. The repeated use of fungicides (mainly boscalid) has resulted in the emergence of resistant microorganisms such as Botrytis cinerea (B. cinerea). However, boscalid resistance was never observed in E. necator. In this study, a large-scale survey of French grapevine field populations of E. necator revealed many field populations with low sensitivity to boscalid. Single spore strains originating from collected resistant populations showed Half maximal effective concentration (EC50) values greater than 100 mg L-1, and strains originating from boscalid sensitive populations showed EC50 values lower than 1 mg L-1. The complete nucleotide sequences of the EnSdhB succinate dehydrogenase of sensitive and resistant single spore strains revealed that H242R and H242Y substitutions in the EnSdhB succinate dehydrogenase subunit conferred E. necator resistance to boscalid. No cross-resistance of E. necator strains bearing H242R and H242Y substitutions in EnSdhB succinate dehydrogenase to fluxapyroxad and fluopyram was noticed. Therefore, our results highlight the emergence of resistance to boscalid activity in French vineyards and warrant the need of the implementation of risk assessment strategies to maintain effective grapevine protection against powdery mildew.
3. Fungicide Resistance Assessment, Mitigation and Extension (FRAME) Network - https://framenetworks.wsu.edu/
Highlights. This FRAME Network (Fungicide Resistance Assessment, Mitigation and Extension Network) will develop tools that can be applied to other specialty crops facing fungicide resistance challenges. The integration of this project’s objectives address: Where fungicide resistance is currently; Improve how we detect and monitor fungicide resistance and improve application efficiency; Predict where and when fungicide resistance will arise; and Develop strategies that help growers, educators, and manufactures answer what they need to do to mitigate resistance development and manage resistance that has already developed. and manage resistance that has already developed.
For additional reading, please refer to American Vineyard Foundation and FRAME Research. Your thoughts, opinions, and observations are much appreciated. Please feel free to email Dr. Walt Mahaffe or call (800)506-9852.