Evaluation of Quinoxyfen Resistance of Erysiphe necator (Grape Powdery Mildew) in a Single Virginia Vineyard.

The protectant fungicide quinoxyfen has been used against grape powdery mildew (Erysiphe necator) in the United States since 2003. In 2013, isolates of grape powdery mildew with reduced quinoxyfen sensitivity (here designated as quinoxyfen lab resistance or QLR) were detected in a single vineyard in western Virginia, USA. Field trials were conducted in 2014, 2015, and 2016 at the affected vineyard to determine to what extent quinoxyfen might still contribute to disease control. Powdery mildew control by quinoxyfen was similar to, or only slightly less than, that provided by myclobutanil and boscalid in all three years. In 2016, early- versus late-season applications of quinoxyfen were compared to test the hypothesis that early-season applications were more effective, but differences were small. A treatment with two early quinoxyfen applications, at bloom and 2 weeks later, followed by a myclobutanil-boscalid plus a low dose of sulfur rotation provided slightly better control of foliar disease incidence than treatments containing four quinoxyfen applications or two midseason or two late quinoxyfen applications supplemented by myclobutanil and boscalid applications; severity differences were small and nonsignificant. Metrafenone and benzovindiflupyr generally provided excellent powdery mildew control. The frequency of QLR in vines not treated with quinoxyfen slowly declined from 65% in 2014 to 46% in 2016. Further research is needed to explain how, despite this QLR frequency, quinoxyfen applied to grapes in the field was still able to effectively control powdery mildew.

Co-Occurrence of Two Allelic Variants of CYP51 in Erysiphe necator and Their Correlation with Over-Expression for DMI Resistance.

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.

Sensitivity of Erysiphe necator and Plasmopara viticola in Virginia to QoI Fungicides, Boscalid, Quinoxyfen, Thiophanate Methyl, and Mefenoxam.

The sensitivity of downy mildew (DM, Plasmopara viticola) and powdery mildew (PM, Erysiphe necator) of grape (Vitis sp.) to commonly used nondemethylation inhibitor, single-site fungicides in and near Virginia was determined from 2005 to 2007, with more limited additional sampling in subsequent years. In grape leaf disc bioassays, 92% of the P. viticola isolates were quinone outside inhibitor (QoI, azoxystrobin) resistant but none were resistant to mefenoxam. In all, 82% of the E. necator isolates were QoI resistant. Most of the QoI-resistant P. viticola and E. necator isolates contained >95% of the G143A point mutation, which confers high levels of QoI resistance. In contrast, QoI-sensitive P. viticola isolates contained less than 1% of G143A. In total, 1 of 145 and 14 of 154 QoI-resistant P. viticola and E. necator isolates (able to grow on azoxystrobin concentration ≥1 µg/ml), respectively, contained <1% G143A. In total, 61 E. necator isolates from 23 locations were tested against thiophanate methyl, and the majority grew well on leaf tissue treated with 50 and 250 µg/ml. Through 2012, none of the E. necator isolates were resistant to boscalid and quinoxyfen. However, in 2013, quinoxyfen-resistant E. necator was detected in one vineyard experiencing difficulties with powdery mildew control. No 50% effective concentration value could be calculated but these isolates tolerated labeled rates with only limited inhibition. QoI (E. necator and P. viticola) and benzimidazole (E. necator) resistance were widespread in Virginia, rendering these materials inadvisable for control of these diseases. The practical importance and current distribution of quinoxyfen resistance needs further investigation.

Geographic distribution of cryptic species of Plasmopara viticola causing downy mildew on wild and cultivated grape in eastern North America.

The putative center of origin of Plasmopara viticola, the causal agent of grape downy mildew, is eastern North America, where it has been described on several members of the family Vitaceae (e.g., Vitis spp., Parthenocissus spp., and Ampelopsis spp.). We have completed the first large-scale sampling of P. viticola isolates across a range of wild and cultivated host species distributed throughout the above region. Sequencing results of four partial genes indicated the presence of a new P. viticola species on Vitis vulpina in Virginia, adding to the four cryptic species of P. viticola recently recorded. The phylogenetic analysis also indicated that the P. viticola species found on Parthenocissus quinquefolia in North America is identical to Plasmopara muralis in Europe. The geographic distribution and host range of five pathogen species was determined through analysis of the internal transcribed spacer polymorphism of 896 isolates of P. viticola. Among three P. viticola species found on cultivated grape, one was restricted to Vitis interspecific hybrids within the northern part of eastern North America. A second species was recovered from V. vinifera and V. labrusca, and was distributed across most of the sampled region. A third species, although less abundant, was distributed across a larger geographical range, including the southern part of eastern North America. P. viticola clade aestivalis predominated (83% of isolates) in vineyards of the European winegrape V. vinifera within the sampled area, indicating that a single pathogen species may represent the primary threat to the European host species within eastern North America.

Fitness of Erysiphe necator with G143A-Based Resistance to Quinone Outside Inhibitors.

Management of grape powdery mildew (Erysiphe necator) using quinone outside inhibitors (QoIs) has eroded in an increasing number of regions due to resistance development. To determine persistence of resistance when QoIs 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, QoI resistance persisted at high frequency for 4 weeks. Resistant populations were also found to persist in one vineyard without QoI application for four consecutive years. The frequency was still about 25% in the fourth year, with higher frequency (36%) in a hotspot section. QoI-resistant populations with >5% G143A also harbored Y136F in the cyp51 gene that confers some resistance to sterol demethylation inhibitors, another fungicide class for powdery mildew control. Double resistance could have been partly responsible for persistence of QoI resistance at this location.

Sensitivity of Erysiphe necator to Demethylation Inhibitor Fungicides in Virginia.

Grape powdery mildew (Erysiphe necator) isolates were collected from 2005 to 2007 from vineyards mostly in Virginia but also some in Maryland, North Carolina, and Pennsylvania. Using a leaf disc assay, the isolates were tested against five demethylation inhibitor (DMI) fungicides. Most isolates exhibited reduced sensitivity to the five DMIs when compared with a sensitive group (n = 12) and compared with unexposed populations reported from other areas. The median resistance factor (RF) was highest for tebuconazole (RF = 399) and myclobutanil (RF = 378), followed by triflumizole (RF = 70), triadimefon (RF = 62), and fenarimol (RF = 44). The sensitive group used as the basis for comparison appears to have been more sensitive than unexposed isolates in New York and California. Our finding that the greatest resistance shift occurred with tebuconazole and myclobutanil contrasts with earlier reports from New York and California, where the greatest resistance shift was observed with triadimefon or triadimenol. Sensitivities to all five DMI fungicides were strongly correlated (pairwise r values of 0.70 to 0.87) but our data suggest that some may retain greater utility than others.