Search

Information on the presence and severity of grape powdery mildew (GPM), caused by Erysiphe necator , has long been used to guide management decisions. While recent advances in the available molecular diagnostic assays and particle samplers have made monitoring easier, there is still a need for more efficient field collection of E. necator . The use of vineyard worker gloves worn during canopy manipulation as a sampler (glove swab) of E. necator was compared with samples identified by visual assessment with subsequent molecular confirmation (leaf swabs) and airborne spore samples collected by rotating-arm impaction traps (impaction traps). Samples from United States commercial vineyards in Oregon, Washington, and California were analyzed using two TaqMan qPCR assays targeting the internal transcribed spacer regions or cytochrome b gene of E. necator . Based on qPCR assays, visual disease assessments misidentified GPM up to 59% of the time with a higher frequency of misidentification occurring earlier in the growing season. Comparison of the aggregated leaf swab results for a row ( n = 915) to the row's corresponding glove swab had 60% agreement. The latent class analysis (LCA) indicated that glove swabs were more sensitive than leaf swabs in detecting E. necator presence. The impaction trap results had 77% agreement to glove swabs ( n = 206) taken from the same blocks. The LCAs estimated that the glove swabs and impaction trap samplers varied each year in which was more sensitive for detection. This likely indicates that these methods have similar levels of uncertainty and provide equivalent information. Additionally, all samplers, once E. necator was detected, were similarly sensitive and specific for detection of the A-143 resistance allele. Together, these results suggest that glove swabs are an effective sampling method for monitoring the presence of E. necator and, subsequently, the G143A amino acid substitution associated with resistance to quinone outside inhibitor fungicides in vineyards. Glove swabs could reduce sampling costs due to the lack of need for specialized equipment and time required for swab collection and processing ., Competing Interests: The author(s) declare no conflict of interest.

Grapevine powdery mildew (GPM), caused by the fungus Erysiphe necator , is a constant threat to worldwide production of grape berries, requiring repeated use of fungicides for management. The frequent fungicide applications have resulted in resistance to commonly used quinone outside inhibitor (QoI) fungicides and the resistance is associated with single-nucleotide polymorphisms (SNPs) in the mitochondrial cytochrome b gene ( cytb ). In this study, we attempted to detect the most common SNP causing a glycine to alanine substitution at amino acid position 143 (i.e., G143A) in the cytb protein, to track this resistance using allele-specific TaqMan probe and digital-droplet PCR-based assays. Specificity and sensitivity of these assays showed that these two assays could discriminate SNPs and were effective on mixed samples. These diagnostic assays were implemented to survey E. necator samples collected from leaf and air samples from California and Oregon grape-growing regions. Sequencing of PCR amplicons and phenotyping of isolates also revealed that these assays accurately detected each allele (100% agreement), and there was an absolute agreement between the presence or absence of the G143A mutation and resistance to QoIs in the E. necator sampled. These results indicate that the developed diagnostic tools will help growers make informed decisions about fungicide selections and applications which, in turn, will facilitate GPM disease management and improve grape production systems.

In the Pacific Northwest, chasmothecia formation is not observed in vineyards until the beginning of véraison despite heavy infestations whereby 100% of leaf tissue is covered by Erysiphe necator . Mating type proximity and distribution were sampled from individual lesions (∼71 mm 2 ) on leaf tissue in a stratified sampling from three canopy heights at three times during the 2013, 2014, and 2015 growing seasons. Both mating types were observed at every sampling point and within the same lesions at all sampling dates and canopy heights. Effect of temperature and day length were examined by inoculating seedlings with known mating type 1 and 2 isolates and placed in incubators at different temperatures (5, 10, 15, 20, 25, and 30°C) or different day length changes (long day to long day, long day to short day, short day to short day, and short day to long day). Chasmothecia were produced at all temperatures that E. necator was able to colonize tissue, and the greatest number of chasmothecia were produced at 15 and 20°C ( P ≤ 0.02). Day length shifts from short day (8 h) to long day (16 h) resulted in a significant increase in chasmothecia production ( P < 0.001). End of season plant stress observed in the Pacific Northwest, such as water stress or host senescence, was assessed under naturally infested field conditions by either girdling canes or applying 150 mg·liter -1 abscisic acid solution to vines, respectively, and quantifying chasmothecia production. No differences were observed in chasmothecia production in the plant stress assessment, likely due to the high vigor and ability for plants to overcome stress treatments.

Predictive models have been developed in several major grape-growing regions to correlate environmental conditions to Erysiphe necator ascospore release; however, these models may not be broadly applicable in regions with different climatic conditions. To assess ascospore release in near-coastal regions of western Oregon, chasmothecia (syn. cleistothecia) were collected prior to leaf drop and placed onto natural and artificial grape trunk segments and overwintered outside. Ascospore release was monitored for three overwintering seasons using custom impaction spore traps from leaf drop (Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie [BBCH] 97) until the onset of the disease epidemic in the following growing season. Airborne inoculum was concurrently monitored in a naturally infested research vineyard. Weather and ascospore release data were used to assess previously developed models and correlate environmental conditions to ascospore release. Ascospore release was predicted by all models prior to bud break (BBCH 08), and was observed from the first rain event following the start of inoculum monitoring until monitoring ceased. Previously developed models overpredicted ascospore release in the Willamette Valley and predicted exhaustion of inoculum prior to bud break. The magnitude of ascospore release could not be correlated to environmental conditions; thus, a binary ascospore release model was developed where release is a function of the collective occurrence of the following factors within a 24-h period: >6 h of cumulative leaf wetness during temperatures >4°C, precipitation >2.5 mm, and relative humidity >80%. The Oregon model was validated using field-collected ascospore datasets, and predicted ascospore release with 66% accuracy (P = 0.02). Extant methods for estimating ascospore release may not be sufficiently accurate to use as predictive models in wet, temperate climatic regions.

Plant pathogen detection systems have been useful tools to monitor inoculum presence and initiate management schedules. More recently, a loop-mediated isothermal amplification (LAMP) assay was successfully designed for field use in the grape powdery mildew pathosystem; however, false negatives or false positives were prevalent in grower-conducted assays due to the difficulty in perceiving the magnesium pyrophosphate precipitate at low DNA concentrations. A quantitative LAMP (qLAMP) assay using a fluorescence resonance energy transfer-based probe was assessed by grape growers in the Willamette Valley of Oregon. Custom impaction spore samplers were placed at a research vineyard and six commercial vineyard locations, and were tested bi-weekly by the lab and by growers. Grower-conducted qLAMP assays used a beta-version of the Smart-DART handheld LAMP reaction devices (Diagenetix, Inc., Honolulu, HI, USA), connected to Android 4.4 enabled, Bluetooth-capable Nexus 7 tablets for output. Quantification by a quantitative PCR assay was assumed correct to compare the lab and grower qLAMP assay quantification. Growers were able to conduct and interpret qLAMP results; however, the Erysiphe necator inoculum quantification was unreliable using the beta-Smart-DART devices. The qLAMP assay developed was sensitive to one spore in early testing of the assay, but decreased to >20 spores by the end of the trial. The qLAMP assay is not likely a suitable management tool for grape powdery mildew due to losses in sensitivity and decreasing costs and portability for other, more reliable molecular tools., Competing Interests: The authors declare that they have no competing interests.

Management of grape powdery mildew (Erysiphe necator) and other polycyclic diseases relies on numerous fungicide applications that follow calendar or model-based application intervals, both of which assume that inoculum is always present. Quantitative molecular assays have been previously developed to initiate fungicide applications, and could be used to optimize fungicide application intervals throughout the growing season based on inoculum concentration. Airborne inoculum samplers were placed at one research and six commercial vineyards in the Willamette Valley of Oregon. Fungicide applications in all plots were initiated at the first detection of E. necator inoculum, and all subsequent fungicide application intervals were made based the grower' standard calendar program or based on inoculum concentration. In adjusted-interval plots, fungicides were applied at the shortest labeled application interval when >10 spores were detected and the longest labeled application interval when <10 spores were detected. Fungicide applications in control plots consisted of the growers' standard management practice. An average of 2.3 fewer fungicide applications in 2013 and 1.6 fewer fungicide applications in 2014 were used in the adjusted fungicide application intervals treatment in grower fields without significant differences in berry or leaf disease incidence between treatments.

This article documents the addition of 220 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Allanblackia floribunda, Amblyraja radiata, Bactrocera cucurbitae, Brachycaudus helichrysi, Calopogonium mucunoides, Dissodactylus primitivus, Elodea canadensis, Ephydatia fluviatilis, Galapaganus howdenae howdenae, Hoplostethus atlanticus, Ischnura elegans, Larimichthys polyactis, Opheodrys vernalis, Pelteobagrus fulvidraco, Phragmidium violaceum, Pistacia vera, and Thunnus thynnus. These loci were cross-tested on the following species: Allanblackia gabonensis, Allanblackia stanerana, Neoceratitis cyanescens, Dacus ciliatus, Dacus demmerezi, Bactrocera zonata, Ceratitis capitata, Ceratitis rosa, Ceratits catoirii, Dacus punctatifrons, Ephydatia mülleri, Spongilla lacustris, Geodia cydonium, Axinella sp., Ischnura graellsii, Ischnura ramburii, Ischnura pumilio, Pistacia integerrima and Pistacia terebinthus., (© 2010 Blackwell Publishing Ltd.)