Evaluation of germicidal UV-C light for suppression of grape powdery mildew and Botrytis bunch rot in Western Oregon.

Germicidal ultraviolet light (UV-C) has been shown to effectively suppress several plant pathogens, as well as some arthropod pests. Recent reports describe the efficacy of nighttime applications of UV-C at doses from 100 to 200 J/m2 in vineyards to reduce grape powdery mildew (Erysiphe necator). Our in vitro studies confirmed efficacy of UV-C to inhibit germination of E. necator and Botrytis cinerea conidia, demonstrated a range of tolerances to UV-C within a collection of E. necator isolates, and showed growth stage-specific effects of UV-C on B. cinerea. Nighttime use of UV-C was evaluated at 48 to 96 J/m2 in small plot trials (<1,000 vines) from 2020 to 2023. Once or twice weekly UV-C applications significantly reduced the incidence of foliar powdery mildew compared to non-UV-C-treated controls (P < 0.02). Suppression of powdery mildew on fruit was less consistent, where once or twice weekly UV-C exposure reduced powdery mildew disease severity in 2020 (P = 0.04), 2021 (P = 0.02) and 2023 (P =0.003), but less so in 2022 (P = 0.07). Bunch rot severity was not significantly reduced with UV-C treatment in any year of the study. Application of UV-C until the onset of fruit color change (veraison) also had a minimal effect on the fruit soluble solids, pH, anthocyanins, or phenolics in harvested fruit at any UV-C dose or frequency (P > 0.10). Suppression of powdery mildew by nighttime application UV-C at lower doses in small plots suggests that such treatments merit further evaluation in larger-scale studies in Western Oregon.

It's a Trap! Part I: Exploring the Applications of Rotating-Arm Impaction Samplers in Plant Pathology.

Although improved knowledge on the movement of airborne plant pathogens is likely to benefit plant health management, generating this knowledge is often far more complicated than anticipated. This complexity is driven by the dynamic nature of environmental variables, diversity among pathosystems that are targeted, and the unique needs of each research group. When using a rotating-arm impaction sampler, particle collection is dependent on the pathogen, environment, research objectives, and limitations (monetary, environmental, or labor). Consequently, no design will result in 100% collection efficiency. Fortunately, it is likely that multiple approaches can succeed despite these constraints. Choices made during design and implementation of samplers can influence the results, and recognizing this influence is crucial for researchers. This article is for beginners in the art and science of using rotating-arm impaction samplers; it provides a foundation for designing a project, from planning the experiment to processing samples. We present a relatively nontechnical discussion of the factors influencing pathogen dispersal and how placement of the rotating-arm air samplers alters propagule capture. We include a discussion of applications of rotating-arm air samplers to demonstrate their versatility and potential in plant pathology research as well as their limitations.

It's a Trap! Part II: An Approachable Guide to Constructing and Using Rotating-Arm Air Samplers.

An increasing number of researchers are looking to understand the factors affecting microbial dispersion but are often limited by the costs of commercially available air samplers. Some have reduced these costs by designing self-made versions; however, there are no published sampler designs, and there is limited information provided on the actual construction process. Lack of appropriate reference material limits the use of these self-made samplers by many researchers. This manuscript provides a guide to designing and constructing rotating-arm impaction air samplers by covering (i) environmental considerations, (ii) construction materials and equipment, (iii) the construction process, and (iv) air sampler deployment. Information regarding how to calculate rotational velocity, motor speed, and power supply requirements and to troubleshoot common issues is presented in an approachable format for individuals without experience in electronics or machining. Although many of the components discussed in this guide may change in their availability or be updated over time, this document is intended to serve as a "builder's guide" for future research into air sampling technology for phytopathology research.

Catching Spores: Linking Epidemiology, Pathogen Biology, and Physics to Ground-Based Airborne Inoculum Monitoring.

Monitoring airborne inoculum is gaining interest as a potential means of giving growers an earlier warning of disease risk in a management unit or region. This information is sought by growers to aid in adapting to changes in the management tools at their disposal and the market-driven need to reduce the use of fungicides and cost of production. To effectively use inoculum monitoring as a decision aid, there is an increasing need to understand the physics of particle transport in managed and natural plant canopies to effectively deploy and use near-ground aerial inoculum data. This understanding, combined with the nuances of pathogen-specific biology and disease epidemiology, can serve as a guide to designing improved monitoring approaches. The complexity of any pathosystem and local environment are such that there is not a generalized approach to near-ground air sampler placement, but there is a conceptual framework to arrive at a "semi-optimal" solution based on available resources. This review is intended as a brief synopsis of the linkages among pathogen biology, disease epidemiology, and the physics of the aerial dispersion of pathogen inoculum and what to consider when deciding where to locate ground-based air samplers. We leverage prior work in developing airborne monitoring tools for hops, grapes, spinach, and turf, and research into the fluid mechanics governing particle transport in sparse canopies and urban and forest environments. We present simulation studies to demonstrate how particles move in the complex environments of agricultural fields and to illustrate the limited sampling area of common air samplers.

A Rapid Glove-Based Inoculum Sampling Technique to Monitor Erysiphe necator in Commercial Vineyards.

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.

Development of a PNA-LNA-LAMP Assay to Detect an SNP Associated with QoI Resistance in Erysiphe necator.

The repetitive use of quinone outside inhibitor fungicides (QoIs, strobilurins; Fungicide Resistance Action Committee [FRAC] 11) to manage grape powdery mildew has led to development of resistance in Erysiphe necator. While several point mutations in the mitochondrial cytochrome b gene are associated with resistance to QoI fungicides, the substitution of glycine to alanine at codon 143 (G143A) has been the only mutation observed in QoI-resistant field populations. Allele-specific detection methods such as digital droplet PCR and TaqMan probe-based assays can be used to detect the G143A mutation. In this study, a peptide nucleic acid-locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA-LAMP) assay consisting of an A-143 reaction and a G-143 reaction, was designed for rapidly detecting QoI resistance in E. necator. The A-143 reaction amplifies the mutant A-143 allele faster than the wild-type G-143 allele, while the G-143 reaction amplifies the G-143 allele faster than the A-143 allele. Identification of resistant or sensitive E. necator samples was determined by which reaction had the shorter time to amplification. Sixteen single-spore QoI-resistant and -sensitive E. necator isolates were tested using both assays. Assay specificity in distinguishing the single nucleotide polymorphism (SNP) approached 100% when tested using purified DNA of QoI-sensitive and -resistant E. necator isolates. This diagnostic tool was sensitive to one-conidium equivalent of extracted DNA with an R2 value of 0.82 and 0.87 for the G-143 and A-143 reactions, respectively. This diagnostic approach was also evaluated against a TaqMan probe-based assay using 92 E. necator samples collected from vineyards. The PNA-LNA-LAMP assay detected QoI resistance in ≤30 min and showed 100% agreement with the TaqMan probe-based assay (≤1.5 h) for the QoI-sensitive and -resistant isolates. There was 73.3% agreement with the TaqMan probe-based assay when samples had mixed populations with both G-143 and A-143 alleles present. Validation of the PNA-LNA-LAMP assay was conducted in three different laboratories with different equipment. The results showed 94.4% accuracy in one laboratory and 100% accuracy in two other laboratories. The PNA-LNA-LAMP diagnostic tool was faster and required less expensive equipment relative to the previously developed TaqMan probe-based assay, making it accessible to a broader range of diagnostic laboratories for detection of QoI resistance in E. necator. This research demonstrates the utility of the PNA-LANA-LAMP for discriminating SNPs from field samples and its utility for point-of-care monitoring of plant pathogen genotypes.

Identification of Putative SDHI Target Site Mutations in the SDHB, SDHC, and SDHD Subunits of the Grape Powdery Mildew Pathogen Erysiphe necator.

Succinate dehydrogenase inhibitors (SDHIs) are fungicides used in control of numerous fungal plant pathogens, including Erysiphe necator, the causal agent of grapevine powdery mildew (GPM). Here, the sdhb, sdhc, and sdhd genes of E. necator were screened for mutations that may be associated with SDHI resistance. GPM samples were collected from 2017 to 2020 from the U.S. states of California, Oregon, Washington, and Michigan, and the Canadian province of British Columbia. Forty-five polymorphisms were identified in the three sdh genes, 17 of which caused missense mutations. Of these, the SDHC-p.I244V substitution was shown in this study to reduce sensitivity of E. necator to boscalid and fluopyram, whereas the SDHC-p.G25R substitution did not affect SDHI sensitivity. Of the other 15 missense mutations, the SDHC-p.H242R substitution was shown in previous studies to reduce sensitivity of E. necator toward boscalid, whereas the equivalents of the SDHB-p.H242L, SDHC-p.A83V, and SDHD-p.I71F substitutions were shown to reduce sensitivity to SDHIs in other fungi. Generally, only a single amino acid substitution was present in the SDHB, SDHC, or SDHD subunit of E. necator isolates, but missense mutations putatively associated with SDHI resistance were widely distributed in the sampled areas and increased in frequency over time. Finally, isolates that had decreased sensitivity to boscalid or fluopyram were identified but with no or only the SDHC-p.G25R amino acid substitution present in SDHB, SDHC, and SDHD subunits. This suggests that target site mutations probably are not the only mechanism conferring resistance to SDHIs in E. necator.

Allele-Specific Detection Methods for QoI Fungicide-Resistant Erysiphe necator in Vineyards.

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.

Effect of Fungicide Mobility and Application Timing on the Management of Grape Powdery Mildew.

Grape powdery mildew (GPM) fungicide programs consist of 5 to 15 applications, depending on region or market, in an attempt to achieve the high fruit quality standards demanded by the market. Understanding how fungicides redistribute and targeting redistributing fungicide to critical crop phenological stages could improve fungicide protection of grape clusters. This study evaluated fungicide redistribution in grapevines from major fungicide groups labeled for GPM control. Translaminar and xylem redistribution was examined by placing fungicide-impregnated filter disks on the adaxial or abaxial leaf surface of detached leaves for 10 min and then incubating for 48 h before inoculating the abaxial surface with conidia. Vapor redistribution used Teflon disks sprayed with fungicides and placed on the abaxial leaf surface of detached leaves 48 h before inoculation. Disease development was rated 10 days later. Translaminar movement through calyptra was tested using flowering potted vines. All fungicides tested redistributed through at least one mechanism. Fungicide timing at critical phenological stages (early, mid, and late bloom) was assessed in small plots of cultivar Pinot noir vines. The application of trifloxystrobin, quinoxyfen, or fluopyram at different bloom stages showed that applications initiated at end of bloom resulted in the lowest berry infection probabilities of 0.073, 0.097, and 0.020, respectively. The results of this study suggest that integrating two carefully timed applications of redistributing fungicides initiated at end of bloom into a fungicide program may be an effective strategy for wine grape growers in western Oregon to produce fruit with low GPM infection.

Risk Factors for Bud Perennation of Podosphaera macularis on Hop.

The hop powdery mildew fungus Podosphaera macularis persists from season to season in the Pacific Northwestern United States through infection of crown buds because only one of the mating types needed to produce the ascigerous stage is presently found in this region. Bud infection and successful overwintering of the fungus leads to the emergence of heavily infected shoots in early spring (termed flag shoots). Historical data of flag shoot occurrence and incidence in Oregon and Washington State during 2000 to 2017 were analyzed to identify their association with the incidence of powdery mildew, growers' use of fungicides, autumn and winter temperature, and other production factors. During this period, flag shoots were found on 0.05% of plants evaluated in Oregon and 0.57% in Washington. In Oregon, the incidence of powdery mildew on leaves was most severe and the number of fungicide applications made by growers greatest in yards where flag shoots were found in spring. Similarly, the incidence of plants with powdery mildew in Washington was significantly associated with the number of flag shoots present in early spring, although the number of fungicide applications made was independent of flag shoot occurrence. The occurrence of flag shoots was associated with prior occurrence of flag shoots in a yard, the incidence of foliar powdery mildew in the previous year, grower pruning method, and, in Washington, winter temperature. A census of hop yards in the eastern extent of the Oregon production region during 2014 to 2017 found flag shoots in 27 of 489 yards evaluated. In yards without flag shoots, 338 yards (73.2%) were chemically pruning or not pruned, whereas the remaining 124 (26.8%) were mechanically pruned. Of the 27 yards with flag shoots, 22 were either chemically pruned or not pruned and 4 were mechanically pruned in mid-April, well after the initial emergence of flag shoots. The prevalence of yards with flag shoots also was related to thoroughness of pruning in spring (8.1% of yards with incomplete pruning versus 1.9% of yards with thorough pruning). A Bayesian logistic regression model was fit to the data from the intensively assessed yards in Oregon, with binary risk factors for occurrence of a flag shoot in the previous year, occurrence of foliar mildew in the previous year, and thoroughness of pruning in spring. The model indicated that the median and 95% highest posterior density interval of the probability of flag shoot occurrence was 0.0008 (0.0000 to 0.0053) when a yard had no risk factors but risk increased to 0.0065 (0.0000 to 0.0283) to 0.43 (0.175 to 0.709) when one to all three of the risk factors were present. The entirety of this research indicates that P. macularis appears to persist in a subset of chronically affected hop yards, particularly yards where spring pruning is conducted poorly. Targeted management of the disease in a subset of fields most at risk for producing flag shoots could potentially influence powdery mildew development regionwide.

Early Detection of Airborne Inoculum of Magnaporthe oryzae in Turfgrass Fields Using a Quantitative LAMP Assay.

Gray leaf spot (GLS) is a destructive disease of perennial ryegrass caused by a host specific pathotype of the ascomycete Magnaporthe oryzae. Early diagnosis is crucial for effective disease management and the implementation of Integrated Pest Management practices. However, a rapid protocol for the detection of low levels of airborne inoculum is still missing. We developed a pathogen-specific quantitative loop-mediated isothermal amplification (qLAMP) assay coupled with a spore trap system for rapid detection and quantification of airborne inoculum of the M. oryzae perennial ryegrass pathotype, and tested its suitability for implementation in GLS-infected turfgrass fields. In summer 2015, two perennial ryegrass plots were artificially inoculated with the pathogen, with four continuously running custom impaction spore traps placed in each plot. Sampling units were replaced daily and tested with the developed qLAMP assay, while plots were monitored for symptom development. Results confirmed that the qLAMP assay-trap system was able to detect as few as 10 conidia up to 12 days before symptoms developed in the field. LAMP technology is particularly appropriate for field implementation by nontechnical users, and has the potential to be a powerful decision support tool to guide timing of fungicide applications for GLS management.

The Ebb and Flow of Airborne Pathogens: Monitoring and Use in Disease Management Decisions.

Perhaps the earliest form of monitoring the regional spread of plant disease was a group of growers gathering together at the market and discussing what they see in their crops. This type of reporting continues to this day through regional extension blogs, by crop consultants and more formal scouting of sentential plots in the IPM PIPE network (http://www.ipmpipe.org/). As our knowledge of plant disease epidemiology has increased, we have also increased our ability to detect and monitor the presence of pathogens and use this information to make management decisions in commercial production systems. The advent of phylogenetics, next-generation sequencing, and nucleic acid amplification technologies has allowed for development of sensitive and accurate assays for pathogen inoculum detection and quantification. The application of these tools is beginning to change how we manage diseases with airborne inoculum by allowing for the detection of pathogen movement instead of assuming it and by targeting management strategies to the early phases of the epidemic development when there is the greatest opportunity to reduce the rate of disease development. While there are numerous advantages to using data on inoculum presence to aid management decisions, there are limitations in what the data represent that are often unrecognized. In addition, our understanding of where and how to effectively monitor airborne inoculum is limited. There is a strong need to improve our knowledge of the mechanisms that influence inoculum dispersion across scales as particles move from leaf to leaf, and everything in between.

Identification of a receptor for the sex pheromone of the vine mealybug, Planococcus ficus.

The vine mealybug, Planococcus ficus, is a significant pest of vineyards in all major grape growing regions of the world. This pest causes significant aesthetic damage to berry clusters through its feeding behavior and secretion of "honeydew", which leads to significant decreases in crop marketability. More importantly, the vine mealybug is a vector of several grapevine viruses which are the causal agent of grapevine leafroll disease, one of the most destructive and economically devastating diseases of the grape industry worldwide. As there is no cure for grapevine leafroll disease, the only control measures available to reduce its spread are to remove infected vines whilst simultaneously controlling mealybug populations. Using transcriptomic libraries prepared from male and female mealybugs and a draft genome, we identified and evaluated expression levels of members of the odorant receptor gene family. Interestingly, of the 50 odorant receptors identified from these P. ficus genetic resources, only 23 were found to be expressed in females, suggesting this flightless life stage has a decreased reliance on the olfactory system. In contrast, 46 odorant receptors were found to be expressed in the alate male life stage. Heterologous expression of eight of these receptors, along with the obligate co-receptor, Orco, in HEK293 cells allowed for the identification of two receptors that respond to lavandulyl senecioate, the sole constituent of the sex pheromone used by this species. Interestingly, one of these receptors, PficOR8, also responded to the sex pheromone used by the Japanese mealybug, Planococcus kraunhiae. The data presented here represent the first report of odorant receptor gene family expression levels, as well as the identification of the first sex pheromone receptor, in soft-scale insects. The identification of a receptor for the vine mealybug sex pheromone will allow for the development of novel, species-specific pest control tools and monitoring devices.

Free-Riding in Plant Health: A Social-Ecological Systems Approach to Collective Action.

Plant disease epidemics often transcend land management boundaries, creating a collective-action problem where a group must cooperate in a common effort to maximize individual and group benefits. Drawing upon the social-ecological systems framework and associated design principles, we review variables of resource systems, resource units, actors, and governance systems relevant to collective action in plant health. We identify a need to better characterize how attributes of epidemics determine the usefulness of collective management, what influences actors' decisions to participate, what governance systems fit different plant health threats, and how these subsystems interact to lead to plant health outcomes. We emphasize that there is not a single governance structure that ensures collective action but rather a continuum of structures that depend on the key system variables identified. An integrated social-ecological systems approach to collective action in plant health should enable institutional designs to better fit specific plant health challenges.

A chromosome-scale genome assembly of the grape powdery mildew pathogen Erysiphe necator reveals its genomic architecture and previously unknown features of its biology.

Erysiphe necator is an obligate fungal pathogen that causes grape powdery mildew, globally the most important disease on grapevines. Previous attempts to obtain a quality genome assembly for this pathogen were hindered by its high repetitive DNA content. Here, chromatin conformation capture (Hi-C) with long-read PacBio sequencing was combined to obtain a chromosome-scale assembly and a high-quality annotation for E. necator isolate EnFRAME01. The resulting 81.1 Mb genome assembly is 98% complete and consists of 34 scaffolds, 11 of which represent complete chromosomes. All chromosomes contain large centromeric-like regions and lack synteny to the 11 chromosomes of the cereal PM pathogen Blumeria graminis. Further analysis of their composition showed that repeats and transposable elements (TEs) occupy 62.7% of their content. TEs were almost evenly interspersed outside centromeric and telomeric regions and massively overlapped with regions of annotated genes, suggesting that they could have a significant functional impact. Abundant gene duplicates were observed as well, particularly in genes encoding candidate secreted effector proteins. Moreover, younger in age gene duplicates exhibited more relaxed selection pressure and were more likely to be located physically close in the genome than older duplicates. A total of 122 genes with copy number variations among six isolates of E. necator were also identified and were enriched in genes that were duplicated in EnFRAME01, indicating they may reflect an adaptive variation. Taken together, our study illuminates higher-order genomic architectural features of E. necator and provides a valuable resource for studying genomic structural variations in this pathogen. IMPORTANCE Grape powdery mildew caused by the ascomycete fungus Erysiphe necator is economically the most important and recurrent disease in vineyards across the world. The obligate biotrophic nature of E. necator hinders the use of typical genetic methods to elucidate its pathogenicity and adaptation to adverse conditions, and thus comparative genomics has been a major method to study its genome biology. However, the current reference genome of E. necator isolate C-strain is highly fragmented with many non-coding regions left unassembled. This incompleteness prohibits in-depth comparative genomic analyses and the study of genomic structural variations (SVs) that are known to affect several aspects of microbial life, including fitness, virulence, and host adaptation. By obtaining a chromosome-scale genome assembly and a high-quality gene annotation for E. necator, we reveal the organization of its chromosomal content, unearth previously unknown features of its biology, and provide a reference for studying genomic SVs in this pathogen.

Association of Spring Pruning Practices with Severity of Powdery Mildew and Downy Mildew on Hop.

Downy mildew (caused by Pseudoperonospora humuli) and powdery mildew (caused by Podosphaera macularis) are important diseases of hop in the Pacific Northwest United States, and cultural practices may affect the severity of both diseases. The association of spring pruning quality and timing with severity of downy mildew and powdery mildew was assessed through analysis of survey data collected from commercial hop yards in Oregon and Washington. Among 149 hop yards surveyed, the most common pruning method was chemical desiccation (48% of yards), mechanical pruning (23%), or a combination of these practices (15%). The quality of pruning was assessed using a three-category ordinal scale ("excellent", "moderate", or "poor") based on the amount of foliage remaining on plants following pruning. Excellent pruning quality was attained more often in yards pruned twice (74.6 to 82.1% of yards) versus once (33.8% of yards), independent of pruning method. Seasonal severity of downy mildew in Oregon increased approximately twofold with reduction in pruning quality from excellent to moderate to poor. Pruning quality was not significantly related to levels of powdery mildew on leaves or cones in Oregon. Under more severe disease pressure in Washington, however, seasonal severity of powdery mildew on leaves and the incidence of cones with powdery mildew were significantly greater in yards that had poor pruning compared with excellent pruning. Moreover, yards that had excellent pruning quality received, on average, 1.1 to 1.5 fewer fungicide applications per season for downy mildew or powdery mildew compared with yards that had moderate or poor pruning quality. This savings was associated with delayed initiation of the first application by 7.5 to 14.2 days in yards with excellent pruning quality. Replicated experiments in commercial yards in Oregon quantified the effect of delaying pruning timing 5 to 21 days compared with growers' standard practices on the diseases and yield. Downy mildew suppression by delayed pruning was dependent on cultivar and year of sampling, being significantly reduced fivefold only in 'Willamette' in 2007. Severity of powdery mildew and cone yield was similar between plots that received the delayed or standard pruning timing treatments. Collectively, these studies emphasize that early spring sanitation measures are associated with reduced primary inoculum and are critically important for managing both downy mildew and powdery mildew. A savings of at least one fungicide application per year appears achievable when spring pruning is conducted thoroughly and slightly delayed compared with growers' current practices.

Effect of Temperature on Sporulation and Infectivity of Podosphaera macularis on Humulus lupulus.

Hop powdery mildew, caused by Podosphaera macularis, can result in complete crop loss and requires numerous fungicide applications for effective management. To assess the impact of temperature on the production of infective conidia, 10-day-old sporulating colonies were exposed to 18, 30, 33, 36, 39, and 42°C for 6 h, and then incubated at 18°C for 18 h. Conidia were harvested, inoculated onto hop plants, incubated at 18°C for 10 days, and then lesions/cm2 of leaf area was determined. Disease was significantly reduced at temperatures ≥30°C with a nonlinear response in the production of infective conidia (P < 0.0001). Temperature effects on sporulation of P. macularis were examined using a custom impaction conidia sampler in growth chambers programmed at constant temperatures of 5, 10, 15, 20, 25, 30, and 35°C, or 18°C before and after ramping to 18, 22, 26, 30, 34, and 38°C for 6 h. The effect of constant temperature on sporulation was best described by a nonlinear thermodynamic model (P = 0.0001) with maximal production near 25°C. Exposure to fluctuating temperatures produced a curvilinear response in sporulation (P = 0.0122) with maximum production near 25°C. These data indicate that inoculum availability is reduced when ambient temperature exceeds 30°C and that modeling inoculum availability could help further refine current disease forecasting models.

Development of a quantitative loop-mediated isothermal amplification assay for the field detection of Erysiphe necator.

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.

Detection of Erysiphe necator in Air Samples Using the Polymerase Chain Reaction and Species-Specific Primers.

ABSTRACT A polymerase chain reaction (PCR) assay employing species-specific primers was developed to differentiate Erysiphe necator from other powdery mildews common in the northwest United States. DNA was extracted from mycelia, conidia, and/or chasmothecia that were collected from grape leaves with a Burkard cyclonic surface sampler. To differentiate E. necator from other erysiphaeceous fungi, primer pairs Uncin144 and Uncin511 were developed to select unique sequences of the internal transcribed spacer regions of E. necator. Using these primers in PCR amplifications, a 367-bp amplicon specific to E. necator was generated, but no amplicons were generated from other erysiphaceous species collected from 48 disparate hosts representing 26 vascular plant families. The PCR limit of detection was one to five conidia of E. necator placed directly into reaction mixtures or 100 to 250 conidia placed on glass rods coated with silicon grease. During field studies, this PCR assay facilitated the detection of E. necator inoculum in air samples within hours of sample rod collection and prior to disease onset. Amplification of E. necator DNA did not occur when the PCR assay was conducted on vineyard air samples collected while grapes were dormant or during periods when vine growth occurred but E. necator remained dormant. The initial PCR detection of E. necator of the season occurred during seasonal ascospore releases caused by precipitation events between bud burst and the prebloom period during the 3 years of the study. Detection ceased for 7 to 11 days following ascospore release and then resumed several days prior to the observance of microscopic symptoms and signs of powdery mildew in the field. Results of this study represent the initial step toward the goal of incorporating an inoculum availability component into current and future grapevine powdery mildew risk assessment models.

Sequential Sampling for Estimation and Classification of the Incidence of Hop Powdery Mildew I: Leaf Sampling.

Hop powdery mildew (caused by Podosphaera macularis) is an important disease of hops (Humulus lupulus) in the Pacific Northwest. Sequential sampling models for estimation and classification of the incidence of powdery mildew on leaves of hop were developed based on the beta-binomial distribution, using parameter estimates of the binary power law determined in previous studies. Stop lines, models that indicate that enough information has been collected to estimate disease incidence and cease sampling, for sequential estimation were validated by bootstrap simulations of a select group of 18 data sets (out of a total of 198 data sets) from the model-construction data, and through simulated sampling of 104 data sets collected independently (i.e., validation data sets). The achieved coefficient of variation (C) approached prespecified C values as the achieved disease incidence ( ) increased. Achieving a C of 0.1 was not possible for data sets in which < 0.10. The 95% confidence interval of the median difference between the true p and included zero for 16 of 18 data sets evaluated at C = 0.2 and all data sets when C = 0.1. For sequential classification, Monte-Carlo simulations were used to determine the probability of classifying mean disease incidence as less than a threshold incidence, pt (operating characteristic [OC]), and average sample number (ASN) curves for 16 combinations of candidate stop lines and error levels (α and ß). Four pairs of stop lines were selected for further evaluation based on the results of the Monte-Carlo simulations. Bootstrap simulations of the 18 selected data sets indicated that the OC and ASN curves of the sequential sampling plans for each of the four sets of stop lines were similar to OC and ASN values determined by Monte Carlo simulation. Correct classification of disease incidence as being above or below preselected thresholds was 2.0 to 7.7% higher when stop lines were determined by the beta-binomial approximation than when stop lines were calculated using the binomial distribution. Correct decision rates differed depending on the location where sampling was initiated in the hop yard; however, in all instances were greater than 86% when stop lines were determined using the beta-binomial approximation. The sequential sampling plans evaluated in this study should allow for rapid and accurate estimation and classification of the incidence of hop leaves with powdery mildew, and aid in sampling for pest management decision making.