Detection of Podosphaera macularis in Air Samples by Quantitative PCR.

Detection and quantification of pathogen propagules in the air or other environmental samples is facilitated by culture-independent assays. We developed a quantitative PCR assay for the hop powdery mildew fungus, Podosphaera macularis, for detection of the organism from air samples. The assay utilizes primers and a TaqMan probe designed to target species-specific sequences in the 28S large subunit (LSU) of the nuclear ribosomal rDNA. Analytical sensitivity was not affected by the presence of an exogenous internal control or potential PCR inhibitors associated with DNA extracted from soil. The level of quantification of the assay was between 200 and 350 conidia when DNA was extracted from a fixed number of conidia. The assay amplified all isolates of P. macularis tested and had minimal cross-reactivity with other Podosphaera species when assayed with biologically relevant quantities of DNA. Standard curves generated independently in two other laboratories indicated that assay sensitivity was qualitatively similar and reproducible. All laboratories successfully detected eight unknown isolates of P. macularis and correctly discriminated Pseudoperonospora humuli and a water control. The usefulness of the assay for air sampling for late-season inoculum of P. macularis was demonstrated in field studies in 2019 and 2020. In both years, airborne populations of P. macularis in hop yards were detected consistently and increased during bloom and cone development.

Identification of quantitative trait loci associated with R1-mediated resistance to powdery mildew and sex determination in hop (Humulus lupulus L.).

KEY MESSAGE: Two QTL were identified using linkage mapping approaches, one on hop linkage group 3 (qHl_Chr3.PMR1) associated with powdery mildew resistance and a second on linkage group 10 (cqHl_ChrX.SDR1) associated with sex determination. Hop (Humulus lupulus L.) is a dioecious species cultivated for use in beer. Hop powdery mildew, caused by Podosphaera macularis, is a constraint in many growing regions. Thus, identifying markers associated with powdery mildew resistance and sex provides the opportunity to pyramid R-genes and select female plants as seedlings, respectively. Our objectives were to characterize the genetic basis of R1-mediated resistance in the cultivar Zenith which provides resistance to pathogen races in the US, identify quantitative trait loci (QTL) associated with R1 and sex, and develop markers for molecular breeding-based approaches. Phenotypic evaluation of the population indicated that R1-based resistance and sex are inherited monogenically. We constructed a genetic map using 1339 single nucleotide polymorphisms (SNPs) based upon genotype-by-sequencing of 128 F1 progeny derived from a Zenith × USDA 21058M biparental population. SNPs were assigned to 10 linkage groups comprising a map length of 1204.97 cM with an average density of 0.94 cM/marker. Quantitative trait locus mapping identified qHl_Chr3.PMR1, associated with R1 on linkage group 3 (LOD = 23.57, R2 = 57.2%), and cqHl_ChrX.SDR1, associated with sex on linkage group 10 (LOD = 5.42, R2 = 25.0%). Kompetitive allele-specific PCR (KASP) assays were developed for both QTL and assessed against diverse germplasm. Our results indicate that KASP markers associated with R1 may be limited to materials that are pedigree-related to Zenith, whereas markers associated with sex may be transferable across populations. The high-density map, QTL, and associated KASP markers will enable selecting for sex and R1-mediated resistance in hop.

Evolution of the Genetic Structure of the Didymella tanaceti Population During Development of Succinate Dehydrogenase Inhibitor Resistance.

Emergence of pathogens with decreased sensitivity to succinate dehydrogenase inhibitor fungicides is a global agronomical issue. Analysis of Didymella tanaceti isolates (n = 173), which cause tan spot of pyrethrum (Tanacetum cinerariifolium), collected prior to (2004 to 2005) and after (2009, 2010, 2012, and 2014) the commercial implementation of boscalid in Tasmanian pyrethrum fields identified that insensitivity developed over time and has become widespread. To evaluate temporal change, isolates were characterized for frequency of mutations in the succinate dehydrogenase (Sdh) B, C, and D subunits associated with boscalid resistance, mating type, and SSR genotype. All isolates from 2004 and 2005 exhibited wild-type (WT) Sdh alleles. Seven known Sdh substitutions were identified in isolates collected from 2009 to 2014. In 2009, 60.7% had Sdh substitutions associated with boscalid resistance in D. tanaceti. The frequency of WT isolates decreased over time, with no WT isolates identified in 2014. The frequency of the SdhB-H277Y genotype increased from 10.7 to 77.8% between 2009 and 2014. Genotypic evidence suggested that a shift in the population structure occurred between 2005 and 2009, with decreases in gene diversity (uh; 0.51 to 0.34), genotypic evenness (E5; 0.96 to 0.67), genotypic diversity (G; 9.3 to 6.8), and allele frequencies. No evidence was obtained to support the rapid spread of Sdh genotypes by clonal expansion of the population. Thus, insensitivity to boscalid has developed and become widespread within a diverse population within 4 years of usage. These results suggest that D. tanaceti can disperse insensitivity through repeated frequent mutation, sexual recombination, or a combination of both.

Impact of Infection Timing and Autumnal Fungicide Applications on Perennation of Pseudoperonospora humuli and Severity of Hop Downy Mildew.

Pseudoperonospora humuli, causal agent of hop downy mildew, is known to survive winter as systemic mycelium in the crown and developing buds of hop (Humulus lupulus). Field studies were conducted over three growing seasons to quantify the association of infection timing to overwintering of P. humuli and development of downy mildew. Cohorts of potted plants were inoculated sequentially from early summer to autumn, overwintered, and then evaluated for symptoms of systemic downy mildew in emerging shoots. Shoots with systemic P. humuli developed after inoculation at any time in the previous year, with the most severe disease typically resulting from inoculation in August. Independent of the timing of inoculation, diseased shoots emerged coincident with the emergence of healthy shoots, beginning as early as late February and continuing through late May to early June. Surface crown buds on inoculated plants exhibited internal necrosis associated with P. humuli at rates ranging from 0.3 to 1.2%, whereas P. humuli was detected by PCR on 7.8 to 17.0% of asymptomatic buds depending on the timing of inoculation and year. Four experiments were conducted to quantify the impact of foliar fungicides applied in autumn on downy mildew the following spring. There was a small reduction of disease in only one study. Together, these studies indicate that infection by P. humuli that leads to overwintering can occur over a broad period of time, but delaying infection until autumn tends to reduce disease levels in the following year. However, in established plantings, postharvest application of foliar fungicides appeared to have little impact on severity of downy mildew in the ensuring year.

Temperature Influences on Powdery Mildew Susceptibility and Development in the Hop Cultivar Cascade.

The hop cultivar 'Cascade' possesses partial resistance to powdery mildew (Podosphaera macularis) that can be overcome by recently emerged, virulent isolates of the fungus. Given that hop is a long-lived perennial and that brewers still demand Cascade, there is a need to better understand factors that influence the development of powdery mildew on this cultivar. Growth chamber experiments were conducted to quantify the effect of constant, transient, and fluctuating temperature on Cascade before, concurrent to, and after inoculation as contrasted with another powdery mildew-susceptible cultivar, 'Symphony'. Exposure of plants to supraoptimal temperature (26 and 32°C) before inoculation led to more rapid onset of ontogenic resistance in intermediately aged leaves in Cascade as compared with Symphony. Cascade was overall less susceptible to powdery mildew when exposed to constant temperature ranging from 18 to 32°C directly after inoculation. However, cultivar also interacted with temperature such that proportionately fewer and smaller colonies developed on Cascade than Symphony at supraoptimal yet permissive temperatures for disease. When plants were inoculated and then exposed to high temperature, colonies became progressively more tolerant to temperatures of 26 to 30°C with increasing time from inoculation to exposure, as moderated by cultivar, the specific temperature, and their interaction. Subjecting plants to simulated diurnal temperature regimes at the time of inoculation or 24 h later indicated Cascade and Symphony responded proportionately similarly on days predicted to be marginally unfavorable or marginally favorable for powdery mildew, although Cascade was quantitatively less susceptible than Symphony. In sum, this research indicates that Cascade is overall less susceptible to powdery mildew than Symphony, and supraoptimal temperature before, concurrent to, or after infection may interact differentially to moderate disease risk in Cascade. Therefore, cultivar-specific risk assessments for powdery mildew appear warranted.

Fungicide Physical Mode of Action: Impacts on Suppression of Hop Powdery Mildew.

Understanding of the physical mode of action of fungicides allows more efficient and effective application and can increase disease control. Greenhouse and field studies were conducted to explore the preinfection and postinfection duration and translocative properties of fungicides commonly used to control hop powdery mildew, caused by Podosphaera macularis. In greenhouse studies, applications made 24 h before inoculation were almost 100% effective at suppressing powdery mildew, regardless of the fungicide evaluated. However, percentage control of powdery mildew based on the number of pathogen colonies per leaf varied significantly between fungicides with increasing time from inoculation to application, ranging from 50 to 100% disease control depending on the fungicide. Fluopyram or fluopyram + trifloxystrobin was particularly efficacious, suppressing nearly all powdery mildew development independent of application timing. In translocation studies, fluopyram and flutriafol were the most effective treatments in each of two separate experiments, resulting in zones of inhibition of 1,036 and 246.3 mm2, respectively, on adaxial leaf surfaces when a single droplet of each fungicide was applied to the abaxial surface of leaves. In field experiments, all fungicide treatments provided nearly complete control of powdery mildew infection when applied before inoculation. Levels of disease control decreased with time depending on treatment, showing trends similar to those observed in greenhouse studies. In the 2017 field experiments, high levels of disease control (>75%) were observed at postinoculation time points for all treatments tested, whereas the same fungicides were more sensitive to application timing in a different year. Findings from this research indicate that differences in efficacy between fungicides are small when applications are made preventively, but postinfection activity and translaminar movement of certain fungicides may render some more effective depending on application coverage and preexisting infection.

A Quantitative PCR Assay for Detection and Quantification of Fusarium sambucinum.

Fusarium sambucinum is an ascomycete that has been isolated from a broad range of plant hosts, including hop (Humulus lupulus L.), where it acts as a causal agent of Fusarium canker, a disease that can impact cone quality and yield in severe cases. Current diagnostic methods rely on isolation of the fungus from plant tissue, a time- and resource-intensive process with limited sensitivity, complicated by the potential presence of other Fusarium spp. that have been reported on hop. Our objective was to develop a rapid and sensitive diagnostic tool to detect and quantify F. sambucinum in plant tissues. Using a modified random amplified polymorphic DNA PCR assay, we identified a F. sambucinum-specific marker that serves as the target in a TaqMan (hydrolysis) probe quantitative PCR (qPCR) assay that can be used to detect F. sambucinum DNA in a background of plant DNA. When used to screen 52 isolates of F. sambucinum and isolates representing 13 other Fusarium spp., the assay was robust in detecting F. sambucinum while discriminating between F. sambucinum and closely related Fusarium spp., including F. venenatum. Furthermore, this assay reliably detects as little as 1 pg of F. sambucinum DNA in a background of total DNA from plant tissue. Within-sample comparisons of this qPCR assay with traditional cultural isolation methods demonstrated the greater sensitivity of the qPCR-based method for detection of F. sambucinum. When used to screen 220 asymptomatic stem samples, the qPCR assay detected F. sambucinum in 100 samples (45.5%); by comparison, F. sambucinum was detected in only 3 samples (1.4%) by culturing methods. Moreover, quantification of F. sambucinum DNA was possible for 60 of these samples, indicating the utility of the qPCR assay for early detection. This assay should be useful in diagnostic and epidemiological applications to detect and quantify F. sambucinum from multiple hosts and environmental samples.

A Comprehensive Characterization of Ecological and Epidemiological Factors Driving Perennation of Podosphaera macularis Chasmothecia on Hop (Humulus lupulus).

Hop powdery mildew, caused by the ascomycete fungus Podosphaera macularis, is a consistent threat to sustainable hop production. The pathogen utilizes two reproductive strategies for overwintering and perennation: (i) asexual vegetative hyphae on dormant buds that emerge the following season as infected shoots; and (ii) sexual ascocarps (chasmothecia), which are discharged during spring rain events. We demonstrate that P. macularis chasmothecia, in the absence of any asexual P. macularis growth forms, are a viable overwintering source capable of causing early season infection two to three orders of magnitude greater than that reported for perennation via asexual growth. Two epidemiological models were defined that describe (i) temperature-driven maturation of P. macularis chasmothecia; and (ii) ascosporic discharge in response to duration of leaf wetness and prevailing temperatures. P. macularis ascospores were confirmed to be infectious at temperatures ranging from 5 to 20°C. The organism's chasmothecia were also found to adhere tightly to the host tissue on which they formed, suggesting that these structures likely overwinter wherever hop tissue senesces within a hop yard. These observations suggest that existing early season disease management practices are especially crucial to controlling hop powdery mildew in the presence of P. macularis chasmothecia. Furthermore, these insights provide a baseline for the validation of weather-driven models describing maturation and release of P. macularis ascospores, models that can eventually be incorporated into hop disease management programs.

Transcriptome-Derived Amplicon Sequencing Markers Elucidate the U.S. Podosphaera macularis Population Structure Across Feral and Commercial Plantings of Humulus lupulus.

Obligately biotrophic plant pathogens pose challenges in population genetic studies due to their genomic complexities and elaborate culturing requirements with limited biomass. Hop powdery mildew (Podosphaera macularis) is an obligately biotrophic ascomycete that threatens sustainable hop production. P. macularis populations of the Pacific Northwest (PNW) United States differ from those of the Midwest and Northeastern United States, lacking one of two mating types needed for sexual recombination and harboring two strains that are differentially aggressive on the cultivar Cascade and able to overcome the Humulus lupulus R-gene R6 (V6), respectively. To develop a high-throughput marker platform for tracking the flow of genotypes across the United States and internationally, we used an existing transcriptome of diverse P. macularis isolates to design a multiplex of 54 amplicon sequencing markers, validated across a panel of 391 U.S. samples and 123 international samples. The results suggest that P. macularis from U.S. commercial hop yards form one population closely related to P. macularis of the United Kingdom, while P. macularis from U.S. feral hop locations grouped with P. macularis of Eastern Europe. Included in this multiplex was a marker that successfully tracked V6-virulence in 65 of 66 samples with a confirmed V6-phenotype. A new qPCR assay for high-throughput genotyping of P. macularis mating type generated the highest resolution distribution map of P. macularis mating type to date. Together, these genotyping strategies enable the high-throughput and inexpensive tracking of pathogen spread among geographical regions from single-colony samples and provide a roadmap to develop markers for other obligate biotrophs.

Development of a Diagnostic Assay for Race Differentiation of Podosphaera macularis.

Hop powdery mildew (caused by Podosphaera macularis) was confirmed in the Pacific Northwest in 1996. Before 2012, the most common race of P. macularis was able to infect plants that possessed powdery mildew resistance based on the R-genes Rb, R3, and R5. After 2012, two additional races of P. macularis were discovered that can overcome the resistance gene R6 and the partial resistance found in the cultivar Cascade. These three races now occur throughout the region, which can complicate management and research efforts because of uncertainty on which race(s) may be present in the region and able to infect susceptible hop genotypes. Current methods for determining the races of P. macularis are labor intensive, costly, and typically require more than 14 days to obtain results. We sought to develop a molecular assay to differentiate races of the fungus possessing virulence on plants with R6, referred to as V6-virulent, from other races. The transcriptomes of 46 isolates of P. macularis were sequenced to identify loci and variants unique to V6 isolates. Fourteen primer pairs were designed for 10 candidate loci that contained single nucleotide polymorphisms (SNP) and short insertion-deletion polymorphisms. Two differentially labeled locked nucleic acid probes were designed for a contig that contained a conserved SNP associated with V6-virulence. The resulting duplexed real-time PCR assay was validated against 46 V6 and 54 non-V6 P. macularis isolates collected from the United States and Europe. The assay had perfect discrimination of V6-virulence among isolates of P. macularis originating from the western U.S. but failed to predict V6-virulence in three isolates collected from Europe. The specificity of the assay was tested with different species of powdery mildew fungi and other microorganisms associated with hop. Weak nonspecific amplification occurred with powdery mildew fungi collected from Vitis vinifera, Fragaria sp., and Zinnia sp.; however, nonspecification amplification is not a concern when differentiating pathogen race from colonies on hop. The assay has practical applications in hop breeding, epidemiological studies, and other settings where rapid confirmation of pathogen race is needed.

First report of Powdery Mildew Caused by Golovinomyces ambrosiae on Cannabis sativa in Oregon.

Oregon is the second largest producer of hemp in the United States with 25,900 ha of hemp licensed to growers in 2019, a nearly six-fold increase over the previous year (Perkowski 2019, Capital Press). Industrial hemp has a wide range of uses including textiles to nutritional supplements; in Oregon, hemp has become one of the most economically promising crops and is mainly cultivated for cannabidiol (CBD) production. Between 2018 and 2019, multiple independent greenhouse growers in western Oregon reported powdery mildew-like signs and symptoms on leaves and buds of several Cannabis sativa cultivars, including 'Cherry Wine'. Signs of the disease started as small, white, powdery patches, typically on the adaxial sides of leaves, and progressed to coalescent colonies on leaves, stems, and buds. Fungi present on diseased tissues had unbranched hyaline conidiophores that measured 140 to 250 µm and grew erect from caulicolous and amphigenous mycelium (n = 15). Foot cells were cylindrical, often tapered at one or both ends, and measured 80 to 117 × 9.5 to 11.9 µm (n = 15). Conidia were catenescent, hyaline, ellipsoidal to barrel-shaped, lacked fibrosin bodies, and measured 24 to 34 × 12 to 18 µm (n = 50). No chasmothecia were observed. Morphological observations overlapped with several Golovinomyces spp. Including G. ambrosiae, G. cichoracearum, and G. spadiceus (Braun and Cook 2012). Identification was confirmed by bidirectional sequencing and phylogenetic analysis of 1,457 nucleotides from the concatenated internal transcribed spacer (ITS), 28S large ribosomal subunit, and beta-tubulin (TUB2) regions of two isolates using primer pairs ITS1/ITS4 and NL1/LR5, and TubF1/TubR1 respectively (Mori et al. 2000, Qiu et al. 2020, Vilgalys and Hester 1990, White et al. 1990; GenBank Acc. No.: MW248121 to MW248124, MW265971 to MW265972). The Oregon hemp isolates grouped (bootstrap value = 100) in a monophyletic clade with G. ambrosiae accessions from Qiu et al. (2020). Pathogenicity was confirmed by transferring conidia by leaf rub inoculation onto 2-to 4-week-old 'Cherry Wine' potted plants and incubated outdoors at 12 to 22°C. Control plants were mock-inoculated using healthy leaves. Powdery mildew symptoms developed on inoculated plants approximately 14 to 21 days later; control plants were asymptomatic. Identification was confirmed by morphological characterization and sequencing using the aforementioned primers. The hemp isolates were also able to infect detached leaves of Humulus lupulus 'Symphony' via similar inoculations; however, colony development on 'Symphony' was slow and sporulation sparse as was reported by Weldon et al. (2020). Golovinomyces spp. have also been reported on hemp in Kentucky (Szarka et al. 2019), Ohio (Farinas and Peduto Hand 2020), and New York (Weldon et al. 2020). Although reported as G. spadiceus, these reports are also likely G. ambrosiae according to new taxonomic revision of the genus (Qiu et al. 2020). This is the first known report of Golovinomyces ambrosiae causing powdery mildew on hemp in Oregon (OSC 171893). While powdery mildew on hemp currently appears most severe in protected cultivation, rapid expansion of hemp cultivation and introduction of new CBD varieties throughout Oregon could lead to increased powdery mildew risk in outdoor cultivation.

A Multiplex TaqMan qPCR Assay for Detection and Quantification of Clade 1 and Clade 2 Isolates of Pseudoperonospora cubensis and Pseudoperonospora humuli.

The ability to detect and quantify aerially dispersed plant pathogens is essential for developing effective disease control measures and epidemiological models that optimize the timing for control. There is an acute need for managing the downy mildew pathogens infecting cucurbits and hop incited by members of the genus Pseudoperonospora (Pseudoperonospora cubensis clade 1 and 2 isolates and Pseudoperonospora humuli, respectively). A highly specific multiplex TaqMan quantitative polymerase chain reaction (PCR) assay targeting unique sequences in the pathogens' mitochondrial genomes was developed that enables detection of all three taxa in a single multiplexed amplification. An internal control included in the reaction evaluated whether results were influenced by PCR inhibitors that can make it through the DNA extraction process. Reliable quantification of inoculum as low as three sporangia in a sample was observed. The multiplexed assay was tested with DNA extracted from purified sporangia, infected plant tissue, and environmental samples collected on impaction spore traps samplers. The ability to accurately detect and simultaneously quantify all three pathogens in a single multiplexed amplification should improve management options for controlling the diseases they cause.

Population Diversity and Structure of Podosphaera macularis in the Pacific Northwestern United States and Other Populations.

Powdery mildew, caused by Podosphaera macularis, is one of the most important diseases of hop. The disease was first reported in the Pacific Northwestern United States, the primary hop-growing region in this country, in the mid-1990s. More recently, the disease has reemerged in newly planted hopyards of the eastern United States, as hop production has expanded to meet demands of local craft brewers. The spread of strains virulent on previously resistant cultivars, the paucity of available fungicides, and the potential introduction of the MAT1-2 mating type to the western United States, all threaten sustainability of hop production. We sequenced the transcriptome of 104 isolates of P. macularis collected throughout the western United States, eastern United States, and Europe to quantify genetic diversity of pathogen populations and elucidate the possible origins of pathogen populations in the western United States. Discriminant analysis of principal components grouped isolates within three to five geographic populations, dependent on stringency of grouping criteria. Isolates from the western United States were phenotyped and categorized into one of three pathogenic races based on disease symptoms generated on differential cultivars. Western U.S. populations were clonal, irrespective of pathogenic race, and grouped with isolates originating from Europe. Isolates originating from wild hop plants in the eastern United States were genetically differentiated from all other populations, whereas isolates from cultivated hop plants in the eastern United States mostly grouped with isolates originating from the west, consistent with origins from nursery sources. Mating types of isolates originating from cultivated western and eastern U.S. hop plants were entirely MAT1-1. In contrast, a 1:1 ratio of MAT1-1 and MAT1-2 was observed with isolates sampled from wild plants or Europe. Within the western United States a set of highly differentiated loci were identified in P. macularis isolates associated with virulence to the powdery mildew R-gene R6. The weight of genetic and phenotypic evidence suggests a European origin of the P. macularis populations in the western United States, followed by spread of the pathogen from the western United States to re-emergent production regions in the eastern United States. Furthermore, R6 compatibility appears to have been selected from an extant isolate within the western United States. Greater emphasis on sanitation measures during propagation and quarantine policies should be considered to limit further spread of novel genotypes of the pathogen, both between and within production areas.

High Levels of Insensitivity to Phosphonate Fungicides in Pseudoperonospora humuli.

Phosphonate (phosphite; HPO3-2) is fungicidal against oomycetes and certain other organisms. The Fungicide Resistance Action Committee has deemed phosphonate to be at low risk of resistance development, and reduced sensitivity to phosphonate has been reported only occasionally in plant pathogens. Reduced sensitivity to the fungicide fosetyl-Al was documented in the hop downy mildew pathogen, Pseudoperonospora humuli, in the early 2000s, but disease caused by insensitive isolates could still be managed commercially if the fungicide rate was doubled from 2.24 to 4.48 kg/ha. In this research, we document the occurrence of isolates of P. humuli in Oregon that possess even higher levels of insensitivity to fosetyl-Al and other phosphonate fungicides. The median estimated effective concentration required to reduce infection by 50% (EC50) for isolates collected from two farms reporting disease control failures was 2.7% (vol/vol) phosphonate (range = 1.6 to 164.2), which was 1.6 times (range = 0.9 to 96.0) the maximum labeled rate of the phosphonate fungicide utilized. In contrast, the median EC50 for isolates obtained from experimental plots that have received only a single application of a phosphonate fungicide was 0.6% (vol/vol) phosphonate (range = 0.11 to 2.3) or 0.3 times the maximum allowable rate. Sensitivity of isolates to a phosphorous acid fungicide, fosetyl-Al, and a plant nutrient product containing an unspecified level of phosphorous acid were linearly related. Insensitivity to the maximum allowable rate of a phosphorous acid fungicide was widespread within and among hop farms in Oregon. Among 54 isolates assayed for phosphonate insensitivity, 96% had EC50 values that exceeded the maximum allow rate of the fungicide used in the assays. Field studies conducted in 2 years further demonstrated that a phosphorous fungicide, a nutrient product containing phosphorous acid, and fosetyl-Al failed to provide commercially acceptable suppression of downy mildew when applied at the maximum allowable rates and even double these rates, whereas fungicides with different modes of action provided 91% or greater disease control. The whole of this research indicates that P. humuli has been selected to tolerate fosetyl-Al and other phosphonate fungicides at rates four times greater than those used earlier to obtain satisfactory suppression of downy mildew. This finding has implications for management of the disease not only in Oregon but also, in other production regions should insensitive isolates be introduced on infected planting material.

Prediction of Spread and Regional Development of Hop Powdery Mildew: A Network Analysis.

Dispersal is a fundamental aspect of epidemic development at multiple spatial scales, including those that extend beyond the borders of individual fields and to the landscape level. In this research, we used the powdery mildew of the hop pathosystem (caused by Podosphaera macularis) to formulate a model of pathogen dispersal during spring (May to June) and early summer (June to July) at the intermediate scale between synoptic weather systems and microclimate (mesoscale) based on a census of commercial hop yards during 2014 to 2017 in a production region in western Oregon. This pathosystem is characterized by a low level of overwintering of the pathogen as a result of absence of the ascigerious stage of the fungus and consequent annual cycles of localized survival via bud perennation and pathogen spread by windborne dispersal. An individual hop yard was considered a node in the model, whose disease status in a given month was expressed as a nonlinear function of disease incidence in the preceding month, susceptibility to two races of the fungus, and disease spread from other nodes as influenced by their disease incidence, area, distance away, and wind run and direction in the preceding month. Parameters were estimated by maximum likelihood over all 4 years but were allowed to vary for time transition periods from May to June and from June to July. The model accounted for 34 to 90% of the observed variation in disease incidence at the field level, depending on the year and season. Network graphs and analyses suggest that dispersal was dominated by relatively localized dispersal events (<2 km) among the network of fields, being mostly restricted to the same or adjacent farms. When formed, predicted disease attributable to dispersal from other hop yards (edges) associated with longer distance dispersal was more frequent in the June to July time transition. Edges with a high probability of disease transmission were formed in instances where yards were in close proximity or where disease incidence was relatively high in large hop yards, as moderated by wind run. The modeling approach provides a flexible and generalizable framework for understanding and predicting pathogen dispersal at the regional level as well as the implications of network connectivity on epidemic development.

Cooperation and Coordination in Plant Disease Management.

Scaling of management efforts beyond the boundaries of individual farms may require that individuals act collectively. Such approaches have been suggested several times in plant pathology contexts but rarely have been implemented, in part because the institutional structures that enable successful collective action are poorly understood. In this research, we conducted in-depth interviews with hop producers in Oregon and Washington State to identify their motivations for and barriers to collective action regarding communication of disease levels, coordination of management practices, and sharing of best management practices and other data for powdery mildew (caused by Podosphaera macularis). Growers were generally open to and engaged in communication with neighbors and others on disease status in their hop yards and some evidence of higher levels of information sharing on management practices was found. However, growers who had developed extensive knowledge and databases were reluctant to share information viewed as proprietary. Relationships, trust, and reciprocity were facilitating factors for communication and information sharing, whereas lack of these factors and social norms of independence and pride in portions of the grower community were identified as impediments. Given the heterogeneity of trust, lack of confidence in reciprocity, and weak shared norms, communication of disease risk and coordinated management may be most successful if directed at a smaller scale as a series of neighborhood-based partnerships of growers and their immediate neighbors. Developing a disease reporting system and coordinated disease management efforts with more producers and at larger spatial extents would require formalized structures and rules that would provide assurance that there is consistency in disease data collection and reporting, reciprocation, and sanctions for those who use the information for marketing purposes against other growers. Given the analyses presented here, we believe there is potential for collective action in disease management but with limitations on the scope and nature of the actions.

Genotyping-by-Sequencing Reveals Fine-Scale Differentiation in Populations of Pseudoperonospora humuli.

Pseudoperonospora humuli is the causal agent of downy mildew of hop, one of the most important diseases of this plant and a limiting factor for production of susceptible cultivars in certain environments. The degree of genetic diversity and population differentiation within and among P. humuli populations at multiple spatial scales was quantified using genotyping-by-sequencing to test the hypothesis that populations of P. humuli have limited genetic diversity but are differentiated at the scale of individual hop yards. Hierarchical sampling was conducted to collect isolates from three hop yards in Oregon, plants within these yards, and infected shoots within heavily diseased plants. Additional isolates also were collected broadly from other geographic regions and from the two previously described clades of the sister species, P. cubensis. Genotyping of these 240 isolates produced a final quality-filtered data set of 216 isolates possessing 25,227 variants. Plots of G'ST values indicated that the majority of variants had G'ST values near 0 and were scattered randomly across contig positions. However, there was a subset of variants that were highly differentiated (G'ST > 0.3) and reproducible when genotyped independently. Within P. humuli, there was evidence of genetic differentiation at the level of hop yards and plants within yards; 19.8% of the genetic variance was associated with differences among yards and 20.3% of the variance was associated with plants within the yard. Isolates of P. humuli were well differentiated from two isolates of P. cubensis representative of the two clades of this organism. There was strong evidence of linkage disequilibrium in variant loci, consistent with nonrandom assortment of alleles expected from inbreeding and/or asexual recombination. Mantel tests found evidence that the genetic distance between isolates collected from heavily diseased plants within a hop yard was associated with the physical distance of the plants from which the isolates were collected. The sum of the data presented here indicates that populations of P. humuli are consistent with a clonal or highly inbred genetic structure with a small, yet significant differentiation of populations among yards and plants within yards. Fine-scale genetic differentiation at the yard and plant scales may point to persistence of founder genotypes associated with planting material, and chronic, systemic infection of hop plants by P. humuli. More broadly, genotyping-by-sequencing appears to have sufficient resolution to identify rare variants that differentiate subpopulations within organisms with limited genetic variability.

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.

Statistical Power in Plant Pathology Research.

In null hypothesis testing, failure to reject a null hypothesis may have two potential interpretations. One interpretation is that the treatments being evaluated do not have a significant effect, and a correct conclusion was reached in the analysis. Alternatively, a treatment effect may have existed but the conclusion of the study was that there was none. This is termed a Type II error, which is most likely to occur when studies lack sufficient statistical power to detect a treatment effect. In basic terms, the power of a study is the ability to identify a true effect through a statistical test. The power of a statistical test is 1 - (the probability of Type II errors), and depends on the size of treatment effect (termed the effect size), variance, sample size, and significance criterion (the probability of a Type I error, α). Low statistical power is prevalent in scientific literature in general, including plant pathology. However, power is rarely reported, creating uncertainty in the interpretation of nonsignificant results and potentially underestimating small, yet biologically significant relationships. The appropriate level of power for a study depends on the impact of Type I versus Type II errors and no single level of power is acceptable for all purposes. Nonetheless, by convention 0.8 is often considered an acceptable threshold and studies with power less than 0.5 generally should not be conducted if the results are to be conclusive. The emphasis on power analysis should be in the planning stages of an experiment. Commonly employed strategies to increase power include increasing sample sizes, selecting a less stringent threshold probability for Type I errors, increasing the hypothesized or detectable effect size, including as few treatment groups as possible, reducing measurement variability, and including relevant covariates in analyses. Power analysis will lead to more efficient use of resources and more precisely structured hypotheses, and may even indicate some studies should not be undertaken. However, the conclusions of adequately powered studies are less prone to erroneous conclusions and inflated estimates of treatment effectiveness, especially when effect sizes are small.

Susceptibility of Hop Crown Buds to Powdery Mildew and its Relation to Perennation of Podosphaera macularis.

In the Pacific Northwestern United States, the hop powdery mildew fungus, Podosphaera macularis, survives overwintering periods in association with living host tissue because the ascigerious stage of the pathogen is not known to occur in this region. Field experiments were conducted over a 5-year period to describe the overwintering process associated with crown bud infection and persistence of P. macularis. Surface crown buds increased in abundance and size beginning in early July and continuing until mid-September. Buds of varying sizes remained susceptible to powdery mildew until late September to early October in each of 3 years of experiments, with susceptibility decreasing substantially thereafter. Potted plants were inoculated sequentially during early summer to autumn, then evaluated in the following year for development of shoots colonized by the powdery mildew fungus (termed flag shoots) due to bud perennation. Emergence of flag shoots was asynchronous and associated with shoot emergence and elongation. Flag shoots emerged over a protracted period from late February to early June, year dependent. In all 4 years of experiments, some infected buds broke and produced flag shoots after chemical desiccation of shoots in spring, a common horticultural practice in hop production conducted to set training timing and eliminate initial inoculum. Flag shoots were most numerous when plants were inoculated with P. macularis in early summer and, consequently, when powdery mildew was present throughout the entire period of crown bud development. The number of flag shoots produced was reduced from 6.8- to 46.6-fold when comparing the latest versus earliest inoculation dates. However, all inoculation timings yielded flag shoots at some level, suggesting that bud infection that occurs over an extended period of time in the previous season may allow the fungus to perennate. In studies in two commercial hop yards in Washington State, fungicide applications made after harvest reduced the level of powdery mildew on leaves in the current year but did not significantly reduce flag shoots in the following year. Given that bud infection occurred over a 10-week period, flag shoots developed even when plants were exposed to inoculum in October and some flag shoots survived chemical pruning practices, management efforts seem best directed to both preventative measures to reduce the likelihood of bud infection and remedial practices to physically eliminate infected crown buds in the ensuing year.