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.

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.

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.

Black Leg and Chlorotic Leaf Spot Occurrence on Brassicaceae Crop and Weed Hosts.

Black leg (caused by Plenodomus lingam and P. biglobosus) and chlorotic leaf spot (caused by Pyrenopeziza brassicae) are economically important fungal diseases of Brassicaceae crops. Surveys of seed fields and weed hosts were conducted to understand the distribution and prevalence of these diseases in Oregon after black leg and chlorotic leaf spot outbreaks occurred in Brassicaceae crops in 2014. Postharvest black leg ratings were conducted in seed fields of canola, forage rape, and turnip in 2015 and 2016. The incidence of black leg was greater for turnip (51%) than for canola (29%) and forage rape (25%). The overall average disease incidence was greater for seed crops harvested in 2015 (46%) than for crops harvested in 2016 (28%). A disease survey of wild Brassicaceae plants was conducted along Interstate 5 in Oregon. Brassicaceae weed population sites were identified and 40 sites were sampled for these diseases. Black leg and chlorotic leaf spot were present in 60 and 45%, respectively, of the sampled sites. Both species of Plenodomus were detected in weed populations, with P. lingam being the predominant species recovered (95%). The northernmost sample site with black leg was <32 km from the Oregon-Washington border, and the southernmost site with black leg was within 32 km of the Oregon-California border. Chlorotic leaf spot was detected <32 km from the Oregon-Washington border, whereas the southernmost site where it was detected was approximately 164 km from the Oregon-California border. Based on this study, infected crop residues and weed hosts may facilitate the persistence and spread of these pathogens.

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.

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.