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) prior to inoculation led to more rapid onset of ontogenic resistance in intermediately aged leaves in Cascade as compared to 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 similar 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.

First Report of Powdery Mildew Caused by Podosphaera macularis on Hemp in Oregon

In Oregon, hemp (Cannabis sativa) production has increased substantially after cultivation was legalized in the 2014 and 2018 Farm Bills. Typically, hemp species are affected by powdery mildew caused by Golovinomyces species. This paper reports on hop powdery mildew caused by Podosphaeria macularis, found colonizing hemp in natural conditions. The occurrence of the hop powdery mildew fungus on hemp may have management implications for both the hemp and hop industry.

Green Strategies of Powdery Mildew Control in Hop: From Organic Products to Nanoscale Carriers

Humulus lupulus L. is a long-lived, perennial, herbaceous, and dioecious climbing plant. The foremost producers in the European Union are Germany, the Czech Republic, Poland, Slovenia, and Spain. The Spanish cultivated area is concentrated in the province of León. Powdery mildew, caused by Podosphaera macularis, menaces hop production and quality in all hop growing regions located in the Northern hemisphere, colonizing leaves, petioles, inflorescences, and finally cones. In this work, powdery mildew control was monitored, comparing nine fungicide strategies: five organics, two integrated disease management (IDM)-based, with and without Nutragreen® nanoscale carrier, and two conventional treatments (CON) with and without Nutragreen® nanoscale carrier. The organic treatments were able to diminish P. macularis on leaves, but no effect was observed in cones. CON treatments reduced the infection on leaves and cones and increased the cone quantity and quality. Likewise, IDM-based treatments provided satisfactory results as they diminished powdery mildew on leaves and cones. Finally, dose reduction using a Nutragreen® nanoscale carrier showed beneficial effects in the control of powdery mildew compared to the commercial dose. Hence, the use of nanoscale carries permits a 30% reduction in pesticide dose, which optimizes yield and hop quality, reduces risks linked to pesticides, and aids in compliance with public and international policy demands.

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 presumed to discharge ascospores 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 the duration of leaf wetness and prevailing temperatures. Podosphaera 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 that can eventually be incorporated into hop disease management programs.

Characterization of Podosphaera macularis Derived from the Hop Cultivar ‘Strata’ and Strata’s Resistance to Powdery Mildew in Oregon

Powdery mildew is one of the most destructive diseases of hop. Host resistance is a most efficient means of managing the disease, but resistance may not be durable. The cultivar Strata was developed by Oregon State University, and the patent application stated the plant was resistant to powdery mildew based on initial greenhouse screening and 8 years of observations at three locations in western Oregon. In 2019, powdery mildew colonies were reported in a commercial yard of Strata near Woodburn, Oregon. Two clonal isolates of Podosphaera macularis were isolated via single-chain transfers from colonies and characterized by inoculating each of seven differential cultivars. Cultivars with Rb, R3, and R5 were susceptible to the isolates. The susceptibility of Strata to powdery mildew was characterized by inoculating seven isolates of P. macularis with known virulences onto leaves of Strata. Sporulation appeared on Strata leaves with all of the differential isolates independent of their specific virulence, but only at trace levels with isolates possessing V6-virulence. Strata-derived isolates were phenotypically indistinguishable from Cascade-adapted isolates when inoculated onto the cultivars Cascade, Strata, and Symphony, as only Strata-derived and Cascade-adapted isolates could cause severe disease on Cascade and Strata. Like Cascade-adapted isolates, Strata-derived isolates also lack virulence on hop plants possessing the R-gene R6. These findings should inform both disease risk assessment in the field and future breeding tactics and strategies involving Strata. Further characterization of Strata is warranted to determine if its resistance is under the same genetic control as Cascade.

First report of Podosphaera macularis causing hop (Humulus lupulus) powdery mildew in Brazil

The hop (Humulus lupulus L.) is a dioecious perennial climbing plant grown commercially worldwide. Wild hops are widely distributed throughout the Northern Hemisphere, Europe, Asia, and North America (Neve, 1991). In the Southern Hemisphere, some of the leading hop-producing countries include South Africa, Australia, and New Zealand. Brazil began hop production less than 5 years ago. In January 2019, amphigenous white powdery circular fungal colonies were observed on the leaves and stems of hop plants (cultivar Chinook) within a 900m2 hop garden in Lages municipality, Santa Catarina State, southern Brazil. The incidence of the disease was present on almost 100 per cent of “Chinook” cultivar plants and diseased foliage was collected to identify the pathogen and used to inoculate healthy plants. Hop powdery mildew lesions with hyaline and septate mycelium with chains of unicellular conidia (n =100) hyaline, barrel-shaped, mean of length/width ± standard deviation 25–27 × 13–18 µm ± 0.980, with fibrosin bodies, and conidiophores erect with cylindrical foot cells, were visible within 10 days. The causal agent was identified as Podosphaera macularis (Wallr.:Fr.) Lind (synonym S. humuli (DC.) Burrill) on the basis of conidial shape, size and host range (Royle 1978; Braun 1987; Mahaffee et al., 2009), complemented with the present molecular analysis. Chasmothecia have not been observed in the field to date. A conidial suspension of 200 ml at concentration of 1.4 x 105 was mixed with 5ul of Tween® 20 for the pathogenicity assay. Ten plants of 9-month-old of hop “Chinook” cultivar, were inoculated with 5 ml of the conidial suspension using a manual spray. The control plot was only sprayed with water. The inoculated plants were maintained at 22ºC ± 1ºC with a 12-hour photoperiod and 65% relative humidity. White mycelia were visible first on the adaxial leaf surfaces of the inoculated younger leaves after 10 days and the disease severity reached between 2 to 5%. No symptoms were observed at the control plot. P. macularis infected most aerial plant tissues of the inoculated plants and caused approximately 50% of cones losses. P. macularis conidia were collected from the infected leaf tissue with a sterile soft camel-hair brush and DNA was extracted using a Wizard Genomic DNA extraction kit. The primers ITS1/ITS4 (White et al., 1990) were used to amplified and sequenced a fragment of the ITS region. PCR products were subjected to Sanger Sequencing to confirm sample species. The resulting 522-bp sequence was deposited into GenBank (accession n°. MN630490). BLASTn showed a 99.81% sequence identity with the CT1 isolate of P. macularis from H. lupulus (MH687414). The presence and identification of P. macularis in hop production regions is a new challenge to growers in Brazil. Research related to the knowledge of the disease cycle, epidemiology, and control strategies for the integrated management should be conducted, as there are no registered fungicides for powdery mildew on hop in Brazil. To our knowledge, this is the first report of P. macularis in Brazil, as well as in South America. References Braun, U. (1987) A Monograph of the Erysiphales (Powdery Mildews). J. Cramer, Berlin, German Democratic Republic. p 113. Mahaffee, W. F., Pethybridge, S.J., Gent, D.H (2009) Compendium of hop diseases and pests. The American Phytopathological Society Press, Saint Paul, Minnesota. Neve R. A (1991). Hops. Chapman and Hall: London. Royle, D. J (1978). Powdery mildew of the hop. Pages 381-409 in: The Powdery Mildews. D. M. Spencer, ed. Academic Press, New York. White, T. J., Bruns, T., Lee, S., and Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. pp. 315-322 in: PCR Protocols: A Guide to Methods and Applications. M. Innis, D. Gelfand, J. Sninsky, and T. White, eds. Academic Press, San Diego.

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 non-specific amplification occurred with powdery mildew fungi collected from Vitis vinifera, Fragaria sp., and Zinnia sp.; however, non-specification 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.

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.