Final report for GNC15-204
In 2016 112 wild hop accessions were screened for their response to Pseudoperonospora humuli, the causal organism of hop downy mildew and a obligate parasite of hops. These 112 accessions originated from private collections or USDA-sponsored collection trips and encompassed the native distribution of hop within North America. Private collections were made from multiple locations within Minnesota, Michigan, Wisconsin, and Vermont. Additionally, USDA-collected materials originated from Kazahkstan, Manitoba and Sakatchewan, CA, and Arizona, Colorado, Missouri, New Mexico, and North Dakota.
Preliminary experiments indicated that botanical varieties significantly differ in their tolerance to hop downy mildew, which may be related to exposure to environmental factors that are conducive for the establishment of the disease in a field setting and not directly to their ability to tolerate infection. Continued experiments with a larger collection indicated there are significant overlaps in tolerance to hop downy mildew across botanical varieties, indicating that resistance or tolerance may be independent of climatic factors and primarily composed of genetic factors.
In 2017, assessments of the sub-sampled germplasm collection were completed by using a whole plant inoculation method. Individual plants from the sub-sampled collection were selected based on geographic origin, presumed genetic diversity (based on geographic origin), and their respective response to infection by P. humuli. Additionally, we demonstrate that for the purposes of breeding downy mildew tolerant or resistant lines, use of foliar assessments may be indicative of field performance in the future based on correlation analysis of publicly available phenotypic data.
- Collect wild hop germplasm for phenotypic evaluation
- Screen wild hop germplasm for downy mildew resistance
Hop germplasm collection. Locale data were aggregated from the USDA Germplasm Resources Information Network (GRIN) and the University of Minnesota Bell Museum of Natural History, St. Paul, MN (MN). We also collected local landowner observations since certain collections occurred on private property. Seed lots from GRIN were selected to randomly survey hop populations across the known range and included sampling of all three native botanical varieties, a single non-native botanical variety (var. lupulus), and the non-native annual species, H. japonicus. Seeds were surface-sterilized with a solution of 20% bleach for 15 minutes and rinsed with sterile distilled water three times before being placed into 90 mm Petri dishes containing sterile moistened sand. Seeds were stratified at 4 °C for a period of 8 weeks prior to placement in a growth chamber for germination at 20 °C under a 12 h photoperiod. Germinated seedlings were then transplanted into LC8 potting media (SunGro Horticulture, Bellevue, WA) in 50-cell flats and allowed to grow for a period of 3 weeks in a greenhouse under a 16 h photoperiod at 22 °C (± 3 °C) before being transplanted into 1 gal pots. Locations identified via herbaria and landowner observations were visited once in fall (September - December) in 2015 or spring in 2016. Rhizomes were collected from mature plants and if present, inflorescences containing fruits from female plants were sampled to obtain seeds. Rhizomes were washed free from contaminating soil and transplanted into a 1 gal container with LC8 potting media and maintained in a greenhouse devoid of P. humuli under a 16 h photoperiod at 22 °C (± 3 °C) and fertilized once weekly with a solution of 400 ppm N (Peters 20-10-20 NPK, J. R. Peters, Allentown, PA) and irrigated as needed.
Hop botanical variety and germplasm detached-leaf screening. P. humuli inoculum, originated as a composite mixture from isolates collected in Michigan, Minnesota, Oregon, and Wisconsin, was maintained on detached leaves of the susceptible cv. Pacific Gem. For experimental purposes, P. humuli inoculum comprised of sporangial suspensions was prepared by collecting heavily infected leaves of the downy mildew susceptible cv. Pacific Gem and shaking vigorously in a 50 mL Falcon tube with 30 mL of sterile water. Inoculum concentration was estimated and standardized to 50,000 spores/mL with the aid of a hemocytometer.
In an initial set of experiments, we randomly-selected a single genotype from each of three hop botanical varieties, H. l. var. lupuloides, var. lupulus, and var. neomexicanus, and the related annual species Japanese hops (H. japonicus) for resistance screening. A single replicate of five leaves from each plant was collected in the early morning and each leaf was placed individually abaxial surface up onto a sterile paper towel inside of a 90 mm Petri dish wetted with 1.5 mL sterile water. The abaxial leaf surface was misted using a handheld spray bottle with approximately 1 mL of inoculum (US Plastics, Lima, OH). Plates were then placed in a growth chamber (Model #E15, Controlled Environments Ltd., Winnipeg, MB, Canada) at 20 °C with a 14 h photoperiod and incubated for seven days post inoculation (DPI). The leaves were then digitally scanned using a Cannon LiDE 1100 flatbed scanner (Cannon USA, Melville, NY) using default settings on a white background. Images were uploaded into APS ASSESS v2.0 (American Phytopathological Society, St. Paul, MN) and the percent diseased leaf area was determined using standard settings. This experiment was arranged in a randomized-complete block design and repeated six times.
Based on our preliminary results a subsequent experiment was carried out on 112 randomly selected genotypes that were sampled from the same germplasm collection. A single replicate of five leaves from each plant was collected as previously described and screened in an identical manner. This experiment was repeated eight times.
Whole plant screening. The abaxial leaf surfaces of three-week old rooted-cuttings of six selected accessions (‘1006.02’, cv. Centennial, ‘Hohnke’, cv. Pacific Gem, cv. Teamaker, and ‘Waldenheimer’) were inoculated using inoculum prepared as described above by lightly misting the abaxial surfaces of the leaves and incubating for 24 h at 20 °C after placing the whole plant in a plastic bag out of direct sunlight in a greenhouse. Following the incubation period, plants were removed from the plastic bags and placed into a greenhouse devoid of P. humuli with a 16 h photoperiod at 22 °C (± 3 °C) and fertilized once weekly with a solution of 400 ppm N (Peters 20-10-20, J. R. Peters, Allentown, PA) and irrigated as needed. Two weeks following inoculation, five leaves were randomly selected from each of three plants (replicates) and digitally-scanned using the previously described methods. This experiment was repeated six times.
Correlation analysis of the hop downy mildew phenotype. We collected trait mean data from previously published studies reporting the percentage of foliar disease and proportion of systemically-infected shoots or basal spikes (Kralj et al., 1998; Woods and Gent, 2016). Briefly, Woods and Gent (2016) assessed 110 hop cultivars for their proportion of infected shoots from 2005 to 2007 in an unsprayed hopyard that was chemically pruned in both 2006 and 2007 for horticultural reasons. Assessments were made on 14 day intervals over three or four assessment periods during each year. Kralj et al. (1998) assessed 100 hop cultivars and breeding lines in an unsprayed hopyard. Assessments were made twice, during May and June, on leaves up to one meter of height on ten plants and foliar severity was estimated and the degree of infection was calculated. We selected common genotypes from each dataset (N = 44) and conducted a correlation analysis of the combined dataset with the goal of identifying predictive relationships between resistance phenotypes to use in subsequent evaluations of germplasm.
Statistical analysis. Data from our three screening experiments were analyzed independently as mixed effects models as a randomized complete block design. In the first experiment in which we assessed the differences between taxa, taxon was considered a fixed effect and replicate as a random effect. In the second experiment in which we evaluated a larger germplasm collection, country of origin was considered a fixed effect and replicate and genotype nested within country of origin as a random effect. In our third experiment in which we assessed a subset of whole plants, accession was considered a fixed effect and replicate was considered a random effect. In all three experiments, percent foliar disease was the response variable and was log-transformed for subsequent analyses. All mixed effects models and correlation analyses were performed using JMP Pro 13 (SAS Institute Inc., Cary, NC). Additionally, means separation was performed using Tukey’s HSD (α=0.05).
Hop botanical variety and germplasm detached-leaf screening. Our initial characterization of the hop downy mildew resistant phenotype based upon experimental trials of interspecific groups of hop support previous observations made by Hoerner (1940) and Mancino (2013) in which limited, if any, disease develops on the annual species H. japonicus. Our results further indicate that there are significant differences (F= 22.567, P= <0.0001) within the sub-taxonomic groups of hop, with H. lupulus var. lupuloides potentially being a source of novel resistance to P. humuli, though we cannot conclude if this effect was due to the specific genotype being evaluated since only one accession was chosen to represent each taxon.
To further test this hypothesis, we evaluated an expanded set of germplasm from diverse locations which represented four of the five sub-taxonomic groups in Humulus spp., as well as accessions of uncharacterized subtaxonomic status. Statistical analysis of percent foliar disease from 112 accessions indicated there were significant differences (F= 3.4989, P= 0.0337) in percent foliar disease, with accessions originating from Canada being more highly-susceptible than those originating from the United States. Since inoculum used in these assays originated from regions where commercial production occurs in the United States, we cannot conclude if inoculum originating from Canada or Eurasia would give different results though others have suggested this may not play a significant role (Gent et al., 2017; Summers et al., 2015). Previous results have indicated that material from North America may contain limited sources of resistance but these reports have primarily been biased towards breeding varieties that contain significant population structure due to historical introgression events (Woods and Gent, 2016). The introgression of native North American hops with European hops is typified by the wild Manitoban hop, ‘BB1’, and several H. lupulus var. neomexicanus accessions, which was the primary foundation of the English breeding program (Darby, 2006). Given the differences of sub-taxonomic status and country of origin in resistance, we suggest the importance of distinguishing accessions of H. lupulus var. lupuloides recovered from the north central United States, specifically Minnesota and North Dakota for use in breeding hops for Minnesota and proximal production areas, as opposed to southern regions in the Canadian provinces, Saskatchewan and Manitoba, where hop has been collected from. These accessions possessed higher levels of foliar resistance to P. humuli when compared to their more northern (or southern) counterparts.
Whole plant screening. There were significant differences (F= 13.659, P= <0.0001) among the six accessions evaluated for whole plant response to hop downy mildew infection. The two wild accessions from Michigan, ‘Hohnke’ and ‘Waldenheimer’, were as susceptible as cv. Pacific Gem, which displayed moderate susceptibility. Henning et al. (2016) report cv. Teamaker to be highly resistant to systemic (“crown”) infection but in this study it performed similarly to moderately susceptible accessions in terms of foliar resistance. The wild accession ‘1006.02’ displayed intermediate levels of foliar resistance, comparable to that of the downy-mildew tolerant genotype cv. Centennial (Kenny and Zimmermann, 1991), although it was not significantly different from cv. Teamaker or the wild Michigan accession ‘Waldenheimer’. Differences in disease resistance across genotypes may be more pronounced under field conditions, where inocula are likely to be more spatially variable and prone to environmental influences compared to the pathogen favorable conditions created with the controlled environment and standardized inoculum levels deployed in our experiments.
Comparison across these studies is complicated by the fact that foliar assays with hop downy mildew are commonly conducted using a subjective ordinal scale (Henning et al., 2015; 2016) whereas we evaluated foliar severity as an objective quantitative measurement (percent foliar disease) using a digital image analysis tool (APS ASSESS). Our results might have benefited from subsequent evaluation of additional disease phenotypes such as determining the proportion of infected shoots as performed by Woods and Gent (2016). However, this extends the length of time necessary to perform evaluations and detracts from developing a high-throughput method for evaluation of large breeding populations, common in most breeding programs. An additional method commonly employed, though not part of this study, is to inoculate small seedlings and score resistance based upon percentage of seedlings that develop terminal shoot infections. This provides family or population level information about the nature of resistance in any given cross and is considered a main method for evaluation of male breeding lines (Darby, 2005; Hoerner, 1932). This assay may have proved useful for evaluation of seedling plants but difficulty in recovering seed from certain locations didn’t provide enough seedlings to allow such assays to take place. An additional concern is that a systemic infection arises following foliar inoculation thus confounding potential differences in the observed phenotypes.
Correlation analysis of hop downy mildew resistance phenotypes. Previous studies have evaluated resistance to hop downy mildew in the field, but consistency across locations and research groups is often problematic due to differences in disease scoring or experimental methods, which may include controlled inoculations or reliance upon natural infestations. A recent study by Woods and Gent (2016) described associations with region of origin of hop and disease resistance but that study evaluated a collection of related cultivars sharing the historical introgression of a wild Manitoban hop referred to as ‘BB1’ and other founding cultivars. Additionally, a number of studies evaluating genetic or metabolic markers associated with resistance to downy mildew describe the likelihood of numerous genetic loci which influence the downy mildew phenotype (Henning et al., 2015; 2016; Kralj et al., 1998; Parker, 2007). Henning et al. (2016) also evaluated multiple disease phenotypes (basal spikes vs. foliar lesions) of a bi-parental population within different environments (field vs. greenhouse) which led to detection of different genetic loci. Henning et al. (2016) demonstrated correlation (r = 0.54-0.57) between their greenhouse and field screenings but resistance phenotypes varied among environments. Results of our analysis of data from two independent studies (Kralj et al., 1998; Woods and Gent, 2016) evaluating two different downy mildew phenotypes suggest there is a significant linear relationship (r = 0.57, P = <0.001) between levels of foliar and crown resistance, which supports claims made by Henning et al. (2016) that the two phenotypes are correlated. Inconsistencies in resistance phenotypes among hop accessions may be related to differences in pathogen isolates though this is unlikely as Summers et al. (2015) recently demonstrated the lack of genetic diversity present in P. humuli and there is no currently published research to suggest differences in virulence among different isolates. Owing to the similarities between our correlation analysis and those conducted previously, it would seem that the phenotypes are relatively stable across time with environments contributing a larger role to disease manifestation and development due to differences in inoculum levels or climatological factors (Johnson et al., 1983; Woods and Gent, 2016). Alternatively, a more important factor that may influence the outcome of our interpretation is the relative vigor or performance of a given hop accession in a given environment acting as a potential source of variation that might contribute to inconsistencies in observed resistance phenotypes.
Data figures can be found in the thesis "Management Strategies for Hop Downy Mildew Utilizing Fungicides and Host Resistance" (2017, Josh Havill)
Educational & Outreach Activities
Results of this work have been disseminated to local groups during presentations (LaCrosse, WI - 2016, St. Paul, MN - 2016, Burlington, VT - 2017) and posters (Tampa, FL - 2016).
Results of this work have been disseminated to regional professionals during presentations (Cleveland, OH - 2017, Wooster, OH - 2017, Shakopee, MN - 2017).
Further work should emphasize characterizing the genetic basis for downy mildew resistance in these wild hop resources and evaluating the agronomic performance of a subset of individuals that originate from distinct geographic regions.