Project : USDA ARS

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Research Project: Breeding, Genomics, and Integrated Pest Management to Enhance Sustainability of U.S. Hop Production and Competitiveness in Global Markets

Location: Forage Seed and Cereal Research Unit

2022 Annual Report

Objectives
The goal of this project is to maintain and enhance the competitiveness of the U.S. hop industry through development of publicly available genetic resources, tools, and knowledge-based pest management systems. This will be accomplished through interdisciplinary research that addresses high priority documented stakeholder needs. Over the next 5 years, the specific objectives to be accomplished are: Objective 1: Develop and release new hop cultivars and germplasm possessing superior disease resistance, yield, and brewing characteristics. (Henning) Objective 2: Identify, characterize, and validate molecular markers associated with qualitative and quantitative resistance to important foliar diseases. (Henning) Objective 3: Identify molecular markers associated with virulence of Podosphaera macularis and use the information to rapidly determine pathogen races. (Gent) Objective 4: Quantify the aggressiveness, fitness, and race of Podosphaera macularis isolates able to overcome partial host resistance and identify new sources of resistance to diverse strains of the pathogen in public germplasm. (Gent) Sub-objective 4A: Characterize the aggressiveness, fitness, and race of Podosphaera macularis virulent on the cultivar Cascade. (Gent) Sub-objective 4B: Identify and quantify the impact of supraoptimal temperature on host susceptibility to and development of powdery mildew on the cultivar Cascade. (Gent) Sub-objective 4C: Characterize publicly available male germplasm for its reaction to multiple strains of Podosphaera macularis. (Gent)

Approach
Objective 1 Research Goal: Develop multiple pathogen resistant germplasm or cultivars. Controlled crosses of cultivars two cultivars will be made using resistant males. Progeny will be screened for disease resistance and phenotypic traits including hop aroma. Selected offspring will be advanced for further evaluation. Objective 2 Research Goal: Identify molecular markers associated with plant resistance to P. humuli and P. macularis. Genetic maps and genome-wide surveys for marker association will be conducted using a bi-parental mapping population derived from a powdery mildew resistant female line and a downy mildew resistant male line. Objective 3 Hypothesis: Markers associated with pathogenic variation in P. macularis can be identified. Isolates of P. macularis from Pacific NW will be collected and race-validated using differential host panels. RNA will be collected from P. macularis isolates and subsequently sequenced using next gen sequencing. SNP markers will be identified from this data. SNPs will be used to fingerprint different isolates and a set of unique markers for each isolate identified. Sub-objective 4A Hypothesis: Strains of P. macularis virulent on Cascade are specifically adapted to this cultivar. Controlled environment experiments will be conducted to determine the aggressiveness and fitness of isolates of P. macularis originating from Cascade to provide fundamental information to guide breeding efforts and disease risk assessment. These races will also be characterized using a differential set of cultivars possessing different resistance genes. Sub-objective 4B Hypothesis: Partial resistance to powdery mildew in the Cascade is modulated by brief exposure to supra-optimal temperature. An extensive set of controlled environment studies will be conducted to define the environmental conditions that moderate infection risk on the Cascade to derive rules for adapting the HOPS powdery mildew risk index to Cascade and similar cultivars. Sub-objective 4C Research Goal: Characterize resistance of USDA males to multiple strains of powdery mildew. A set of 150 individuals –resistant to downy mildew--will be tested for their resistance to multiple races of P. macularis. Resistance to three different isolates-each with unique virulence genes—will be sequentially scored across all male lines. Remaining resistant individuals will be further evaluated to determine the nature of resistance.

Progress Report
In support of Objective 1, ARS scientists in Corvallis, Oregon, completed the third year of evaluation for disease resistance to both powdery mildew and downy mildew in germplasm developed for this objective. Five female Cascade-based crosses were identified as disease-free over the course of three years. Three Centennial-based crosses were free from powdery mildew and exhibited low incidence of downy mildew the third year of evaluations in the field. Of these offspring, only one produced abundant hop cone product with desirable aroma qualities suggesting strong potential for future variety or germplasm release. While not listed in the project plan, additional crosses using two USDA female germplasm lines possessing resistance to both diseases were crossed with male USDA 64037M and simultaneously evaluated for both diseases, cone aroma, and for production potential. Four additional female offspring exhibited no disease symptoms, high yield potential and excellent hop cone aroma. In support of Objective 2, ARS scientists completed all quantitative trait locus (QTL) studies for both powdery and downy mildew resistance in a population segregating for quantitative resistance to both diseases. The journal article covering quantitative resistance to downy mildew was submitted for publication while the journal article covering quantitative resistance to powdery mildew is in preparation. Downy mildew resistance was scored and shown to be polygenic in nature with small effects for each loci. Five minor disease loci on chromosomes 1, 2 and 5 were identified. Gene content of these loci is under investigation. Quantitative resistance to powdery mildew also appears to be under polygenic control with one major and three minor disease loci controlling expression. The major disease locus is located on chromosome 7 and covers a 5-Mb region consisting of nine clusters of putative R-genes along with downstream disease or stress-response genes. Breeder-friendly molecular markers for quantitative resistance to powdery mildew are currently under development and validation. In support of Objective 3, ARS scientists published a journal article describing the development, validation, and use of a simple and rapid diagnostic assay that can differentiate between two widely prevalent pathogenic races of the hop powdery mildew fungus. From a single diseased leaf, we can now predict pathogen virulence in hours, which is critical for knowing which cultivars may be susceptible to infection by a particular isolate of the pathogen. We used the diagnostic assay to sample hundreds of powdery mildew samples from hop and hemp to determine which strains of the fungus occur on widely grown cultivars to understand disease risk at the landscape level. In support of Sub-objective 4A, ARS scientists completed studies on the cultivar ‘Cascade’ to characterize new variants of the hop powdery mildew fungus that emerged in the United States, and which cause severe disease on this cultivar. We expanded this research to another cultivar ‘Citra’, which is now the most widely grown hop cultivar in the industry, to understand what strains of the fungus occur on Citra and if the fungus is becoming more aggressive on this cultivar. We found that multiple strains of the hop powdery mildew fungus may cause disease on Citra. We did not find support of Citra possessing a known resistance gene to powdery mildew. Rather, all strains of the fungus evaluated were able to grow on Citra at some level. Citra-derived strains and strains representing the three predominant variants of the hop powdery mildew found in the Pacific Northwest were similarly aggressive on this cultivar. These results show that isolates of the fungus found on Citra are varied, yet at this time are neither locally adapted for increased virulence to this particular cultivar, nor generally adapted for enhanced virulence on other susceptible cultivars. This has important implications for how, when, and where host resistance is developed and deployed in the landscape to maximize the durability of genetic disease resistance. In support of Sub-objective 4B, ARS scientists planned, initiated, and conducted field studies to validate a decision aid that estimates the risk of hop powdery mildew on the cultivar Cascade and similarly susceptible cultivars. We also conducted a fourth year of studies to understand the importance of late season fungicide applications on Cascade. Under low disease pressure conditions in 2021, adjusting application intervals based on the revised powdery mildew risk index saved up to two fungicide applications as compared to treating on a set interval. We again found no benefit from fungicide applications made on Cascade when applied after bloom. These studies provide guidance on developing effective management programs for powdery mildew on Cascade with minimal inputs. In support of Sub-objective 4C, ARS scientists continued a collaboration with researchers at the University of Minnesota to validate molecular markers associated with an important source of resistance to powdery mildew, called R1. Evaluation of plants that likely contain or likely do not contain this form of resistance was completed. This will enable validation of DNA-based assays that can quickly identify R1-based resistance in plants without the need for laborious and time-consuming methods based on inoculation.

Accomplishments
1. Two new male hop genomes assembled. The hop genome is large and highly heterozygous, and this complexity is compounded by hop having both X and Y sex chromosomes in male lines and only X chromosomes in females. Most crops do not have sex chromosomes and possess flowers that are both male and female on the same plant. Crop genome sequences are useful for researching the genetics and biology of the crop and advancing efforts to breed improved cultivars. Previously, a draft sequence was only available for the female USDA ‘Cascade’ cultivar. No genome sequences for male hops were available. ARS and Oregon State University scientists in Corvallis, Oregon, completed the first sequencing of two male genomes (USDA 19058M, USDA 21375M). Together, these genomes form a three-part genome resource of related hop plants, as USDA 21375M is the offspring from USDA lines ‘Cascade’ and ‘19058M’. These new genomes will provide genomic researchers with valuable information on the genetics of hop as well as provide sequence data for Y-chromosomes and contribute towards our understanding of evolution of plants in the Cannabaceae family.

2. Development of a modified powdery mildew risk index. A strain of the hop powdery mildew fungus has emerged in Western United States. In 2012, the strain was aggressive on the cultivar Cascade, the most widely grown cultivar at the time, which previously was not severely affected by the disease. ARS researchers in Corvallis, Oregon, characterized how this strain of the fungus and Cascade respond to high temperature, and used this knowledge to develop a revised disease risk index to better inform growers. A beta version of a web app and an automated email delivery system of the powdery mildew risk index was developed in partnership with university collaborators for validation and dissemination to stakeholders. In field testing, use of the risk index has reduced the number of fungicide applications required by as much as one-third compared to standard practices in certain years.

U.S. DEPARTMENT OF AGRICULTURE

Forage Seed and Cereal Research Unit: Corvallis, OR