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INVESTIGATOR: Ocamb, C. M.; Gent, DA, .

PERFORMING INSTITUTION:
OREGON STATE UNIVERSITY
CORVALLIS, OREGON 97331

MITIGATING THE INCREASING THREAT POSED BY FUSARIUM CANKER IN HOP

NON-TECHNICAL SUMMARY: Fusarium canker has been an increasingly greater threat to hop production in Oregon, Idaho, and Washington and is a priority issue for US hop producers. A better understanding is needed of the epidemiology and ecology of the Fusarium species involved in the development Fusarium canker and new management strategies are required in order to ensure the economic viability of producers in the Pacific Northwest that produce 98% of U.S. hops. We will characterize the diversity of Fusarium sambucinum isolates recovered from hop plants in commercial yards and propagative rootstock material, and will determine if additional Fusarium species are involved in cankered bines. Cover crops commonly rotated in hop yards will be examined to discern whether they are potential hosts for F. sambucinum in greenhouse evaluations. We will determine the impact of cultural practices related to irrigation drip tube placement and spring pruning methods on development of Fusarium canker. We will broadly communicate results to stakeholders throughout the region. Research outcomes include a better understanding of the pathogen biology and disease epidemiology as well as new management strategies for Fusarium canker in hop. Through the proposed outreach activities, we address multiple levels of needs for the hop industries while reducing Fusarium canker threat for US hop production in the Pacific Northwest, as well as articulated priorities of the CPPM "Plant Protection Tools and Tactics" program and the National Roadmap for IPM.

OBJECTIVES: We will characterize the diversity of Fusarium sambucinum isolates recovered from hop plants in commercial yards and propagative rootstock material, and will determine if additional Fusarium species are involved in cankered bines. Cover crops commonly rotated in hop yards will be examined to discern whether they are potential hosts for F. sambucinum in greenhouse evaluations. We will determine the impact of cultural practices related to irrigation drip tube placement and spring pruning methods on development of Fusarium canker. We will broadly communicate results to stakeholders throughout the region.Objective 1. Resolve basic aspects of the epidemiology and ecology of Fusarium canker.Characterize the diversity of Fusarium spp. associated with cankered hop stems.Clarify the host range of F. sambucinum derived from hop on common cover crop spp.Objective 2. Develop non-chemical management approaches for Fusarium canker that reduce the impact of the disease.Quantify the impact of cultural practices related to irrigation placement and spring pruning method on Fusarium canker.Characterize the prevalence, incidence, and population differentiation of F. sambucinum and other Fusarium spp. associated with planting rootstock.Objective 3. Broadly communicate results to stakeholder partners to accelerate learning and adoption of best management practices developed in this project.

APPROACH: We will establish replicate plots in OR and WA to evaluate Fusarium canker development. Pathogen colonization on stems will be quantified using culture-based methods and the quantitative PCR assay that we developed for F. sambucinum. We will collect the basal portions of stems from up to 10 plants per plot, preferentially sampling bines that exhibit basal swelling, aseptically remove the lower 2.5 cm of the stem, split the pieces in half longitudinally, and freeze one half of the tissue at -80C until DNA extraction/processing. Bines on the hills sampled will be visually examined on each collection dates for signs of reproduction of Fusarium spp. on hop stems during the summer. DNA levels of F. sambucinum per unit area of the stem will be quantified by qPCR. We will use one half piece of stem tissue for plating onto a Fusarium-selective medium. Fusarium colonies will transferred to potato dextrose agar and carnation leaf agar for identification to species. Species identification will be confirmed for representative isolates by sequencing of one or more of three barcoding regions amplified by primers targeting the internal transcribed spacer ITS regions, elongation factor gene EF1-α7, or aminoadipate reductase gene lys2.We will use purified F. sambucinum isolates obtained from hop to test for pathogenicity on rye, common vetch, daikon, and crimson clover. Inoculation method will depend on the host. We will include five isolates derived from hop on each cover crop species, with proper positive and negative controls. Within each cover crop type, disease incidence or severity will be analyzed in a generalized linear mixed model framework to determine relative susceptibility of each host and isolate × host interactions.Studies will be conducted in two commercial hop yards in OR and WA to quantify how pruning practices in spring impact the incidence of canker. We will establish replicated plots in commercial yards of 'Citra' and investigate spring pruning by both mechanical and chemical methods. Each pruning treatment will be replicated eight times, with each replicate plot consisting of one row having at least 100 plants. Plot space and associated production costs will be provided by collaborating farms. Treatments will be applied to the same plots during both years of study to understand multi-year treatment effects. The incidence of wilted plants will be rated monthly during May to Aug. The date of the first swelling of the basal portion of the stem will be noted through regular assessment of a subset of plants and pathogen recovery by isolation for confirmation of the causal agent. Data will be analyzed using a generalized linear mixed model appropriate for repeated measures to quantify pruning impacts on disease severity.We will establish replicated plots in a commercial yard of 'Citra' and compare the impact of one vs. two drip lines placement on the incidence of Fusarium canker. Each irrigation treatment will be replicated seven times in a randomized complete block with each replicate plot consisting of one row having at least 100 plants. Plot space and associated production costs will again be provided by Perrault Farms. Treatments will be applied to the same plots during both years of the study to understand multi-year treatment effects. Emitters in the single irrigation line will be 0.52 gallons per hour; emitters in the plots with two drip lines will be 0.26 gallons per hour. Irrigation timing and duration will be per the standard practices of the cooperating grower, which in this yard will be irrigation sets started and ended manually. Therefore, duration of irrigation is constrained by logistics and typically will be sets of 4, 8, or 12 hours, depending on weather and crop stage. Incidence of canker will be quantified over time and analyzed as described above.We will obtain rootstock of three hop cultivars, with one being Citra, from three different hop propagators located in OR and WA. Replicate plant samples for each cultivar × propagator will be evaluated for pathogens by using culture-based methods, sequencing of diagnostic loci, and the quantitative PCR assay that we developed for F. sambucinum as described above. Data will be analyzed by standard mixed model approaches.A basic understanding of population diversity and structure is central to understanding how a pathogen population may be reproducing, spreading, and differentiated based on factors such as geographic region or cultivar. For instance, genetic diversity studies with the hop powdery mildew fungus showed that populations on cultivated and wild hop plants are distinct, and that strains from the PNW have been disseminated across the U.S. in association with planting material. Similar insights could be gleaned from understanding the genetic diversity and differentiation of populations of F. sambucinum obtained from hop rootstock.We will collect up to 180 isolates of F. sambucinum from across OR and WA using a hierarchical sampling approach with levels of the hierarchy including state, source of planting material, each cultivar from each source, and isolates within each yard. We will also collect approximately 20 isolates of F. sambucinum obtained from other hosts and regions to understand the relatively diversity of the isolates from hop.Purified isolates will be prepared for DNA extraction. We recently deeply sequenced two isolate F. sambucinum obtained from potato and hop on each of two PacBio SMRT cells and assembled the reads into two reference genomes using Canu. We will re-sequence the isolates we describe above on an Illumina HiSeq 3000 as 150 bp paired-end reads. Reads will be mapped to one of the reference genomes and variants called using a variant calling program such as GATK. Variants will be quality filtered to omit samples that failed sequencing, had excessively low or high coverage, unacceptably high missingness, or unusual patterns of depth of sequencing.The degree of genetic differentiation among sources of planting material and cultivar within each propagator will be quantified by AMOVA and measures of genotypic diversity using standard approaches. Ordination plots based on principal coordinates analysis and a matrix of pairwise distances will be constructed to visualize the differentiation of isolates from within each of the sources of planting material and cultivar. These analyses will provide evidence of genetic differentiation at multiple spatial scales and whether planting material is a probable source of F. sambucinum in newly established yards.Outreach and educational efforts are embedded in how we conduct research because the goal of this research is to aid growers, industry, and policy makers in reaching informed and appropriate management decisions. Several levels of outreach activities will transfer knowledge to stakeholders. We will focus on stakeholders in the primary hop productions in WA, OR, and ID, but the information will be readily accessible to others through public-facing websites and social media. Emerging research and recommendations will be shared with growers nationwide through on-farm research, field days, industry and university websites. Timely updates and highlights will be disseminated to friends of the Northwest Hop Information Facebook page. Science-based information will be presented directly to farm managers and workers through field days, grower meetings/conferences including annual meetings hosted by OR Hop Growers Commission, WA Hop Growers Commission, and the Hop Research Council, and agricultural consultant company grower meetings through the PNW. Critical information will be published in the "Pacific Northwest Plant Disease Management Handbook". The scientific community will be engaged through presentations at national scientific meetings and through scientific journal articles.

PROGRESS: 2022/09 TO 2023/08
Target Audience:Presentations to the Hop Industry Gent, D. H. 2023. 2023 disease management considerations. Oregon Hop Commission. March 16, St. Paul, Oregon. Gent, D. H. 2023. Nitrogen fertility, pest management, and hop quality factors. Marion Ag Annual Hop and Hazelnut Grower Meeting. March 7, St. Paul, Oregon. Borland, T., and Gent, D. H. 2023. Muliphasic evaluation of pathogenicity, mycotoxin potential, and management of the Fusarium canker fungus. Winter Meeting of the Hop Research Council. January 25, Santa Rosa, California. Wiseman, M. S., and Gent, D. H. 2023. Lack of susceptibility is the new resistance: a multi-omics informed investigation into hop powdery mildew susceptibility genes. Winter Meeting of the Hop Research Council. January 25, Santa Rosa, California. Borland, T., and Gent, D. H. 2023. Putting together the puzzle pieces: accumulating insights on Fusarium Canker. Washington Hop Industry Annual Meeting. January 5. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training was provided to the post-doctoral research associate (RA) via the PI and post-doctoral RA working side-by-side, on rating hop seedlings for canker and root rot as well as conducting isolations from symptomatic tissue samples. Undergradates in the laboratory were trained in preparing hop propagants for processing and conducting subculturing of putative Fusarium isolates for classical identification. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?For Objective 1a,additional Fusarium isolates obtained will be identified by morphological characters and sequencing of DNA barcoding loci. For Objective 1b, study runs will be set up for determination of Fusarium colonization of common cover crops. For Objective 2a, field studies in Oregon and Washington that quantify the impact of irrigation placement and spring pruning method on the incidence of Fusarium canker will continue through the 2024 growing season. For Objective 2b, another set of propagation material (900 plants) will be obtained in the spring/early summer for evaluation of Fusarium presence on rooted cuttings. An estimated 90 other isolates will be sequenced are planned for sequencing in spring 2024 for population evaluations. For Objective 3, presentations will be made on results in Obj. 2a to the hop industry over the 2023-24 winter months at regional and national meetings.

IMPACT: 2022/09 TO 2023/08
What was accomplished under these goals? Objective 1. Resolve basic aspects of the epidemiology and ecology of Fusarium canker. Characterize the diversity of Fusarium spp. associated with cankered hop stems. We developed an isolate collection from 18 hop yards of the cv. 'Citra' across Idaho, Oregon, and Washington to assess the population diversity and genetic structure of F. sambucinum. We described a complex of Fusarium species may be associated cankered stems, with F. sambucinum being the most common but F. solani and F. oxysporum also frequently recovered. Additional collections were made from six additional cultivars from eight yards (five from Oregon; three from Washington). There will be at least 200 additional isolates that have been purified to a single spore culture, and are awaiting identification by morphological characters and sequencing of DNA barcoding loci. b) Clarify the host range of F. sambucinum derived from hop on common cover crop spp. A preliminary study using rye, common vetch, daikon, and crimson clover and two isolates of Fusarium sambucinum was conducted during the summer months to evaluate experimental set-up parameters including seeding rate, inoculum levels, length of study, and approach to sampling the cover crop study. Additional isolates, including from seedlings, are being increased for study runs that will be set up this fall and winter. Objective 2. Develop non-chemical management approaches for Fusarium canker that reduce the impact of the disease. Quantify the impact of cultural practices related to irrigation placement and spring pruning method on Fusarium canker. We conducted field studies in Oregon and Washington in 2023 to quantify the impact of irrigation placement and spring pruning method on the incidence of Fusarium canker. We found a small yet significant effect of mechanical versus chemical pruning in spring on subsequent disease development in both. In three studies conducted in Washington (two farms) and Oregon (one farm), we did not detect a significant effect of drip tube configuration on the severity of disease. However, at one location in Washington where yield was measured, we recorded an 18.4% increase in yield with the double drip tube configuration. The accumulating knowledge indicates that mechanical pruning tends to have a small yet significant effect on later season wilting of bines associated with Fusarium canker. Effects of drip tube configuration on disease development have been non-detectable to date. Characterize the prevalence, incidence, and population differentiation of F. sambucinum and other Fusarium spp. associated with planting rootstock. We obtained 100-plant samples of three different hop varieties from each of three propagators. Plants were examined for canker and root rot symptoms, with symptomatic tissues sampled for the presence of Fusarium spp. by plating onto a selective medium as well as for PCR testing for the presence of F. sambucinum. Analyses of disease data shows that there are differences among propagators and hop varieties. By propagator (across varieties), incidence of canker ranged between 20 and 26% while root rot incidence was 17 to 62%. There was a significant difference in overall root rot levels between two propagators that provided plants of the same three varieties (17 vs 62%). Six varieties of hop were examined, and one variety had the highest incidence of disease (53% of plants had canker; 92% had root rot). The other two varieties that were provided by the propagator with the most-diseased variety, had the lowest incidence of canker. Isolations for Fusarium from stems and roots of plants with symptoms consistent with canker or root rot yielded Fusarium spp. at lower levels compared to disease incidence levels with many isolations being Trichoderma and/or Penicillium spp. Identification of isolates to species is underway. By propagator (across varieties), frequency of Fusarium spp. in isolations from plants ranged between 3 and 28%. Frequency of Fusarium spp. in isolations from plants ranged between 3 and 22% among the six hop varieties examined. DNA from symptomatic tissue has been isolated and screened for F. sambucinum. Preliminary data identifies >60 plant samples as being infected with F. sambucinum according to PCR testing. Two high quality genomes of the pathogen were assembled and annotated as references for the population diversity aspects of this research. We identified a DNA extraction protocol that yielded a suitable yield of high quality DNA to support Illumina sequencing of F. sambucinum. DNA from 90 isolates of F. sambucinum was extracted and sequenced bidirectionally by Illumina NextSeq 2000 P2 as 150 bp inserts. Data analysis is currently underway. An estimated 90 other isolates will be sequenced are planned for sequencing in spring 2024. Objective 3. Broadly communicate results to stakeholder partners to accelerate learning and adoption of best management practices developed in this project Presentations were made to the hop Industry (see previous section).

PUBLICATIONS (not previously reported): 2022/09 TO 2023/08
Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Abstracts Presented at Professional Meetings Borland, T., E. Lopez, S. Massie, C. M. Ocamb, W. J. Thomas, and D. H. Gent. 2023. CSI: Hop Yard - collecting evidence to solve the Fusarium canker mystery. American Phytopathological Society, 2023 Annual Meeting, Denver, CO, Aug 12-16.