Progress 09/01/23 to 08/31/24
Outputs
Target Audience:Presentations were made to the Hop Industry members at meetings of the Hop Research Council, which represents industry members across the globe, as well presentations made at the Idaho Hop Commission and the Washington Hop Industry Annual Meeting. Presentations were also given at professional meetings to fellow scientists and researchers. Changes/Problems:We encountered difficulties with obtaining hop propagants from hop propagators during 2024; one of three propagators shut down and the other two propagators were concerned about our discovery of F. sambucinum on a subset of their young plants in 2023 samples. One propagator was reassured when we conveyed that the results from propagant examinations were be publicized only in scientific journals and would not presented at industry meetings, since the identity of the propagators is difficult to protect as there are only a few propagators and all cultivars we examined had a subset of plants that were found to have F. sambucinum associated with roots and/or stems. We are still working on obtaining propagants from the second propagator. We found that the PCR primers used for standard PCR testing of tissue samples from hop propagants were less sensitive than the primers we developed for realtime PCR, so propagant testing of 2024 samples will be done by only realtime PCR. The realtime PCR results for the 2023 propagant samples as well as the incidence of root rot was much greater than we anticipated when drafting the proposal, which lead to delays in conducting investigations on for Objective 1b, where we are investigating the colonization of cover crops. What opportunities for training and professional development has the project provided?Further training was provided to the post-doctoral research associate (RA) via the PI and RA working side-by-side, setting up cover crop studies in the greenhouse and rating hop propagants for canker and root rot as well as conducting isolations from symptomatic tissue samples. Two new undergraduates in the laboratory were trained in preparing hop propagants for processing, extraction of nucleic acids, running standard PCR reactions, and subculturing of putative Fusarium isolates for classical taxonomic identification. One undergraduate was trained in realtime PCR assays. A graduate student (PHD candidate) supported on different dollars conducted the genomic examinations of Fusarium sambucinum for the evaluations of population diversity, learning about the science behind population genetics as well as visiting another USDA lab in Peoria, IL, to be trained in mycotoxin production. How have the results been disseminated to communities of interest?Ocamb (PI) and Gent (Co-PI) were interviewed by OSU Extension and an article 'OSU studies the effects of pathogen attacking Pacific Northwest hop fields' was published on-line in June 2024 outlining the disease problem and aspects of research with an acknowledgement of support provided by NIFA-CPPM. https://extension.oregonstate.edu/news/osu-studies-effects-pathogen-attacking-pacific-northwest-hop-fields What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Resolve basic aspects of the epidemiology and ecology of Fusarium canker. 1a. Characterize the diversity of Fusarium spp. associated with cankered hop stems. Develop a publication on the diversity of Fusarium spp. associated with cankered hop stems. 1b. Clarify the host range of F. sambucinum derived from hop on common cover crop spp. Complete the additional runs using F. sambucinum on cover crops in the greenhouse. Conclude quantitative PCR testing of cover crop root samples. Potentially conduct studies in a growth room with cover crop seedlings growing in test tubes. Objective 2. Develop non-chemical management approaches for Fusarium canker that reduce the impact of the disease. 2a. Quantify the impact of cultural practices related to irrigation placement and spring pruning method on Fusarium canker. Collect 2024 yield measurements, analyze data, and begin publication on effects of irrigation placement and spring pruning method on Fusarium canker. 2b. Characterize the prevalence, incidence, and population differentiation of F. sambucinum and other Fusarium spp. associated with planting rootstock. Obtain propagants from a second propagator for evaluations. Analyze data and initiate publication on the prevalence and incidence of F. sambucinum and other Fusarium spp. associated with hop propagants. Conclude data analysis to quantify population differentiation based on state of origin, source of planting material, hop yards, and isolate within a hop yard. Develop publication on this information as well as the information developed on the biosynthetic gene clusters common across the F. sambucinum genomes. Objective 3. Broadly communicate results to stakeholder partners to accelerate learning and adoption of best management practices developed in this project. Additional presentations will be given to the hop industry in 2025.
Impacts
What was accomplished under these goals?
Objective 1. Resolve basic aspects of the epidemiology and ecology of Fusarium canker. 1a. Characterize the diversity of Fusarium spp. associated with cankered hop stems. We completed an isolate collection from 18 hop yards of the cv. 'Citra' across Idaho, Oregon, and Washington and 8 other yards representing six distinct cultivars to assess the diversity of Fusarium species associated with cankered stems and the population diversity and genetic structure of F. sambucinum. Isolates were identified to species by morphological characters and confirmed by sequencing of the TEF-1 alpha gene. We demonstrated that a complex of at least 11 Fusarium species may be associated cankered stems, with F. sambucinum being the most common. Fusarium oxysporum, F. solani, and F. clavum occurred less frequently but were recovered consistently. We found that species diversity was similar across cultivars, although greater species diversity was found in Washington as compared to the other states. 1b. Clarify the host range of F. sambucinum derived from hop on common cover crop spp. During late winter and early summer in 2024, replicated studies were set up using rye, common vetch, daikon, and crimson clover and two isolates of F. sambucinum and have been sampled; the first of two runs evaluating two additional isolates of F. sambucinum was also conducted and sampled. Root samples were collected for quantitative PCR testing and extractions of nucleic acids from the samples are underway. Objective 2. Develop non-chemical management approaches for Fusarium canker that reduce the impact of the disease. 2a. Quantify the impact of cultural practices related to irrigation placement and spring pruning method on Fusarium canker. We initiated a final year of fields studies in Oregon and Washington in 2024 to quantify the impact of irrigation placement and spring pruning method on the incidence of Fusarium canker. The impact of irrigation delivery with one versus two drip irrigation tubes is being evaluated at three locations and spring pruning method is being evaluated at two locations. Data collection is ongoing. Yield measurements, data summary, and analysis are planned for later in 2024. 2b. 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 in 2023. 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. Confirmation of Fusarium species is still underway but a range of Fusarium species were isolated form propagants sampled in 2023 and include F. oxysporum, F. solani, F. equiseti/F. incarnatum, F. avenaceum, and F. citri but interestingly, no F. sambucinum was isoalted from propagants. 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. Standard PCR testing was conducted at least twice on each tissue sample and followed by realtime PCR screening (which is more sensitive). The latter testing has not been concluded but based on the standard PCR tests, % propagants that had F. sambucinum ranged from 2 to 50% among the nine sets of 100-plant samples. No variety had a total absence of F. sambucinum. Realtime PCR testing completed to date reveals additional positives for plant samples that tested negative for F. sambucinum by standard PCR testing, approximately 50% of the 2023 plants remain to be tested by realtine PCR. Propagants were obtained from one propagator so far in 2024. The 300 plants (100 plants from each of three varieties) have been examined for rot and diseased tissue samples were plated onto amended Nash-Snyder medium for Fusarium isolations as well as placed in the freezer for realtime PCR testing. We are attempting to obtain propagants from a second propagator; the third propagator has shutdown and gone out of business. Two high quality genomes of the pathogen were assembled and annotated as references for the population diversity aspects of this research. Sequence data and annotations were submitted GenBank for public availability. DNA from 170 other isolates of F. sambucinum obtained from hop, potato, and other representative others hosts was obtained and submitted for bidirectionally sequencing using Illumina NextSeq 2000 P2 as 150 bp inserts. Data analysis is underway to call variants and quantify population differentiation based on state of origin, source of planting material, hop yards, and isolate within a hop yard. We identified primary biosynthetic gene clusters common across the F. sambucinum genomes analyzed to date. These include six biosynthetic gene clusters associated with mycotoxin production. The trichothecene profile of 33 isolates was measured following in vitro growth. We demonstrated that isolates of F. sambucinum from hop produce diverse trichthecenes that may include didecalonectrin, decalonectrin, DAS, and T2-toxin. However, most isolates from hop did not produce DAS or T2-toxin whereas most isolates from potato did. The absence of DAS production in hop-derived isolates was consistently associated with a deletion in the TRI13 gene in the 19 F. sambucinum genomes that were available at the time of these studies. These results may suggest chemotypes exist within F. sambucinum and that chemotype diversity varies by host of origin. Objective 3. Broadly communicate results to stakeholder partners to accelerate learning and adoption of best management practices developed in this project. Presentations made to industry: Borland, T., and Gent, D. H. 2024. Population diversity and genomic insights into the biology of the Fusarium canker pathogen. Hop Research Council. August 6, Corvallis, Oregon. Gent, D. H. 2024. Nitrogen fertility as a determinant of pest outcomes and hop quality factors. Idaho Hop Commission. March 14, Caldwell, Idaho. Borland, T., and Gent, D. H. 2024. Getting to the root of the problem: Updates on Fusarium canker. Hop Research Council. January 17, Frisco, Texas. Borland, T., and Gent, D. H. 2024. Digging deep for answers: Updates on Fusarium canker research. Washington Hop Industry Annual Meeting. January 4. Gent, D. H. 2023. Best practices for priority plant health issues. Summer Meeting of the Hop Research Council. August 2, Boise, Idaho.
Publications
- Type:
Conference Papers and Presentations
Status:
Awaiting Publication
Year Published:
2024
Citation:
Borland, T., Nunnemacher, H., Cauldron, N. C., Grunwald, N., Ocamb, C. M., and Gent, D. H. 2024. Comparative genomic insights into an emerging adversary of hop, Fusarium sambucinum. Plant Health 2024. July 27-30, Memphis, Tennessee. Phytopathology In press.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Borland, T. G., Cauldron, N. C., Nunnemacher, H. A., Grunwald, N. J., Ocamb, C. M., and Gent, D. H. 2024. Exploration of secondary metabolite genetic diversity in Fusarium sambucinum through comparative genomic approaches. Fungal Genetics. March 12-17, Pacific Grove, California.
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Progress 09/01/22 to 08/31/23
Outputs
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.
Impacts
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
- 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.
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