Progress 07/01/24 to 06/30/25
Outputs
Target Audience:The target audience for this proposed research includes researchers, students, crop consultants, Extension specialists, farmers, stakeholders, industry professionals, USDA-ARS (sunflower unit) researchers, and the broader community. This research is particularly important given the critical role that sunflowers play in U.S. agriculture. Approximately 80% of sunflower production occurs in the U.S. states of Minnesota, North Dakota, and South Dakota, making it a vital crop in these regions. While fungicides are essential tools in managing Phomopsis stem canker in sunflower, it is crucial to address the potential risks associated with their overuse. Excessive application of fungicides can lead to selection pressure on fungal pathogens, potentially resulting in the emergence of resistant strains of D. helianthi and D. gulyae--the causal agents of Phomopsis stem canker. These resistant strains may continue to cause disease, undermining the effectiveness of fungicides and posing a long-term threat to sunflower production. To effectively communicate our findings related to fungicide resistance, we presented our research at the Sunflower Annual Forum, a key event organized by the National Sunflower Association, which brings together farmers, industry professionals, and researchers from the university and the USDA-ARS. The forum, which attracted more than 100 attendees from across the U.S., provided an excellent platform for sharing our insights with the farming community and stakeholders. In addition, webinars and field days were conducted in Minnesota, North Dakota, and Nebraska (a total of over 200 attendees), as well as one-on-one meetings with farmers in South Dakota to help crop consultants and agriculture professionals identify Phomopsis stem canker. Additionally, the research was published in the Sunflower Magazine, a widely circulated publication from the National Sunflower Association, further extending the reach of our findings to a broader audience. By disseminating this information through these channels, we aim to raise awareness and promote sustainable practices in the management of sunflower diseases, ultimately benefiting both agricultural professionals and the agricultural community as a whole. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?As part of the project, one graduate student received specialized training in conducting and interpreting fungicide resistance assays, including the use of PCR techniques to detect resistance mutations in fungal pathogens such as Diaporthe. This hands-on experience not only contributed to the student's academic development but also built NDSU's capacity for ongoing diagnostics and fungicide resistance monitoring in Diaporthe species. In addition, we shared findings from this research and provided guidance on how to identify Phomopsis stem canker in the field, along with other common sunflower diseases. This information was disseminated to farmers, researchers, and industry professionals--including agronomists and crop consultants--through a variety of Extension and outreach activities such as webinars, field days, trade magazine articles, the National Sunflower Association Board of Directors meeting, and one-on-one engagement with farmers. These outreach and Extension efforts not only raised awareness but also encouraged stakeholders to take a more proactive role inadoptingIPM practices which includes the use of disease-tolerant hybrids. How have the results been disseminated to communities of interest?The results of the project were widely disseminated through multiple channels to ensure that the results reached both the scientific community and the broader agricultural industry. These included presentations at the Sunflower Forum organized by the National Sunflower Association (sunflower commodity group), webinar oranized by the North Central IPM Center, field days organized by Nuseed and University of Nebraska (Scottsbluff, NE),where key findings were shared with farmers, researchers, and industry professionals. Additionally, the results were communicated during a meeting with the National Sunflower Association Board of Directors in Fargo, ND. Further, the findings were published in The Sunflower, the official trade magazine of the National Sunflower Association, which is widely read by farmers, agronomists, and other stakeholders in the cropindustry. What do you plan to do during the next reporting period to accomplish the goals?For the next reporting period, we plan to establish field trials in North Dakota and Nebraska to evaluate the efficacy of fungicides containing Succinate Dehydrogenase Inhibitors (SDHIs) as an alternative to QoI fungicides, specifically targeting Diaporthe-resistant sunflower hybrids. In addition to the field trials, we will conduct to survey for Diaporthe-resistant strains, and field training sessions focused on the identification of Phomopsis stem canker and other common sunflower diseases. These training sessions will also cover the importance of understanding and managing fungicide resistance. During these sessions, we will emphasize Integrated Pest Management (IPM) practices that can be adopted when fungicide-resistant strains of Diaporthe are identified.
Impacts
What was accomplished under these goals?
Issue or Problem Addressed: The study aims to identify and understand the occurrence of Diaporthe species causing Phomopsis stem canker in sunflower fields, particularly their resistance to quinone outside inhibitor (QoI) fungicides, to inform better management practices for sunflower growers in the U.S. states of Minnesota, North Dakota, South Dakota, and Nebraska. Major activities completed as part of Objective 1 (Determine the prevalence of QoI-resistant strains ofD. helianthiandD. gulyaecausing Phomopsis stem canker in MN, ND, NE, and SD): The primary audience for this research includes researchers, farmers, crop protection professionals, and the broader community. The survey provided insights into the prevalence of Diaporthe species and their potential resistance to QoI fungicides. In 2024, a total of 48 sunflower fields were surveyed across North Dakota (n = 31, from 12 counties) and South Dakota (n = 17, from 5 counties). Additionally, sunflower stalks from one field each in Minnesota and Nebraska were included in the study. A total of 113 Diaporthe isolates were recovered through direct isolation from sunflower plants exhibiting symptoms of Phomopsis stem canker, characterized by dark brown stem lesions ranging from 15 to 20 cm in length. Commercial sunflower fields were randomly selected, ensuring that they were at least five kilometers apart within each state. Four to five plant samples were collected from each field, and Diaporthe species were isolated from the stems and leaves using standard isolation procedures. First, the plant tissues were rinsed with tap water and then cut into 10-mm segments. These segments were surface sterilized with 0.05% sodium hypochlorite and 70% ethanol for one minute each, followed by three rinses with sterile distilled water (30 seconds per rinse), and dried on autoclaved paper towels. Afterward, the segments were plated on potato dextrose agar (PDA) supplemented with 0.03% streptomycin sulfate (Sigma-Aldrich, St. Louis, MO) to reduce bacterial contamination. The plates were incubated at 22°C for 7 to 10 days under alternating 12-hour light and dark cycles. Hyphal-tipped Diaporthe colonies were sub-cultured onto fresh PDA plates. A Polymerase Chain Reaction (PCR) assay was performed to detect QoI-resistant isolates of D. gulyae and D. helianthi based on mutations in the cyt b gene region. Since cyt b gene sequences for these species were unavailable, primers were designed through primer walking based on the cyt b sequences of D. longicolla, a related fungus. The primers used were: Dan1L - 5'- CAC GCT AAC ACT GCT TCA GC-3' (forward) and Dan1R - 5'-TGG AGC CAT AGG GAT TCT ATC A-3' (reverse). Forty-four isolates of D. helianthi (Minnesota [n = 1], Nebraska [n = 3], North Dakota [n = 35], South Dakota [n = 5]) and 14 isolates of D. gulyae (North Dakota [n = 3], South Dakota [n = 11]) were randomly selected from the various counties surveyed. These isolates were cultured in potato dextrose broth. The genomic DNA of these isolates was extracted using the CTAB method, and molecular identities were confirmed using quantitative PCR assays (Elverson et al. 2020) before further analysis. The cyt b gene of each confirmed isolate was amplified using the primers mentioned above. The PCR reaction was carried out on at least 100 ng/µL of genomic DNA from D. gulyae and D. helianthi. The 25-µL reaction mixture was prepared, containing 2 µL of fungal DNA (10 ng/µL), 0.75 µL of each primer (forward and reverse, 10 µM), 12.5 µL of 2× Taq PCR Master Mix (Qiagen, Valencia, CA), and 9 µL of sterile nuclease-free water. The PCR cycling conditions were: initial denaturation at 95°C for 5 minutes, followed by 30 cycles of 95°C for 30 seconds, 54.8°C for 30 seconds, and 72°C for 1 minute, with a final extension at 72°C for 10 minutes. A water control was included in each PCR run. PCR products were visualized under UV light on 1.0% agarose gels stained with GelRed® Nucleic Acid Gel Stain (Biotium Inc., Fremont, CA, USA). The amplified products were sent for sequencing at a commercial sequencing facility (Functional Biosciences, Madison, WI). The obtained nucleotide sequences were translated into amino acid sequences using the bioinformatics tool "Translate" (https://www.bioinformatics.org/sms2/translate.html). The sequences were aligned with reference protein sequences from the National Center for Biotechnology Information (NCBI) database to analyze the presence of amino acid substitutions (G143A, F129L, or G137R). Sequencing of the 550-bp coding region of the cyt b gene was performed on 58 isolates to detect mutations associated with QoI fungicide resistance. No target site point mutations associated with amino acid substitutions (G143A, F129L, or G137R) in the cyt b gene were found in any D. gulyae or D. helianthi isolates. Key Outcomes or Accomplishments: During this reporting period, the project revealed a high prevalence of D. helianthi in North Dakota and D. gulyae in South Dakota, suggesting regional differences in the species composition of Diaporthe. Furthermore, the absence of QoI resistance mutations in the Diaportheisolates suggests that current fungicide resistance is not widespread in the fields surveyed. This work contributes to improving pest management by providing critical information on pathogen distribution and QoI fungicide resistance patterns.
Publications
- Type:
Other
Status:
Published
Year Published:
2025
Citation:
Mathew, F., Markell, S., Harveson, R., McCaghey, M., Kovacs, P., and Mohan, K. 2025. Phomopsis Continues to Challenge Dakotas: Fungicide Strategies Show Promise. The Sunflower. February 2025.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Mohan, K., Markell, S., Scherer, J., Hansen, B., Mathew, F., McCaghey, M., Aspholm, P., Kovacs, P., Passone, N., Harveson, R., and Rickey, A. 2025. Fungicides against Phomopsis in sunflowers. 47th National Sunflower Association Research Forum, Fargo, ND. January 8-9, 2025.
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