Source: NORTH DAKOTA STATE UNIV submitted to NRP
ESTABLISHING ALFALFA IN INTERCROPPING WITH SUNFLOWER AND SORGHUM TO IMPROVE ALFALFA YIELD AND PROFITABILITY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1029204
Grant No.
2022-70005-38225
Cumulative Award Amt.
$587,671.00
Proposal No.
2022-05762
Multistate No.
(N/A)
Project Start Date
Sep 1, 2022
Project End Date
Aug 31, 2025
Grant Year
2022
Program Code
[AFRP]- Alfalfa and Forage Program
Recipient Organization
NORTH DAKOTA STATE UNIV
1310 BOLLEY DR
FARGO,ND 58105-5750
Performing Department
(N/A)
Non Technical Summary
Alfalfa is a key crop to sustainable agriculture that contributes positively to farm profitability, soil health, and the environment. In both states, ND and SD, sunflower and alfalfa are part of the crop rotation in the central and western part of both states. Alfalfa forage yield in the seeding year is low, thus establishing alfalfa with a cash crop in the seeding year provides an additional revenue for the farmer in the second year to offset the low income in the seeding year. The overall goal is to determine the productivity, economics, and environmental benefits of establishing alfalfa by intercropping with sunflower or forage/grain sorghum in an effort to promote the inclusion of alfalfa in crop rotations. Alfalfa will be intercropped with sunflower planted at 76- and 152-cm row spacing in ND and SD, and with forage sorghum in ND and grain sorghum in SD. Transcriptomic changes will determine, at the molecular level, targets and mechanisms involved in interspecies competition that impact alfalfa establishment, nitrogen fixation, and yield. An economic and environmental sustainability analysis will be conducted to determine profitability and to quantify ecosystem services of establishing alfalfa in intercropping with sunflower compared with establishing alfalfa the year after sunflower or sorghum is harvested. Outcomes of the project include: 1) increased knowledge of management of crops in intercropping; 2) Improved soil health, reduced soil erosion, and nutrient losses; and 3) increased profitability. In the long-term, we expect to increase alfalfa acreage and profitability by increasing adoption of intercropping alfalfa with sunflower.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2051640106060%
1021844102040%
Knowledge Area
102 - Soil, Plant, Water, Nutrient Relationships; 205 - Plant Management Systems;

Subject Of Investigation
1640 - Alfalfa; 1844 - Sunflower;

Field Of Science
1060 - Biology (whole systems); 1020 - Physiology;
Goals / Objectives
The overall goal of the proposal is to determine the productivity, economics, and environmental benefits of establishing alfalfa by intercropping with sunflower or forage/grain sorghum. This will likely drive more growers to plant alfalfa in rotation with other crops in their system and increase the number of alfalfa acres. Increasing the acres of alfalfa will increase net returns to the farmers, decrease soil erosion and nutrient run-off to water sources, decrease soil organic carbon losses, reduce greenhouse gases emissions, and increase soil carbon sequestration and nitrogen supply to the cropping system.Objectives1. Evaluate the impact that intercropping alfalfa with sunflower or forage/grain sorghum planted at various row spacings has on alfalfa stand establishment, forage yield and nutritive value, nitrogen fixation and N crop utilization, and weed suppression.2. Evaluate the impact that integrating alfalfa into multi-cropping systems has on the physiological and molecular mechanisms associated with plant-plant interactions leading to altered alfalfa establishment, crop yield, nitrogen fixation and crop utilization.3. Assess the sustainability and impact of alfalfa intercropping systems to the environment and grower profitability.
Project Methods
Objective 1. Field intercropping experimentsExp.1: Alfalfa-sunflower intercropping experiments will be initiated at Prosper and Hickson, ND and Sturgis, SD, in May 2023. Each experimental location will have five treatments: alfalfa alone, sunflower planted at 60- and 30-inch row spacing without alfalfa, and sunflower intercropped with alfalfa at both rows spacing . Each experimental unit will have four rows of sunflower and/or 16 rows of alfalfa.Exp.2: The alfalfa-sorghum experiments will be conducted in two locations in ND with alfalfa intercropped with forage sorghum at 12-inch row spacing and two harvest systems (1-cut at the end of the season or 2 cuts, first one at 4.5 ft of plant height). In SD,alfalfa will be intercropped with grain sorghum . Row spacing of sorghum will be 30-inches apart and alfalfa will be drilled at the same time as sorghum.In Year 2, a spring-seeded alfalfa treatment will be seeded on the plots that had sunflower at 30 and 60-inch row spacing the previous year or sorghum, to simulate the business-as-usual sequences (cash crop followed by spring-seeded alfalfa). Alfalfa established in intercropping system during Year 1 will be evaluated in Year 2 .Soil collected from 0-6 inches will be analyzed for NO3-N, P, K, pH, and organic matter.Soil organic carbon (SOC) will be analyzed in Year 1 before establishing the experiment and in Year 3 at the end of the experiment.Soil from 6-24 inches depth will be analyzed for NO3-N. Gravimetric water content (0-6 inches) will be collected throughout the season in experiment both years.This project will employ the use of 15N-natural abundance, considered the most reliable method, which provides a time-integrated value of nitrogen fixation. Ground samples from all crops and treatments will be sent to a lab with radioisotopes evaluation capabilities to determine nitrogen stable-amount isotope 14N/15N ratio.Normalized difference vegetation indexand photosynthetically active radiationintercepted by the crop(s) will be evaluated. Biomass yield and forage nutritive value data will be collected the first year only in the sole alfalfa treatment (1-2 cuts).Stand counts of alfalfa will be taken in every plot in the fall of Year 1 and spring and fall of Year 2.Sunflower seed will be harvested with a plot combineand seeds will be dried and analyzed for oil and protein content, with aXDS Near Infrared (NIR) Rapid Content Analyzer. Grain sorghum will be harvested with a plot combine (Wintersteiger) at a moisture content of 18%, Sunflower seed will be harvested at the end of the season, at physiological maturity, with a plot combine (Hege) . Forage sorghum will be harvested twice in season.In Year 2 and 3, all alfalfa treatments will be harvested with a plot harvester (3-4 cuts are expected). The sample will be ground through 1-mm mesh. The samples will then be analyzed for forage nutritive value (ash, crude protein, neutral detergent fiber , acid detergent fiber, acid detergent lignin, neutral detergent fiber digestibility. Sunflower seeds will be analyzed for oil and protein content.Data collected in field experiments will be analyzed separately by location with the procedure MIXED of SAS 9.4 with the repeated measures function for each harvest within a season according to the experimental design. For mean separation test LSD at 5% significance will be used.Objective 2. Molecular studies of alfalfa in intercroppingFor molecular studies, samples will be collected fromfield sites and placed directly into liquid N and stored at -80Cprior for laboratory extraction of RNA and development of RNA-seq libraries for sequencing. Sequence data will be analyzed and compared to identify molecular processes that could be manipulated to improve alfalfa establishmentin multi-cropping systems, improve alfalfa N2 fixation and crop utilization, and reduce crop yield losses resulting from inter-species competition.Leaf and root samples will be collected from alfalfa alone or intercropped with sunflower and sorghum in Year 1. Tissue samples will also be collected from sunflower and sorghum alone or intercropped with alfalfa when 50% of the control sunflower plants reach the R5 stage of crop development. Total RNA from all samples will be extracted and used to construct in-house made Illumina libraries, which will be sequenced by a commercial sequencing facility to a depth of >15 million reads per library. Sequence data obtained from the libraries will be processed using in-house resources and either de novo assembled or mapped to available reference genomes of alfalfaand sunflower using the latest methods for transcript unit assembly and counting. Differential expression among genes will be identified by the latest bioinformatics methods and subjected to gene set enrichment and network analyses to identify overrepresented ontologies among biological pathways and molecular mechanisms.The raw and cleaned sequence data will be stored in the CyVerse Discovery Environment. Raw sequence data will be deposited in the NCBI SRA database as soon as it is analyzed.Objective 3. Environmental and Economic Sustainability Assessment Life cycle assessment (LCA): Field data, crop management, and lab results will be used as inputs in a crop simulation model in order to estimate ecosystems services provided by the cropping systems studied. Nitrogen and carbon cycles, soil water management and erosion will be quantified by the process-based cropping system model DNDC. The results of the model simulation will be validated using field data to ensure the reliability of the estimates produced. A further online and user-friendly crop simulation model (COMET-farm) will be run and the results compared to the DNDC to assess the reliability of the former.The output of the model, along with field data, will be used as an input for the LCA model using the SimaPro 9.1 software. The system boundary will be set from 'cradle to farm gate' including all farm inputs , field operations, crop outputs, and farm direct emissions to air and water . In the LCA process, the impact assessment methodology TRACI II will be employed, which allows a quantification of multiple impacts.An uncertainty analysis will be run on the results of the crop simulation model and the LCA. The results of the environmental assessment (crop simulation model + LCA) will provide an estimate of multiple impacts and ecosystem services, in particular those, which are of primary interest for farmers and the market: carbon footprint, field nitrogen and phosphorus management, soil health and soil organic carbon variation, field water management and erosion control.Economic analysis and economic sustainability assessment: (Hanson) Economic analysis will summarize forage and grain yield, returns and costs for 2-year sequences. Alfalfa budgets will be created for the third year of the experiments where all experiments only will have alfalfa.Whole farm budgets and partial budgets will be developed using rates and financial information for the crops according to crop budget calculations for each state. All budgets will have two consecutive years. Enterprise budgets will be calculated similarly to the budgets published by NDSU Extension .An economic assessment using the crop budgets described above, field data, and literature will be performed in order to compare the economic performance of the cropping systems studied. Sensitivity analyses will be conducted to determine how the economic viability of the different experimental pairings may change with fluctuations in yield, output prices, and input prices. This information will support decision makers (farmers and industry) in choosing farming practices that can reduce the environmental impact of cropping systems by minimizing the economic investment. Sensitivity analysis varying yield penalty to cash crops and crop prices will be included.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:Farmers, crop advisors, seed and chemical companies reps, undergraduate and graduate students, NRCS and USDA/ARS personnel. People from all the groups listed above participated in our field day in Prosper, ND on 14 August 2024 andFargo, ND on 17 September 2024 or in Sturgis , SD field days in 2024. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project is currently providing the funding for training of two MS students and one PhD student at North Dakota State University: 1. Franklin Omeye, MS, Intercropping of alfalfa and sainfoin with forage sorghum, Expected graduation May 2025 2. Shazzadul Islam, MS, Intercropping of alfalfa and sainfoin with forage sorghum, Expected graduation May 2025. 3. Maria Mazala, PhD Transcriptomic and Gene Expression in Alfalfa Intercropped with Sunflower under Field Conditions. Expected graduation December 2026. How have the results been disseminated to communities of interest?A field day and tour was conducted in Prosper, ND on August 14, 2024, 35 people attended. Dr. Berti hosted the NCCC31 Forages committee in Fargo, forage researchers and graduate students from 11 states (OR, MD, WI, TX, TN, MN, FL, MI, ND, OH, IA, WY) had the opportunity to see the sunflower and sorghum alfalfa intercropping research plots and an on-farm sunflower alfalfa intercropping system in Leonard, ND, from a farmer using alfalfa as a perennial cover in his organic alfalfa production. In addition, all students in the project presented posters at the NCCC31 annual meeting. A second field day was conducted on September 17, 2024 in Fargo, ND, there were 45 attendees to the event, farmers crop consultants, graduate students, faculty and staff. Tow field days were organized in Sturgis, SD; 18 June, 2024- 30 participants and 26 June- 13 participants. We did plots tours for both and discussed the potential merits of intercropping as well as some of the practical issues of implementing such a system. In addition, research results have been presented at several conferences by the PI or graduate students: ASA-CSSA-SSSA 2023 and 2024, Midwest Forage Association 2024, NAAIC 2024; SWCS 2024, and AFGC 2024. What do you plan to do during the next reporting period to accomplish the goals?Objective 1 1. Alfalfa and sainfoin forage sorghum intercropping We willobtain weather report data for the stations close to Prosper and Fargo, where trials were conducted to do a thorough analysis to verify the interplay between the weather parameters and other yield and biodiversity parameters studied in this trial. The data for the samples that were sent to the stable isotope facility are being analysed. All data collected in this experiment in ND and SD will be analyzed and reported in next year's period and published in a peer review. 2. Alfalfa and sainfoin sunflower intercropping Data collection from SAIC-23 and SAIC-24 will continue until winter begins in November 2024. All collected data including metrics such as gravimetric water, insect traps, PAR, forage yields from both 2023 and 2024 treatments, nutritive value, sunflower grain yield, oil and fatty acid content, biomass analysis, 15N transfer, will be analyzed and compiled into a manuscript for an MS defense in Spring 2025, with plans to publish this research in reputable journals. Objective 2 In early 2025, the graduate student will start preparing RNAseq libraries from the leaf samples collected at time points one and three. The leaf RNAseq libraries will be sequenced and analyzed as previously described. In addition, the samples taken in 2024 will be extracted, prepared for sequencing and analyzed. Objective 3 From September 2024 until August 2025, we plan to examine which practices are most environmentally friendly by subjecting all the data to life cycle analysis (LCA) both for the alfalfa-sorghum and alfalfa- sunflower intercropping systems. Also, we will examine the financial advantages of the individual intercropping system studies in collaboration with Dr. Erik Hanson, an NDSU agrobusiness faculty member participating in this project. Our goal is to create a model that incorporates and best explains each of the factors.

Impacts
What was accomplished under these goals? ?Objective 1 Alfalfa or sainfoin sunflower intercropping Alfalfa and sainfoin were intercropped with sunflower (SAIC-23) in Hickson and Prosper, ND, in a randomized complete block design with 7 treatments and 4 replicates. Results showed no significant impact on sunflower grain yield (1,800-3,400 kg/ha) at both 40 and 80 N kg/ha fertilizer rates. Alfalfa maintained expected forage yields (1.2 tons/acre) for the first cut, while sainfoin lagged in intercropping but excelled as a monocrop (1.5 tons/acre). No significant forage nutritional differences in total digestible nutrients (TDN) and crude protein (CP) were found between mono and intercropped treatments. Soil gravimetric water data indicated no significant differences in water use patterns across treatments. Fall bulk density measurements and insect trap data collection concluded, identifying 19 insect families. Intercropping enhanced biodiversity at both sites, though sainfoin plant density and forage yields were severely impacted in intercropping. Sunflower oil and fatty acid content showed no significant variation across treatments. In 2024, (SAIC-24); the same experiment was planted at Prosper on 7 June 2024 and in Fargo on 10 June 2024, though Fargo plots were destroyed by rabbits. MAP and urea fertilizers were applied at both sites. Additionally, spring-seeded alfalfa was planted in Treatment 4 after last year's sunflower harvest. In 2024, the nitrogen isotope study was started in ND with the advice of Dr. Graham from SDSU: 15N nitrogen was applied to 1 m² microplots to assess N transfer from alfalfa to sunflower, comparing it with non-nodulating alfalfa. Biomass and seed samples were taken, ground and sent for radioisotope analysis. By August 2024, two cuts were completed from last year's alfalfa treatments, and data collection on gravimetric water, insect traps, PAR, and other metrics is ongoing. In South Dakota, the year effect for sunflower seed yield was much stronger than that of sorghum. However, intercropping with alfalfa did not affect overall sunflower seed yield. Overall, yields were less than half of the 2023 yield in 2024 at 2,224 kg ha-1 vs. 943 kg ha-1, respectively. For both studies, years combined, alfalfa/sunflower compared with alfalfa alone averaged 1,713 kg ha-1 vs. 1,674 for 30" row spacing and 1294 kg ha-1 and 1654 kg ha-1, respectively for 60" row spacing. Alfalfa or sainfoin forage sorghum intercropping The statistical analysis of the 2023 data, showed no discernible difference in the yield of forage sorghum across all treatments. This suggests that adding these perennial legumes (alfalfa or sainfoin) has no effect on the yield of forage sorghum and increasing the fertilizer rate from 40 kg N ha-1 to 80 kg N ha-1 is not sustainable or cost-effective because it did not increase the yield of forage sorghum. As one of the many benefits of incorporating alfalfa into our cropping system, the trial in 2023 also saw a rise in beneficial insects introduced in the plot where alfalfa was interplanted, validating the increase in biodiversity hotspot. Compared to the standard practice of sowing alfalfa in the spring, which rarely produced more than two cuts, we were able to obtain four cuttings from the alfalfa that was sown in 2023, which promoted yield stability. In 2024, the experiment was repeated in Fargo and Prosper, ND, and in summary intercropping enhances forage quantity and quality in comparison to monocultures. Sorghum improved overall forage quality and yield stability by providing biomass and structural support. But weed control was found to be difficult, especially in the early stages of the growing season. However, data gathering procedure in this project is currently ongoing. Isotopic 15N study was started in 2024 and samples have been sent for radioisotope ratio, to estimate amount of fixed N2 from alfalfa to the sorghum. In South Dakota, across two years of field trials, Sorghum forage yield averaged 7,138 kg ha-1 with no statistical differences between intercrop treatments or harvest year. This is due in large part to the wide standard errors observed between treatments, particularly in 2023. Notably, in the drier year (2024), both treatments with 60" (152-cm) row spacing, average forage yields were approximately 50% of the treatments on 30" (76 cm) spacing. If analyzed as a single year, this would be a statistically different result. Year 2 Alfalfa Forage Yield- In South Dakota Similar to Year 1 yields, second year alfalfa was not affected by intercropping with either sorghum or sunflower. Second year alfalfa was harvested on 6/24/24 for the first cutting and 9/7/24 for the second cutting. Across all treatments, alfalfa forage yield following sunflower intercropping averaged approximately 4,650 kg ha-1 compared to just 690 kg ha-1 for the second cutting. Rainfall significantly affected alfalfa regrowth, where no precipitation was recorded for the month of July and just over 1" of rainfall was received in August. Likewise, following sorghum intercropping, Year 2 alfalfa yields were not affected by intercropping treatment or row spacing. However, overall average yields were significantly lower than in the sunflower plots. Across all treatments, alfalfa forage yield following sorghum intercropping was 70% of the sunflower plots, averaging approximately 3,230 kg ha-1 in the first cutting compared to just 505 kg ha-1 for the second cutting (Figure AA). This difference is likely due to differences in location of the plots rather than previous crop since the solo alfalfa plots averaged 4,500 kg ha-1 and 3,210 kg ha-1 for the sunflower and sorghum plots, respectively. Objective 2 For the sunflower and alfalfa intercropping study, final sample collection from field plots in Prosper and Hickson, North Dakota in 2021 and from Hickson, ND and Red Lake Falls MN in 2022 produced a total of 336 root and 336 leaf samples. Samples were collected at three time points during the growing season of 2021 and 2022. At each time point and location, 28 root and 28 leaf samples were collected. Although RNA was extracted from all 672 samples, as of November 8, 2024, only RNA extracted root tissues collected at time points one (late July) and three (late August) have been used to develop RNAseq libraries for sequencing. Because 11 of the 224 library preparations failed, after multiple attempts, the final number of root libraries sequenced by Fulgent BioPharma from time points one and three will be 213. The Ph.D. graduate student working on this project has started analyzing sequence data for the first set of 96 RNAseq libraries and will continue to analyze sequence data as it is received from Fulgent BioPharma. Additional samples were taken from the Prosper study at two growth stages in 2024 season for further analysis. ?Objective 3 Economic data and life cycle inventory data has been collected to start the LCA and cost analysis of both alfalfa-sunflower and sorghum sunflower intercropping in January 2025.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Berti, M.T. 2024. What is the value of alfalfa in a diverse multicrop rotation? ND Soil and Water Conservation Society Annual Meeting. Dickinson, ND, 19 September, 2024 Invited speaker
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Omeye, F., Berti, M.T., Mosqueda, H., Lindell, H., Islam, Md.S., Morocho-Lema, M., and Igboke, O. 2024. Establishing alfalfa or sainfoin in an intercropping system with forage sorghum. Annual meeting NCCC31 Forage committee and R-CAP, Fargo ND, 12-15 August, 2024.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Mosqueda H., M.T. Berti, S. Bibby, A. Kurth, and H. Lindell. 2023. Integrating Alfalfa into Corn or Forage Sorghum to Increase System Diversity and Perenniality. [Abstract] ASA-CSSA-SSSA International Annual Conference, St, Louis, MO, 29 Oct-1 Nov, 2023.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Islam, Md.S., Berti, M.T., Omeye, F., Mosqueda, H.Igboke, O., and Grijalba, M. 2024. Establishing alfalfa and sainfoin through intercropping with sunflower. Annual meeting NCCC31 Forage committee and R-CAP, Fargo ND, 12-15 August, 2024.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Mazala, M., Tabert, M., Islam, Md.S., Anderson, J.V., Horvath, D.P. and Berti, M.T. 2024. Transcriptomic analysis of alfalfa-sunflower intercropping. Annual meeting NCCC31 Forage committee and R-CAP, Fargo ND, 12-15 August, 2024
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Omeye, F., Berti, M.T., Mosqueda, H., Kurth, A., Lindell, H., and Islam, Md.S. 2024. Establishing alfalfa or sainfoin in an intercropping system with forage sorghum. Soil Water Conservation Society Annual Conference, Myrtle Beach, NC 21-24 July 2024.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Islam, Md. S. and M.T. Berti. 2024. Establishing alfalfa and sainfoin thorough intercropping with sunflower. Soil Water Conservation Society Annual Conference, Myrtle Beach, NC 21-24 July 2024.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Islam, Md.S., and M.T. Berti. 2024. Intercropping alfalfa and sainfoin with sunflower boosts forage production, soil health, and biodiversity. North American Alfalfa Improvement Conference, Pasco, WA, 24-26 June 2024.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Berti, M.T.* 2024. Intercropping sunflower with alfalfa. Midwest Forage Association Annual conference, Wisconsin Dells, WI, 19-21 February 2024.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Omeye, F. M.T. Berti, H. Mosqueda, A. Kurth, H. Lindell. And Md S. Islam. 2024. Increased Forage Yield and Nutritive Value of Forage Sorghum Grown in Intercropping with Alfalfa. American Forage Grassland Conference, Mobile AL January 8-10, 2024.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Omeye, F., M.T. Berti, H. Mosqueda, H. Lindell, Md S. Islam, M. Morocho-Lema, and O. Igboke 2024. Intercropping perennial legumes with a warm-season grass to improve forage sustainability. [Abstract] ASA-CSSA-SSSA International Annual Meeting, San Antonio, TX, November 10-13, 2024
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Islam, Md S., M.T. Berti, F. Omeye, H. Lindell, H. Mosqueda, A. Kurth, O. Igboke, M. Morocho, and M. Grijalba. 2024. Intercropping alfalfa and sainfoin with sunflower: Boosts forage production, soil health, and biodiversity {Abstract] ASA-CSSA-SSSA International Annual Meeting, San Antonio, TX, November 10-13, 2024.


Progress 09/01/22 to 08/31/23

Outputs
Target Audience:Farmers, crop advisors, seed and chemical companies reps, undergraduate and graduate students, NRCS and USDA/ARS personnel. People from all the groups listed above participated in our field day in Fargo and Hickson ND on September 19, 2023. Changes/Problems:Soil Health Extension Agronomist, Dr. Abbey Wick, left NDSU on May 2023.The extension portion of the project is being handled by the PI with the assistanceof Extension Crops Specialist Dr. Clair Keene in the Department of Plant Sciences at NDSU. What opportunities for training and professional development has the project provided?The project is currently providing the funding for training of two MS students at North Dakota State University: 1. Franklin Omeye, Intercropping of alfalfa and sainfoin with forage sorghum, Expected graduation May 2025 2. Shazzadul Islam, Intercropping of alfalfa and sainfoin with forage sorghum , Expected graduation May 2025. In addition MS student Mikayla Tabert started her MS in 2021 with this project finishing in May 2023. Thus the last year of her research was funded thorugh this project. How have the results been disseminated to communities of interest?A field day was organized in Fargo and Hickson, ND, September 18, 2022 and September 19, 2023. In both years, students showed farmers the alfalfa sunflower intercropping trials getting a lot of interest from them. Some farmers attending the 2022 field day implemented alfalfa -corn intercropping in their field headlines. Now they have alfalfa established which will cut for hay. A field day was organized in Sturgis , SD on July 11.2023 with approximately 20 attendees to show both sorghum-alfalfa and sunflower-alfalfa intercropping. In addition, research results have been presented at several conferences by the PI or graduate students: ASA-CSSA-SSSA 2022 and 2023, AFGC 2023 and MFA 2023. What do you plan to do during the next reporting period to accomplish the goals?Objective 1 Data from 2023 season experiments not available for this report will be included in next year's report. This includes: gravimetric water, PAR, soil moisture, insect diversity, biomass composition and seed oil content in North Dakota and yield, forage quality and oil content in SD. The experiments established in 2023 will be followed up evaluating alfalfa and sainfoin establishment and forage yield and quality in 2024 in North Dakota and forage yield in South Dakota. The same experiments will be established again in 2024. Both experiments established in 2023 with intercropping of sunflower with alfalfa or forage sorghum with alfalfa will be repeated in 2024 at same locations. In addition to same evaluations in 2023, we will evaluate 15N isotope in sunflower, forage sorghum and alfalfa crops biomass, with and without intercropping. Objective 2 RNAseq Library Preparation and Sequencing: The graduate student assigned to this project has extracted total RNA from 84 root samples collected in Hickson, North Dakota and is expected to have RNA extracted from all root and leaf samples collected from Hickson in 2022 completed by mid-January 2024. The graduate student is also learning to isolate mRNA for the preparation of RNAseq libraries. Once RNAseq libraries have been developed and pass quality control standards, they will be sent out on contract for sequencing. While RNAseq libraries prepared from samples collected from Hickson, ND are being sequenced, the graduate student will extract the root and leaf samples collected from Red Lake Falls, Minnesota and repeat the process of preparing RNAseq libraries for sequencing. This staggered approach will allow the graduate student to conduct a phased approach to accomplishing the sequencing portion of this project. Staggering the project by locations will allow the student to repeat steps involved in the development of RNAseq libraries, which should enhance the learning process. Data Analysis: The graduate student will also be trained by inhouse bioinformatics specialists to filter, assemble and annotate the sequencing data once it is returned from the sequencing facility. The resulting sequence data will be run through high-capacity software and hardware designed to handle big-data analyses and the output will be used to determine the impact that establishing alfalfa with sunflower planted at difference row spacings has on molecular mechanisms and gene regulation in each species. Analysis of all RNAseq libraries prepared from both locations in 2022 should be completed by the end of 2024 and will be compared to RNAseq studies completed with samples collected from replicated studies done at Hickson and Prosper, North Dakota in 2021. The outcomes from this project will help us to build testable hypothesis on signaling and molecular mechanisms involved in plant-plant interactions/competition, which will be presented to customers, stakeholders and other scientists at conferences and workshops. Objective 3. All information on inputs and equipment used in Experiments in Objective 1 will be collected in each system and at all locations. This information and yield will be used to do a cost analysis to determine profitability of each system. Soil organic C and bulk density samples were taken at the beginning of the experiments as baseline and will be taken again at the end of the experiments estab;lished in 2023. Greenhouse gasesemissions from input and processes from 'cradle to gate' will be calculated to use in an life cycle assessment (LCA) of each system.

Impacts
What was accomplished under these goals? Objective 1 This objective was started before the grant was funded and the original experiment as described in the grant is already completed and preliminary results are indicated below. The 2023 experiments design was modified to include not only alfalfa but sainfoin (Onobrychis vicifolia). Sainfoin is a drought tolerant perennial legume that adapts well to grow in dry environments where forage sorghum adapts as well. In addition, sainfoin reduces methane production in cows which is of interest to reduce the environmental impact of beef production. In addition, two N rates were included to evaluate the system with minimum external inputs and the ability of the legume to provide for some of the N to the forage sorghum while reduce the greenhouse gases emissions associated with chemical nitrogen fertilizers and nitrous oxide production. Alfalfa sunflower intercropping a) This project started in 2021, before the grant was awarded. This project was submitted but not granted the first year, but we started the research as indicated in the grant proposal. This study has been completed and the MS student graduated.The main objective was to compare alfalfa intercropped with sunflowers at two row spacings, with spring-seeded alfalfa alone in a two-year system. The specific objectives were 1) to determine the economic impact of establishing alfalfa intercropped with sunflower in a two-year production system to skip the low yielding seeding year; 2) to determine yield losses due to alfalfa competition with sunflower planted at 76- and 152-cm row spacings; 3) To determine sunflower yield losses due to different seeding dates of cover crop competition at 76-cm sunflower row spacing and 4) to determine the yield and forage nutritive value of alfalfa coming from intercropping in the seeding year compared with spring-seeded alfalfa monoculture. Results indicated that intercropping is a way to utilize available resources more efficiently and profitably. Alfalfa did not impact sunflower seed yield at 76-cm row spacing and had a slightly lower yield the following year due to intercropping, yet it was the most profitable two-year cropping system evaluated. Intercropping sunflower improves ecosystem services to improve future farm sustainability. b) In 2023, a replicated experiment was established in Prosper and Hickson, ND. The experimental design was an RCBD with 4 replicates. Treatments included:1) alfalfa alone; 2) alfalfa-sunflower + 40 kg N/ha; 3) alfalfa-sunflower + 80 kg N/ha; 4) sainfoin alone; 5) sainfoin-sunflower + 40 kg N/ha; 6) sainfoin-sunflower +80 kg N/ha and 7) Sunflower alone + 80 kg N/ha. Sunflower seed yield and alfalfa and sainfoin forage yield were collected as well as insect visitation in each treatment using sticky traps and soil traps.Sunflower yield was the same in all treatments as in the 2021 and 2022 experiment, indicating intercropping with either alfalfa or sainfoin is feasible. Nitrogen rates did not have an effect at either location. c)In South Dakota, alfalfa was intercropped with sunflower at 76-cm and 152-cm row spacing similar to the experiment conducted between 2021 and 2022 in two locations in North Dakota. As expected yield of alfalfa was greater for alfalfa alone compared with alfalfa intercropped at the two different row spacings. Sunflower seed was harvested in October 2023, but data has not been analyzed yet. 2. Forage sorghum and corn intercropping with alfalfa or sainfoin a) Cropping system diversification by including perennial legumes such as alfalfa can improve soil and increase overall yields. However, despite the benefits it can have on soil health, alfalfa production is declining because its establishment year is often less productive, resulting in declining interest from producers to introduce the perennial into their annual cropping systems. The objectives of this three-year study are to reduce the economic loss to farmers in the seeding year and determine whether intercropping corn or sorghum with alfalfa during establishment can increase overall yield and quality of the perennial system. Nine treatments consisting of corn (C), sorghum (S), and alfalfa (A) alone or intercropped (corn (CA) or sorghum (SA) with alfalfa) were planted in two North Dakota locations (Hickson and Prosper) in 2022 using different management strategies: 0.6-m gaps every 1.8-m within corn rows (G, corn gaps; GA, corn gaps with alfalfa) or a two-harvest system of forage sorghum 45 and 90 days after planting (S2, two-harvest sorghum; SA2, two-harvest sorghum with alfalfa). The experiment was repeated in 2023 at same locations but data is not yet available. Light interception, soil characteristics, insect diversity, biomass and corn grain yield, and forage nutritive value were measured. Forage protein concentration was significantly greater in S2 (94 g/kg) and SA2 (90 g/kg) than S (72 g/kg) and SA (61 g/kg). Dry matter was similar across all sorghum treatments, and the reduction of grain yield in G and GA was less than 17% as compared with C and CA. Alfalfa failed to establish under SA, however, it maintained similar stands in SA2 compared with A. Yield and nutritive value for alfalfa in year two were no different between A, CA, and GA, only the S2 treatment reduced the alfalfa yield in the second year. b) The experiment was planted in Fargo and Prosper, ND on 31 and 30 May 2023, respectively. The experimental design was an RCBD with four replicates. Treatments included:1) alfalfa alone; 2) alfalfa-sorghum + 40 kg N/ha; 3) alfalfa-sorghum 80 kg N/ha; 4) sainfoin alone; 5) sainfoin-sorghum + 40 kg N/ha; sainfoin-sorghum + 80 kg N/ha and 7) forage sorghum alone. Forage sorghum biomass yield is not yet available. c) InSouth Dakota, the forage sorghum intercropping experiment was planted on June 7, 2023 on wheat stubble. The experiment was and RCBD with 4 replicates and five treatments; alfalfa alone, sunflower planted at 76- and 152-cm row spacing without alfalfa, and sunflower intercropped with alfalfa at both rows spacing. As expected the yield for alfalfa was much greater for alfalfa alone than that intercropped into forage sorghum; however, forage sorghum yield was not significantly higher when planted alone at 76-cm row spacing (~9 Mg/ha dry matter) or intercropped with alfalfa at 76-cm row spacing. Forage sorghum planted at 152-cm row spacing whether alone or intercropped with alfalfa had significantly lower yield than at 76-cm row spacing. Evaluation of N2 fixation and nitrogen contribution of alfalfa to forage sorghum or sunflower was not possible in 2023. The 15N isotope was ordered but arrived too late in June to be able to use it this season. All experiments will be replicated in 2024 and 15N isotope studies will be conducted as planned. Weed suppression was not evaluated in 2023, since weeding was apriority to collect good establishment and yield data. Objective 2 Sample collection: In 2022, leaf and root samples were collected from field grown oilseed hybrid sunflower 'Falcon' and alfalfa 'Vamoose, RR' planted in a randomized complete block design at Hickson, North Dakota and Red Lake Falls, Minnesota. Root and leaf tissue from sunflower planted in 76-cm or 152-cm row spacing and inter-seeded with or without alfalfa were collected at both locations on July 25 - 26, August 2 - 3, and August 19 -20, 2022. Root and leaf samples were also collected from the inter-seeded alfalfa or from alfalfa planted alone on the same sampling dates. In total, four replicates/treatment produced 96 root and 72 leaf samples at each location for both sunflower and alfalfa (336 samples total). All samples collected in the field were immediately frozen in liquid nitrogen and stored at -80 C prior to being ground to a fine powder for total RNA extraction using established laboratory protocols. Objective 3 Nothing to report

Publications

  • Type: Other Status: Published Year Published: 2022 Citation: Berti, M.T., and M. Tabert. 2022. Establishing alfalfa while growing sunflower. Forage Focus Magazine. August 2022. Midwest Forage Association, St. Paul, MN. p. 2-3
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Berti, M.T., M. Tabert, and J.V. Anderson. 2023. Alfalfa-sunflower intercropping is a profitable way to establish alfalfa. 34th Annual Conference Association for the Advancement of Industrial Crops, Corvallis , OR, 27-30 August 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Berti , M.T. 2023. Establishing alfalfa in intercropping with sunflower. Midwest Forage Association Annual Symposium, Wisconsin Dells, 21-22 February 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Tabert, M. M.T. Berti, J.V. Anderson, S. Bibby, M. Neubauer, K. Mozea, A. Kurth, and H. Mosqueda. 2023 Establishing alfalfa through sunflower intercropping. American Forage Grassland Council Conference, Winston-Salem, NC, 8-11 January, 2023.