Research Project: Novel Weed Management Solutions: Understanding Weed-Crop Interactions in Northern Climates

Location: Sunflower and Plant Biology Research

2019 Annual Report

Objectives
Objective 1: Identify, at the genome and physiological levels, plant-plant interactions that impact plant growth and lead to crop yield losses, especially crop-weed interactions that occur during the critical weed-free period, and interactions that occur between the different crops inter-planted in relay cropping systems, such as corn, soybeans, or sunflowers relayed with camelina, ryegrass, or canola. [NP304, Component 2, Problem Statement 2A3] Sub-objective 1.A: Determine the parameters for evaluating the impacts of winter annual cover crops on corn, sunflower, and Amaranthus spp. productivity. Sub-objective 1.B: Identify physiological and molecular mechanisms that control interactions between cover crops and corn, sunflower, and Amaranthus spp. Sub-objective 1.C: Evaluate impacts of candidate genes on cover crop-relay crop and cover crop-weed interactions. Objective 2: Determine the molecular and physiological mechanisms by which winter annual cover crops suppress weeds in northern temperate agroecosystems, and identify genes that will enhance weed suppression in these crops, such as genes associated with weed-tolerance, cover-crop tolerance, and cold hardiness. [NP304, Component 2, Problem Statement 2A3] Sub-objective 2.A: Identify genetic markers for improving the weed-suppressing trait of winter hardiness in winter canola and/or camelina varieties. Sub-objective 2.B: Evaluate the weed-suppressing traits of winter-hardy canola and camelina in the field.

Approach
Weeds are major pests of agro-ecosystems that reduce production of the nation’s food, feed, fiber and fuel crops. The industry-adopted practice of rotating crops with engineered tolerance to a limited set of herbicides continues to put selection pressure on the evolution of herbicide-resistant weeds. As part of a holistic and sustainable approach to managing weeds in temperate agro-ecosystems, we propose to identify cover crop-relay crop interactions to enhance relay crop productivity, cover crop-weed interactions to enhance weed suppression, and identify winter-hardy annual cover crops that suppress weeds in relay cropping systems. In this proposal, winter canola will serve a dual purpose as both a cover crop for evaluating weed suppression, and as a surrogate weed for weed-relay crop interactions. Our model relay cropping system consists of inter-seeding a commodity crop (corn or sunflower) into an established cover crop (winter canola) such that their lifecycles overlap. Currently, no winter-hardy annual broadleaf cover crops are economically suited for weed suppression in relay cropping systems in the upper Midwest (UMW) and Northern Great Plains (NGP). Consequently, the objectives of this project are: (1) elucidating regulatory signals and pathways associated with cover crop-relay crop and cover crop-weed interactions that impact plant productivity, and (2) identifying economically suited winter-hardy broadleaf cover crops that suppress weed establishment in inter-seeded relay crops. These objectives will be accomplished through the use of physiological, molecular, and genomic approaches, with the long-term goals of revolutionizing weed management practices.

Progress Report
Sub-objective 1.A: Determine the parameters for evaluating the impacts of winter annual cover crops on corn, sunflower, and Amaranthus spp. productivity. Phenological data for corn and sunflower have been collected and analyzed. The work on corn has been submitted for peer review. Due to a critical vacancy in the unit, work on Amaranthus has not been accomplished. Sub-objective 1.B: Identify physiological and molecular mechanisms that control interactions between cover crops and corn, sunflower, and Amaranthus spp. In FY18, we published a paper identifying Salicylic Acid (SA) signaling as a probable regulator of weed-induced yield loss in corn under field conditions. In FY19, we submitted another manuscript that strengthened the hypothesis that SA regulates corn’s response to weed pressure and identified several robust weed-responsive genes in corn. The necessary datasets have been obtained for analyzing and submitting a manuscript describing mechanisms associated with weed-induced yield losses in sunflower, and that help sunflower detect and respond to both above- and below-ground weed signals. Finally, we have obtained the necessary datasets to identify the corn signaling mechanism responsive to below-ground signals from weeds. We expect to have the full dataset analyzed for generating a peer-reviewed manuscript by the end of 2019. Sub-objective 1.C: Evaluate impacts of candidate genes on cover crop-relay crop and cover crop-weed interactions. Two weed-inducible genes have been identified, and constructs are being developed to test the hypothesis that sequences within the promoter of these weed-inducible genes can regulate the expression of a reporter gene when weeds are present. If the promoter successfully results in weed-induced transcription of the reporter gene, the promoter will be modified to identify the portion responsible for its specific weed-inducibility. Additionally, because salicylic acid has been identified as a probable regulator of weed-induced yield loss in corn, constructs are currently being designed to determine if weed-induced expression of a known SA degradation gene can be achieved, and if so, will it protect corn against weed-induced yield loss. Sub-objective 2.A: Identify genetic markers for improving the weed-suppressing trait of winter hardiness in winter canola and/or camelina varieties. Two separate freezing experiments using the Kansas State University winter canola diversity panel (413 winter canola accessions) have been completed. After fully cold-acclimating the winter canola accessions (5 C for 8 weeks), phenotypic data was collected based on three different damage measures including: 1) visual damage scores two weeks following freezing (-15 C for 4 hours), 2) photosynthetic efficiency obtained by fluorometer readings at three-days after freezing, and 3) photosynthetic efficiency seven-days after freezing. Each freezing experiment included three replicated runs (with three technical reps/run for a total of 9 plants) and the freezing experiments were replicated twice for an overall total of 18 plants evaluated. To identify loci associated with freezing tolerance in the winter canola diversity panel, a genome-wide association study (GWAS) was conducted. Thirty-two significant genetic loci were identified among both experimental runs. Candidate genes within the associated loci have been identified and a manuscript is currently under development for peer review in FY19. Because the Kansas State University winter canola diversity panel was developed in a region of the U.S. classified as zones 6a-6b (-23.3 to -17.8 C), the germplasm may not be practical for the winter hardiness zones of North Dakota, which are classified as zones 3b-4a (-37.2 to -31.7 C). Thus, we obtained an additional 222 winter canola accessions from Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) in Germany and have completed genotyping these accessions in collaboration with North Dakota State University. We are currently phenotyping these mostly European lines for freezing tolerance under controlled environmental conditions. We have completed the first round of freezing experiments (222 line with 3 reps/line) and a second and third freezing experiment will be accomplished in August and October of 2019. Once the phenotyping for freezing tolerance is accomplished on the IPK lines of winter canola, we will conduct a genome wide association study to identify genetic loci associated with freezing tolerance. This information will help to determine if the GWAS results obtained from the IPK lines correlate well with our previous results obtained using the Kansas State University diversity panel and/or may identify additional significant loci associated with freezing tolerance in winter canola. Using a genome wide association study approach, four genomic loci associated with freezing tolerance in winter canola were also identified after three days of cold-deacclimation. Additionally, RNAseq datasets obtained from control (no cold acclimation), cold-acclimated (5 C for 8 weeks) and following 1 and 2 weeks of deacclimating conditions (10C), identified over 13,000 genes that are differentially-expressed under these conditions in winter canola. Combined, these studies allowed the identification of ten candidate genes within the four loci that may be involved in altering the time required for cold-deacclimation to occur. Methods have also been developed to analyze photographic data from the canopy of arabidopsis plants to assess freezing damage. Two arabidopsis mutant lines for each candidate gene identified by the cold-deacclimation genome wide association study were obtained. We are currently testing the impact of these gene mutations on arabidopsis’ ability to alter the deacclimation period, or to cold-acclimate and/or grow under cold conditions. To confirm the involvement of these candidate genes in freezing tolerance and cold deacclimation processes, we are first developing CRISPR technology protocols for functional evaluations in winter canola and camelina. To establish a working protocol and transformation process, six binary vector CRISPR constructs with modified FLOWERING LOCUS C (FLC) and MADS-AFFECTING FLOWERING 2 (MAF2) have been developed and transformed into camelina cultivars CO46 (a spring biotype) and Joelle (a winter biotype) to evaluate the efficiency of the protocols and their impact on flowering- and vernalization-related processes. Screening for potential transgenic lines by phenotypic alteration of flowering and vernalization is ongoing. Once these protocols are in place, we will move forward with testing the impact of candidate genes on freezing tolerance and cold deacclimation processes in winter canola and camelina. Sub-objective 2.B: Evaluate the weed-suppressing traits of winter-hardy canola and camelina in the field. The 222 IPK lines obtained from the European collection of winter canola were fall planted in two replicated field plots and allowed to over-winter. Although good germination and stand establishment was observed in the fall, none of the accessions survived the winter conditions of 2018-2019 in Fargo, North Dakota. In the fall of 2017, two replicated field plots were set up to test the ability of the top 5 most freezing tolerant (4 hours at -15 C) accessions from a Kansas State University winter canola diversity panel. Again, fall stand establishment was good but the major plant species identified in all plots in the spring of 2018 (winter survival) was field pennycress. These native populations of field pennycress were allowed to mature and their ability to suppress weeds and retain nutrients was evaluated in 2018 and 2019. A strong correlation was observed between the field pennycress canopy cover and canopy cover of other weeds, indicating that field pennycress has superior weed suppressing traits. Chemical analysis obtained from the whole plant suggests that allelopathic chemicals could also be playing some role in the weed suppressing traits of field pennycress, but further analysis will be needed to confirm that hypothesis. Additionally, field pennycress plants retained significant levels of essential nutrients (nitrogen, phosphorus, potassium and sulfur) from the soil; thus, reducing nutrient runoff from the soil. The outcomes of this research and the ecosystem benefits of developing field pennycress as a winter hardy oilseed cover crops for relay- and double cropping system of cold northern agroecosystems were presented at the Weed Science Society of America Annual Meeting in 2019. A manuscript related to the outcomes of this field study will also be submitted in 2019 and will include data related to seed oil quality and other whole plant allelopathic properties.

Accomplishments
1. Field pennycress is a beneficial oilseed cover crop for suppression of weeds in the Northern Great Plains. Winter hardy oilseed cover crops for the North Central U.S. are lacking. Recently, ARS scientists in Fargo, North Dakota, observed native populations of field pennycress as the dominant species in replicated field plots early in the growing season. These native populations were allowed to grow and were evaluated for beneficial ecosystem services. Field pennycress canopy cover and stand establishment had significant correlations with suppression of other weeds in the field plots and the plants retained significant levels of essential soil nutrients. These results demonstrate the potential for field pennycress to be developed into a useful winter hardy oilseed cover crop for managing weeds and reducing nutrient runoff in northern agroecosystems.

2. Identifying genes of Camelina sativa that control winter hardiness. Cultivars of Camelina sativa differ significantly in their freezing tolerance. ARS scientists in Fargo, North Dakota, studied freezing traits in individuals from crosses between a freezing-tolerant and a freezing-sensitive genotype and discovered that a small number of genes impart freezing tolerance to the resistant line. Unique genetic lines developed to identify the genomic locations of these freezing tolerance genes will help breeders improve winter survival in Brassicaceae oilseed cover crops suitable for the North Central U.S.

3. Identifying genes and signals regulating response of corn to weeds. Weeds induce yield losses in corn through early season signaling processes. ARS scientists in Fargo, North Dakota, determined that salicylic acid and phytochrome signaling are probable regulators of these weed-induced yield losses. Two corn genes consistently induced by weeds in both greenhouse and field studies were identified and provide the basis for deciphering the signaling process by which corn detects and responds to weeds. These regulatory sequences will allow testing of the hypothesis that corn can be made weed tolerant by blocking salicylic acid signaling when weeds are present.

4. Identifying genes associated with and controlling cold acclimation processes in winter canola. Winter hardy plants must be exposed to a period of cold temperatures to cold acclimate and become freezing tolerant. Because of global climate change, plants may become more susceptible to freezing damage due to either insufficient cold acclimation or due to brief warm spells that cause plants to cold deacclimation. ARS scientists in Fargo, North Dakota, identified four chromosomal locations associated with altered cold deacclimation rates in winter canola and ten candidate genes were identified within these regions. Using the model plant, Arabidopsis thaliana, mutations in several of these genes altered freezing tolerance following cold-deacclimation, and one reduced damage even in plants that were not cold-acclimated. This work provides the tools needed for breeders to develop crops more tolerant to climate change-related temperature fluctuations.

5. Identifying molecular markers associated with freezing tolerance in winter canola. Few winter canola lines can consistently survive the winter conditions of the Northern Great Plains. ARS scientists in Fargo, North Dakota, completed a genome wide association study and identified 32 chromosomal locations associated with freezing tolerance in winter canola and more than 20 candidate genes were identified within these regions. Markers associated with these chromosomal regions provide a starting point for breeders to integrate freezing tolerance into elite breeding lines of both winter- and spring types of canola, which should help to increase the production of winter and spring canola in the U.S.

6. Identifying molecular markers to differentiate summer and winter biotypes of camelina. Many accessions of camelina are misclassified as winter- or summer-annuals. ARS scientists in Fargo, North Dakota, identified molecular markers to accurately (100%) differentiate summer- and winter-biotypes of camelina using a simple polymerase chain reaction technique. These results have been used to accurately re-classify existing accessions of summer- or winter-biotypes within USDA-ARS collections of camelina. This molecular marker technique has been published and will be useful for future classification of camelina germplasm prior to being marketed for production.

7. Improved methods for evaluating agronomic traits of camelina oilseed. Determining the agronomic traits of oilseeds can be time consuming and expensive. ARS scientists in Fargo, North Dakota, in collaboration with scientists from North Dakota State University, developed calibration equations to determine crude protein, total oil, fatty acid profiles, and the summer and winter biotype of camelina seed using near infrared spectrometry. The results of this work will be useful as a rapid, non-destructive, high-throughput method for determining agronomically important traits in camelina seed.

Review Publications
Ma, G., Zhang, W., Liu, L., Chao, W.S., Gu, Y.Q., Qi, L., Xu, S.S., Cai, X. 2018. Cloning and characterization of the homoeologous genes for the Rec8-like meiotic cohesin in polyploid wheat. Biomed Central (BMC) Plant Biology. 18:224. https://doi.org/10.1186/s12870-018-1442-y.
Fiebelkorn, D., Horvath, D., Rahman, M. 2018. Genome-wide association study for electrolyte leakage in rapeseed/canola (Brassica napus L.). Molecular Breeding. 38:129. https://doi.org/10.1007/s11032-018-0892-0.
Chao, W.S., Wang, H., Horvath, D.P., Anderson, J.V. 2019. Selection of endogenous reference genes for qRT-PCR analysis in Camelina sativa and identification of FLOWERING LOCUS C allele-specific markers to differentiate summer- and winter-biotypes. Industrial Crops and Products. 129:495-502. https://doi.org/10.1016/j.indcrop.2018.12.017.
Carvalho, L.J.C.B., Anderson, J.V., Da Silva, J.P., Chen, S., De Souza, C.R.B. 2019. Protein content in cassava storage root is associated with total abundance of carotenoids. International Research Journal of Plant Science. https://doi.org/10.14303/irjps.2019.003. 10(1):1-10.
Wittenberg, A., Anderson, J.V., Berti, M.T. 2019. Winter and summer annual biotypes of camelina have different morphology and seed characteristics. Industrial Crops and Products. 135:230-237. https://doi.org/10.1016/j.indcrop.2019.04.036.
Liu, D., Horvath, D., Li, P., Liu, W. 2019. RNA sequencing characterizes transcriptomes differences in cold response between northern and southern Alternanthera philoxeroides and highlight adaptations associated with northward expansion. Frontiers in Plant Science. 10:24. https://doi.org/10.3389/fpls.2019.00024.
Anderson, J.V., Wittenberg, A., Li, H., Berti, M.T. 2019. High throughput phenotyping of Camelina sativa seeds for crude protein, total oil, and fatty acids profile by near infrared spectroscopy. Industrial Crops and Products. 137:501-507. https://doi.org/10.1016/j.indcrop.2019.04.075.
Horvath, D., Anderson, J.V., Chao, W.S., Zheng, P., Buchwaldt, M., Parkin, I.A.P., Dorn, K. 2019. Genes associated with chloroplasts and hormone-signaling, and transcription factors other than CBFs are associated with differential survival after low temperature treatments of Camelina sativa biotypes. PLoS One. 14(5):e0217692. https://doi.org/10.1371/journal.pone.0217692.