Research Project: Biology of Weed-Crop Interactions to Improve Weed Management Strategies in Northern Agro-ecosystems

Location: Weed and Insect Biology Research

2025 Annual Report

Objectives
Objective 1: Determine the nature of inter and intraspecific competition and extent of crop yield loss among relay or double cropped agricultural plant species in comparison to natural weed competition. Sub-objective 1A: Identify, under field conditions, the genes that are differentially regulated by natural weed populations, cover crops, and intra-specific competition in sunflower (Helianthus annuus). Sub-objective 1B: Examine the impact of intra-specific competition on sunflower and corn (Zea mays L.) yield loss and gene expression under controlled conditions Objective 2: Identify genetic or biochemical signals associated with interspecific competition and determine the associated biological mechanisms that can be used as targets for genetic manipulation. Sub-objective 2A: Create constructs from corn promoters to identify the transcription factor(s) binding sites regulating weed- and/or cover crop-inducible genes. Sub-objective 2B: Test if changes in salicylic acid levels corresponds to weed perception in corn. Sub-objective 2C: Utilize the weed inducible promoter from corn to suppress the salicylic acid signaling during weed-crop or crop-cover crop interactions under controlled greenhouse conditions. Objective 3: Functionally characterize specific targets impacting interspecific competition for genetic manipulation of weed tolerance, winter survival, early maturation, and/or response to bioherbicides. Sub-objective 3A: Identify winter hardy canola and camelina germplasm that also have an early maturity trait for reducing competition between the cover crop and the relay-crop. As a first step, we will map early maturation Quantitative Trait Loci (QTLs) in a segregating Recombinant Inbred Line (RIL) population of Camelina sativa. Sub-objective 3B: Determine if the freezing tolerance genes identified from winter camelina will increase freezing tolerance in canola (Brassica napus L.). Sub-objective 3C: Functionally characterize the weed-induced PIF3 genes in soybean (Glycine max (L.) Merr).

Approach
Integrated weed management (IWM) is considered the most effective approach for managing weeds. In the northern Great Plains, incorporation of winter-hardy crops or cover crops as components of IWM systems are gaining popularity as an approach for managing weeds and the spread of herbicide resistant weeds. However, just like weeds, inter-specific competition with winter crops or cover crops, when used in multi-cropping systems, results in yield losses in major commodities. In this project, multi-cropping refers to fall-planting of oilseed cover crops that overwinter and are terminated or harvested prior to planting a primary summer commodity crop (double-cropping) or a primary commodity crop inter-seeded into the cover crop such that their life cycles overlap (relay-cropping). Factors impacting competition-induced yield losses have only been evaluated in a limited number of traditional multi-cropping systems under field conditions and this gap in knowledge needs to be addressed as new cropping and IWM systems suitable for the northern Great Plains are developed. To generate new knowledge for regionally-appropriate IWM approaches, the goals of this project are to: 1) understand how major commodity crops perceive and respond to inter- and intra-specific competition, 2) identify genes regulating winter survival and early maturity that can be manipulated to improve these traits in winter crops and cover crops, and 3) identify targets for mitigating competition-induced yield losses through breeding or genetic manipulation. Being able to multi-crop major commodities with winter-hardy crops or cover crops without resulting in yield loss, or mitigating weed-induced yield losses in general, would provide new IWM options. Thus, the objectives of this project will address gaps in our knowledge that limit the ability to develop sustainable IWM approaches appropriate for agricultural intensification in the northern Great Plains.

Progress Report
Objective 1: As part of this objective, field treatments included intra-cropping (sunflower-sunflower) and inter-cropping (sunflower-natural weeds or alfalfa- sunflower). Under field conditions in South Dakota, analysis of RNAseq data associated with intra- (sunflower-sunflower) or inter-specific (sunflower-natural weed populations) competition only identified one differentially expressed gene (DEG) of unknown function between treatments. Thus, it was impossible to develop any testable hypotheses on that data alone. Analysis of RNAseq data from samples collected over time and space in Minnesota and North Dakota related to intra- (sunflower-sunflower) and inter-specific (alfalfa-sunflower) competition are still being sequenced and analyzed. However, preliminary results from root samples of alfalfa in competition with inter-cropped sunflower identified 47 DEGs that overlap among the study treatments. Gene Set Enrichment Analysis identified two overrepresented gene ontologies associated with defense response and heme binding, and highlighted pathways involved in secondary metabolism including phenylpropanoid, xenobiotic, isoflavonoid, staurosporine, benzoxazinoid and carotenoid biosynthesis as key biological processes associated with the roots of alfalfa inter-cropped with sunflower. Two transcription factors containing binding sites known as TRITERPENE SAPONIN BIOSYNTHESIS ACTIVATING REGULATOR 1 and 2 (TSAR1 and TSAR2) were also overrepresented among the DEGs identified in root tissue of alfalfa, which, in other plant systems, are involved in activating a suite of secondary metabolism genes involving the plant hormone jasmonic acid. These preliminary results provide knowledge for developing testable hypothesis for signaling mechanisms involved in interspecific plant-plant competition. Under greenhouse conditions, time course studies were done to evaluate corn competition with weeds, which indicated that soil soluble signals had a greater impact on corn growth than light quality signals. The soil soluble signals impacted photosynthesis in leaves and cell wall production in the roots, whereas the light quality signal did not have a consistent impact on leaves but did impact protein turnover and cell growth in roots. These studies also identified overrepresented gene ontologies associated with oxidative stress signaling in corn throughout the time of weed exposure. Gene ontologies associated with nitrogen use and transport, and abscisic acid (ABA) signaling were also observed in corn competing with weeds, with defense responses being enriched at later time points. Many of the genes included specific sequence motifs known to bind with transcription factors such as far-red-impaired response 1 and several APETALA2/Ethylene Responsive Factors, which are proteins known to regulate gene expression. These gene networks and protein complexes regulate the balance between defense and growth in plants and this knowledge should provide breeders with potential mechanisms for mitigating weed-induced crop yield losses. Objective 2: Studies confirmed that a gene, called DOMAIN-CONTAINING 1 (DC1), was consistently up-regulated in corn in response to competition with weeds, under greenhouse conditions. The plant hormone, salicylic acid (SA), was also significantly greater in corn growing with weeds than under weed-free conditions, suggesting SA may be a potential cause for weed-induced yield loss in crops. The promotor region of the DC1 weed-inducible gene was attached to a reporter gene (Red3) and transferred into corn but the response to weeds, though consistent, was not significant. Transgenic plants containing NahG (a gene coding for salicylate hydroxylase that should reduce SA) under control of the DC1 promoter expressed NahG RNA under control conditions and enhanced expression under weedy conditions. However, homozygous lines containing NahG did not show consistent up-regulation of the reporter gene in all transgenic lines tested. Additionally, the expression of NahG did not significantly inhibit the weed response of the transgenic corn. These transgenic corn lines are important for confirming the functionality of weed-inducible promoters and the role of SA in weed-induced yield losses, but additional work is needed to understand these observations. However, a major outcome from this objective helped to develop new theories regarding plant-plant competition. Objective 3: Winter canola/rapeseed accessions (621 total) were evaluated for freezing tolerance in response to cold acclimation/deacclimation treatments using Genome-Wide Association Studies (GWAS). Candidate genes in the chromosome regions associated with freezing tolerance included SENSITIVE TO FREEZING 2 (SFR2) that is involved in the remodeling of certain cell membranes in response to freezing, and VERNALIZATION INDEPENDENCE 3 (VIP3) that prevents deacclimation when mutated in Arabidopsis thaliana (a mutation in this gene was also identified in our deacclimation insensitive canola accessions). Constructs for canola SFR2 and VIP3 were developed using clustered regularly interspaced short palindromic repeats (CRISPR) technology and are being used to characterize their roles in transgenic plants. A protocol for successful regeneration of winter canola from callus tissue was recently accomplished and is being evaluated for the regeneration of transgenic winter canola plants. Field research using all of our winter canola/rapeseed accessions and a winter hardy camelina check revealed that overwinter survival was correlated with suppression of early season weeds. Nine winter hardy canola/rapeseed lines that survived in both North Dakota and Minnesota were identified, which contained several gene models previously associated with freezing tolerance, including SFR2. Seed from three of these 9 winter canola lines was increased in the greenhouse and is currently being regionally evaluated in North Dakota, Minnesota, and Alaska for overwinter survival under field conditions. Interestingly, the best winter survival in 2024/2025 overwinter trials occurred in Fairbanks, Alaska. This, in part, could be due to the better insulating properties of seasonal snow cover in Alaska, or the poor fall establishment of winter canola in North Dakota and Minnesota in 2024 resulting from a lack of fall precipitation. The field study will be repeated in the fall of 2025 for evaluation of winter survival in 2026. Winter biotypes of camelina (Camelina sativa) have shown good winter survival in the Great Plains, upper Midwest, and Alaska and are an intermediate crop with proven weed-suppressing traits that make an excellent oilseed feedstock for sustainable aviation fuel. However, the development of camelina germplasm with improved agronomic traits is still needed. As part of this objective, a biparental population developed from a winter- by spring-cross of camelina resulted in 254 Recombinant Inbreed Lines (RILs) that were used for Quantitative Trait Loci (QTL) analysis and homozygosity mapping. Chromosome regions of the camelina genome associated with freezing tolerance, flowering time, and other important oilseed traits (seed size, thousand seed weight, total fat, crude protein and oil profile) highlighted potential roles for the transcription factor known as FLOWERING LOCUS C (FLC). A complex locus containing multiple cysteine-rich receptor-like kinase genes and a single plant serine-rich receptor-like gene were also identified by homozygosity mapping and QTL analysis of freezing tolerance in the RILs. To gain better insights into the functional role for each of the three FLC genes (one each in Chromosomes 8, 13, and 20), functional studies with CRISPR knockouts for each of the FLC genes are underway. This work also improved the assembly of the camelina genome and provided a new reference genome for C. sativa, and helped identify candidate genes associated with freezing tolerance, flowering time, and other agronomically important traits of oilseeds. An impactful outcome of this objective was the transfer of seed from the diverse population of 254 RILs of camelina, and the spring type parent (C046) to the North Central Regional Plant Introduction Station in Ames, Iowa (https://npgsweb.ars-grin.gov/gringlobal/cooperator?id=120628), which represents the largest single donation of camelina to the Germplasm Resource Information Network (GRIN). This research and associated resources provide breeders and others with phenotypic and genotypic knowledge for improving traits and provide new options for sustainable intensification of U.S. crop production in cold climates and soils, while also providing farmers, growers, and industry with enhanced integrated pest management options. RNAseq analysis from soybean also implicated two PHYTOCHROME INTERACTING FACTOR 3 (PIF3) genes that may play a role in weed-induced signaling responses. To further explore the role of these genes in soybean- weed interactions, soybean lines with CRISPR-based homozygous knockouts of one or both weed-inducible PIF3 genes were developed. These transgenic lines are currently being evaluated to determine the impact of these knockouts on weed responsiveness, alterations in gene expression, and signal perturbations in soybeans growing under greenhouse conditions with and without canola as the weed competitor.

Accomplishments
1. Competition by kochia does not impact canola growth or yield. Canola growers expressed concerns that herbicide resistant kochia may cause problems for canola yield. To investigate how planting densities of canola impact kochia growth and how kochia density impacts canola growth, ARS scientists in Fargo, North Dakota, investigated the responses of canola and kochia competition under both field and greenhouse conditions. Outcomes indicated that kochia growth and its transcriptome are significantly impacted by even very low densities of canola, but canola was not affected by any of the kochia densities. This information will help growers, and the canola industry better understand the impact of herbicide resistant kochia on canola yields and option for better control.

2. A new germplasm resource for improving agronomic traits in camelina. Camelina (Camelina sativa) suppresses weeds and the oilseed is a feedstock for the sustainable aviation fuel market. Camelina consists of both winter- and spring-biotypes, which provides an opportunity for exploring its genetic diversity for improving desired agronomic traits. Scientists at the USDA-ARS in Fargo, North Dakota, developed a spring by winter biparental population of 254 recombinant inbreed lines (RILs) that were phenotyped and genotyped to help identify regions of the camelina chromosome and underlying genes associated with agronomically important traits. Seeds from the RILs were transferred to the National Plant Germplasm System where they are available on request. This new camelina germplasm collection provides breeders and other researchers with the knowledge and resources for developing camelina lines with desired agronomic traits for farmers, growers and industry needs.

3. A new AI app for quantifying freezing damage in plants. Quantifying freezing damage in crops is often subjective in nature, which limits the ability to accurately access crop performance. To improve quantification of freezing damage in crops, ARS scientists in Fargo, North Dakota, in collaboration with engineers at North Dakota State University developed a new app for quantifying freezing damage in plants. The new app was developed using digital technologies from multiple crop species with varying degrees of freezing damage. Algorithms were also developed and refined to allow quantification of freezing damage using a standard cell phone photo. This new technology will allow growers and insurance adjusters to more accurately access freezing damage in real time and provide breeders with new tools for improving stress tolerance in crops.

4. Identification of spring type Kernza. Farmers and growers have expressed interest in perennial grain crops that also provide environmental benefits and improve soil health. Kernza is an emerging perennial grain and cover crop that generally requires cold treatment (vernalization) to initiate flowering and grain production. Recently, ARS scientists in Fargo, North Dakota, identified Kernza germplasm that enters the reproductive state without vernalization. This new discovery should allow breeders to develop spring type Kernza germplasm for more southern regions of the U.S. that do not receive sufficient cold temperature for inducing the vegetative to reproductive transition. It also provides scientists with new resources to discover the underlying molecular regulation of reproduction in perennial grains.

Review Publications
Long, Y., Zheng, P., Anderson, J.V., Horvath, D.P., Sthapit Kandel, J., Li, X., Rahman, M., Chao, W.S. 2024. A novel strategy to map a locus associated with flowering time in canola (Brassica napus L.). Molecular Genetics and Genomics. https://doi.org/10.1007/s00438-024-02191-w.
Gesch, R.W., Eberle, C.A., Berti, M.T., Ott, M., Anderson, J.V. 2025. Productivity and seasonal water use of double cropped dry bean, proso millet, and sunflower after early maturing winter camelina. Industrial Crops and Products. https://doi.org/10.1016/j.indcrop.2025.120953.
Shaikh, T., Rahman, M., Anderson, J.V., Sthapit Kandel, J., Roy, J., Vaughn, J.N., Smith, T.P., Abernathy, B., Ontano, A., Dobrin, B.H., Dorn, K.M., Horvath, D.P. 2024. QTL mapping to identify loci and candidate genes associated with freezing tolerance trait in camelina sativa. Industrial Crops and Products. 222. Article 119562. https://doi.org/10.1016/j.indcrop.2024.119562.
Berti, M.T., Morocho-Lema, M., Anderson, J.V. 2025. Sensitivity of winter and spring camelina to salinity during germination. Industrial Crops and Products. https://doi.org/10.1016/j.indcrop.2025.121293.
Ontano, A., Dobrin, B.H., Smith, T.P., Abernathy, B., Sthapit Kandel, J., Shaikh, T., Rahman, M., Anderson, J.V., Vaughn, J.N., Horvath, D.P. 2024. Assembly and analysis of sequence from a spring and winter type Camelina sativa by whole genome PacBio Hifi technologies. Industrial Crops and Products. 221. Article 119346. https://doi.org/10.1016/j.indcrop.2024.119346.
Berti, M., Morocho-Lema, M., Anderson, J.V., Lizarazo-Torres, C. 2025. Nitrogen rates affect seed yield and carbon intensity in spring and winter camelina. Industrial Crops and Products. https://doi.org/10.1016/j.indcrop.2025.121473.
Hosain, S., Horvath, D.P., Roy, J., Hoque, A., Arifuzzaman, M., Mukhlesur, R. 2025. Genome-wide association study and genomic prediction for pod-shattering tolerance in a diverse rapeseed/canola germplasm collection. Euphytica. https://doi.org/10.1007/s10681-025-03494-8.
Sunil, G., Khan, A., Horvath, D.P., Sun, X. 2025. Evaluation of multispectral imaging for freeze damage assessment in strawberries using AI-based computer vision technology. Smart Agricultural Technology. https://doi.org/10.1016/j.atech.2025.100851.
Paul, N., Gc, S., Horvath, D.P., Sun, X. 2024. Deep learning for plant stress detection: A comprehensive review of technologies, challenges, and future directions. Computers and Electronics in Agriculture. https://doi.org/10.1016/j.compag.2024.109734.
Hao, X., Tang, J., Chen, Y., Huang, C., Zhang, W., Liu, Y., Yue, C., Wang, L., Ding, C., Dai, W., Yang, Y., Horvath, D.P., Wang, X. 2024. CsCBF1/CsZHD9-CsMADS27, a critical gene module controlling dormancy and bud break in tea plants. Plant Journal. https://doi.org/10.1111/tpj.17165.