Location: Weed and Insect Biology Research
2021 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. A. A medium maturity sunflower hybrid (Falcon) was planted at two field sites in North Dakota (Hickson and Prosper), one site in Minnesota (Red Lake Falls), and one site in South Dakota (Brookings) between May 12-22, 2021. At the North Dakota and Minnesota locations, sunflower was planted at 30 and 60-inch row spacing. At the North Dakota location, sunflower was interseeded with or without alfalfa; however, at the Minnesota location, a cover crop mix was interseeded with sunflower on the planting date (May 22), or at the V2 (vegetative stage 2) (June 9) or V6-V8 (June 19) stages of sunflower development. All field plots at the North Dakota and Minnesota locations were sprayed with herbicide or hand weeded as needed. At the South Dakota location, sunflower was planted at two times the normal planting rate for intra-specific competition. For inter-specific competition studies in South Dakota, sunflower was interseeded with either rapeseed or wild sunflower, or natural weeds were allowed to grow. At the V4 stage of sunflower development, one treatment at the South Dakota location included removal of cover crops or weed as competitors of sunflower. All field sites were planted in a randomized complete block design with 4 replicates per treatment. At the R1 (reproductive 1) stage of development, tissue samples were collected from the distal end of the largest fully expanded healthy leaf and from roots (including tightly bound soil) of two sunflower plants per replicated treatment. Collected samples were immediately frozen in liquid nitrogen and stored at -80 C for future RNA extraction and analysis of leaf nutrient content. Sunflower plant height, stem diameter, and fresh and dry weights is being recorded at various stages of sunflower development at all locations.
Objective 1, B. A medium maturity sunflower hybrid (Falcon) and a corn inbred (B73) were planted in pots and grown under greenhouse conditions. Sunflower and corn were grown in 2-gallon pots with sunshine mix, or in 1-gallon pots (to determine impact of reduced soil volume). All studies were set up in a randomized complete block design with 6 replicates per treatment for sunflower and 3 replicates for corn. Plants were grown under 16:8 hour light:dark conditions and watered daily and fertilized weekly to reduce or eliminate any impact of nutrient availability. Treatments included 1 sunflower plant per pot (either a 2- or 1-gallon pot), 2 sunflower plants per pot, and 4 sunflower plants per pot. At 4 weeks of growth, plant height and stem diameter data were collected. At the R4 stage of sunflower development, plant height, stem diameter, and fresh and dry weight of above ground tissue was recorded. Additionally, tissue samples were collected from a healthy mid-level leaf and from root tissue of each sunflower replicate per treatment at the R4 stage of development. For corn, a single corn seedling was grown in a 2-gallon pot with four additional corn plants grown in cone-tainers in the same pot to prevent root-to-root contact and with opaque above-ground cones to prevent light quality signaling between plants until corn was at the V6 stage of development. At that point, root-to-root contact was re-established by removing the corn plants from their cone-tainers and replanting the corn plant back into the soil in half of the pots. The other half were mock treated by removing the corn in cone-tainers from the potted soil, but then replacing the corn and cone-tainers back into the potted soil as they were. Root material was collected at 0, 1, 2, 3, 7, and 14 days from treated and mock-treated plants. All samples were immediately frozen in liquid nitrogen and stored at -80 C for future RNA extraction.
Objective 2, A. We have amplified a weed-inducible promoter to a gene called DC1, using genomic DNA isolated from corn line B73. The amplified DC1 promoter was cloned into a plant expression vector called pTF101. To verify that the cloned DC1 promoter contained sequences responsible for turning the gene on in response to weed presence, a reporter gene encoding for a red fluorescent protein called DsRed was inserted behind the DC1 promotor, and all the amplified and cloned products were confirmed by sequencing. To verify if the DC1 promoter activity is responsible for regulating gene expression in response to weed pressure or other competitors, corn was transformed with our constructs through a contract with the Iowa State University Plant Transformation Facility. We obtained thirteen unique clones of individual transgenic corn plants (containing the artificially introduced DC1 promoter and DsRed) and we grew the transgenic lines in a greenhouse (1 plant per pot). After one month of greenhouse growth, four canola plants (3-week-old) were transplanted into the corn pot as plant competitors to see if they would mimic weed-induced responses and turn on the DC1 promoter (as controls, transgenic corn did not have a canola treatment). Three weeks after dual growth of corn with canola, leaf tissue was collected from all the transgenic corn plants for RNA extraction and cDNA synthesis. After the collection of leaf tissue from corn, all canola plants were removed from pots containing transgenic corn. We are in the process of determining if the DC1 promoter caused an increase in DsRed gene transcription in response to canola as a competitor. In addition, all transgenic corn plants used in this study are being crossed with corn line B73 to perpetuate the transgenic lines.
To test the hypothesis that salicylic acid signaling may be involved in corn yield losses when exposed to competition by weeds or canola, we also placed a bacterial gene called NahG behind the DC1 weed-inducible promoter. NahG codes for a protein that degrades salicylic acid (a plant hormone involved in regulating plant growth and development). For the NahG construct, the NahG gene was amplified and the octopine synthase terminator was added behind the NahG coding sequence; all constructs were confirmed by sequencing. Multiple clones from fourteen independent transgenic corn plants were obtained and grown in the greenhouse. Growth of transgenic plants, dual growth of corn and canola, sample handling, and screening of transgenes are the same as that of DsRed transgenic plants previously described.
Objective 2, B. Corn (85-day VT double pro hybrid 75k85) was planted at 30- and 60-inch row spacing at field plots in Hickson and Prosper, North Dakota in 2020 and grown with or without interseeded alfalfa. Corn was also planted in 30-inch rows at a field plot in Fargo, North Dakota in 2021 and grown with or without competition from natural weed populations. All field studies were conducted using a randomized complete block design with 4 replicates per treatment. Corn was also grown in pots under greenhouse conditions with 1 corn plant per pot. In the greenhouse, potted corn plants were grown with or without 4 rapeseed plants per pot. For both the field and greenhouse studies, tissue samples were collected from the distal 10 cm of the top-most healthy expanded leaf and from root of each corn replicate per treatment at the V12 stage of development. All samples were immediately frozen in liquid nitrogen and stored at -80 C for future salicylic acid extraction and measurements. Plant height, stem diameter, and fresh and dry weight of two corn plants per replicate and treatment were also recorded at the V12 stage of development.
Due to an incompatibility issue between our High-Performance Liquid Chromatography (HPLC) instrument and an older fluorometer, analysis of salicylic acid levels in the collected corn tissue samples was delayed. However, a new fluorometer compatible with our existing HPLC system was purchased and installed in the summer of 2021 and we are currently conducting quality control on the methodology for extraction and measurements of free and conjugated salicylic acid from corn. By late summer to early fall of 2021, we expect to start measuring salicylic acid levels from collected corn tissue samples.
Objective 3, A. Our F7 Recombinant Inbred Line (RIL) population, consisting of 254 lines, was developed by crossing a winter biotype (Joelle) with a spring biotype (CO46) and advancing selected offspring through 7 generations of single seed descent. The F7 RIL population has been successfully phenotyped for days to first flower and freezing tolerance, and DNA extracted from each individual in the population has been used to genotype for single nucleotide polymophisms (SNPs). Within the RIL population, 38 lines (6 replicate plants/line) did not flower without first receiving a cold (5 C for 56 days) treatment, 35 lines had mixed flowering (among the 6 replicate plants/line) without a cold treatment, and the remainder (181 lines) all flowered without a cold treatment. Among those lines that flowered without a cold treatment, average days to first flower ranged from 32 to 71. When all 254 lines in the RIL population received a cold treatment (5 C for 56 days), all lines and replicate plants flowered - with the days to first flower ranging from 81 to 106 (this includes the 56 days of cold treatment). The results suggest that our vernalization treatment reduces the range for days to first flower by approximately 45%. Genotyping of DNA isolated from each line in the RIL population identified over 3700 high quality SNP markers, which were filtered first for markers homozygous and divergent in the two parents (CO46 and Joelle), and with a major to minor allele ratio between 0.25 and 1.5 covering all contigs with hits to known camelina genes. We are currently mapping the SNPs to the camelina genome and identifying quantitative trait loci linked to early maturity and freezing tolerance.
Accomplishments
1. A genetic resource for improving agronomic traits in Brassica oilseed species. A genetic resource for improving agronomic traits in Brassica oilseed species. Freezing tolerance and early maturity are important agronomic traits of winter hardy oilseed cover and cash crops used for multi-cropping systems in the northern United States. Camelina is an oilseed species related to canola that is gaining popularity as a cover or cash crop option in multi-cropping systems. Identifying winter hardy camelina germplasm with an early maturity trait would reduce competition in multi-cropping systems where the life cycle of cover crops and commodity crops overlap. ARS scientists in Fargo, North Dakota, developed new lines of camelina by crossing a winter- and spring-biotype. The 254 lines in this new population have been used to identify genetic markers and to evaluate their freezing tolerance and flowering time. This new resource will be a valuable tool for marker assisted selection of germplasm and traditional breeding for improving agronomic traits in Brassica oilseeds and will provide new options for enhancing sustainable intensification practices.
2. Deciphering mechanisms involved in weed-induced yield loss in corn. To develop weed tolerant crops, there is a need to understand the signaling processes by which crops perceive and respond to weeds. ARS scientists in Fargo, North Dakota, together with their collaborators at South Dakota State University, identified a gene in corn that is consistently up-regulated in response to weeds and further identified the plant hormone salicylic acid as a potential cause for weed-induced yield loss in crops. Researchers created transgenic corn lines with the promotor from the weed-inducible gene linked to a gene that reduces salicylic acid levels. In the presence of weeds, the weed-inducible promoter in the transgenic corn works as an “on” switch to increase production of the gene that reduces salicylic acid levels. These transgenic lines are an important resource for generating the knowledge needed to develop weed-tolerant corn varieties, to minimize the need for herbicides, and to enhance sustainable intensification practices through multi-cropping systems.
3. Expanding the production range of canola. Canola is an important oilseed crop contributing to the global demand for oil production. Although winter canola generally produces greater yields than spring canola, the range of winter canola is limited by its inability to withstand the winter conditions experienced in many regions of the northern U.S. As a first step towards increasing winter hardiness in canola, ARS scientists in Fargo, North Dakota, have evaluated a population of rapeseed (a progenitor of canola) containing 222 lines collected from Europe. Using genetic approaches, several genome regions associated with freezing tolerance were identified. The genetic markers and genes associated with these chromosome regions will provide breeders with options for integrating freezing tolerance traits into elite breeding lines of both winter- and spring-types of canola, and to expand canola production in the United States.
Review Publications
Olson, D.O., Anderson, J.V. 2021. Review on unmanned aerial vehicles, remote sensors, imagery processing, and their applications in agriculture. Agronomy Journal. 113(2):971-992. https://doi.org/10.1002/agj2.20595.
Wittenberg, A., Anderson, J.V., Berti, M.T. 2020. Crop growth and productivity of winter camelina in response to sowing date in the northwestern corn belt of the USA. Industrial Crops and Products. 158:1-12. https://doi.org/10.1016/j.indcrop.2020.113036.
Chao, W.S., Horvath, D.P., Stamm, M.J., Anderson, J.V. 2021. Genome-wide association mapping of freezing tolerance in canola (Brassica napus L.). Agronomy Journal. 11(2):233. https://doi.org/10.3390/agronomy11020233.
Horvath, D.P., Jiaping, Z., Chao, W.S., Ashok, M., Rahman, M., Anderson, J.V. 2020. Genome wide association studies and transcriptome changes during acclimation and deacclimation in divergent canola varieties. International Journal of Molecular Sciences. 21(23):9148. https://doi.org/10.3390/ijms21239148.
Horvath, D.P., Stamm, M., Talukder, Z., Fiedler, J.D., Horvath, A., Horvath, G., Chao, W.S., Anderson, J.V. 2020. A new diversity panel for winter rapeseed (Brassica napus, L.) genome wide association studies. Agronomy Journal. 10(12):2006. https://doi.org/10.3390/agronomy10122006.
Prasifka, J.R., Ferguson, B., Anderson, J.V. 2020. Fatty acid data and crop surveys indicate sources of red sunflower seed weevil, Smicronyx fulvus LeConte (Coleoptera: Curculionidae), populations and suggest strategies for management. Environmental Entomology. 50(1):154-159. https://doi.org/10.1093/ee/nvaa158.
