The U.S. Department of Agriculture–Agricultural Research Service (USDA-ARS) has been a leader in weed science research covering topics ranging from the development and use of integrated weed management (IWM) tactics to basic mechanistic studies, including biotic resistance of desirable plant communities and herbicide resistance. ARS weed scientists have worked in agricultural and natural ecosystems, including agronomic and horticultural crops, pastures, forests, wild lands, aquatic habitats, wetlands, and riparian areas. Through strong partnerships with academia, state agencies, private industry, and numerous federal programs, ARS weed scientists have made contributions to discoveries in the newest fields of robotics and genetics, as well as the traditional and fundamental subjects of weed–crop competition and physiology and integration of weed control tactics and practices. Weed science at ARS is often overshadowed by other research topics; thus, few are aware of the long history of ARS weed science and its important contributions. This review is the result of a symposium held at the Weed Science Society of America’s 62nd Annual Meeting in 2022 that included 10 separate presentations in a virtual Weed Science Webinar Series. The overarching themes of management tactics (IWM, biological control, and automation), basic mechanisms (competition, invasive plant genetics, and herbicide resistance), and ecosystem impacts (invasive plant spread, climate change, conservation, and restoration) represent core ARS weed science research that is dynamic and efficacious and has been a significant component of the agency’s national and international efforts. This review highlights current studies and future directions that exemplify the science and collaborative relationships both within and outside ARS. Given the constraints of weeds and invasive plants on all aspects of food, feed, and fiber systems, there is an acknowledged need to face new challenges, including agriculture and natural resources sustainability, economic resilience and reliability, and societal health and well-being.

Modern high-throughput molecular and analytical tools offer exciting opportunities to gain a mechanistic understanding of unique traits of weeds. During the past decade, tremendous progress has been made within the weed science discipline using genomic techniques to gain deeper insights into weedy traits such as invasiveness, hybridization, and herbicide resistance. Though the adoption of newer “omics” techniques such as proteomics, metabolomics, and physionomics has been slow, applications of these omics platforms to study plants, especially agriculturally important crops and weeds, have been increasing over the years. In weed science, these platforms are now used more frequently to understand mechanisms of herbicide resistance, weed resistance evolution, and crop–weed interactions. Use of these techniques could help weed scientists to further reduce the knowledge gaps in understanding weedy traits. Although these techniques can provide robust insights about the molecular functioning of plants, employing a single omics platform can rarely elucidate the gene-level regulation and the associated real-time expression of weedy traits due to the complex and overlapping nature of biological interactions. Therefore, it is desirable to integrate the different omics technologies to give a better understanding of molecular functioning of biological systems. This multidimensional integrated approach can therefore offer new avenues for better understanding of questions of interest to weed scientists. This review offers a retrospective and prospective examination of omics platforms employed to investigate weed physiology and novel approaches and new technologies that can provide holistic and knowledge-based weed management strategies for future.

Leafy spurge (Euphorbia esula L.) is an invasive perennial weed infesting range and recreational lands of North America. Previous research and omics projects with E. esula have helped develop it as a model for studying many aspects of perennial plant development and response to abiotic stress. However, the lack of an assembled genome for E. esula has limited the power of previous transcriptomics studies to identify functional promoter elements and transcription factor binding sites. An assembled genome for E. esula would enhance our understanding of signaling processes controlling plant development and responses to environmental stress and provide a better understanding of genetic factors impacting weediness traits, evolution, and herbicide resistance. A comprehensive transcriptome database would also assist in analyzing future RNA-seq studies and is needed to annotate and assess genomic sequence assemblies. Here, we assembled and annotated 56,234 unigenes from an assembly of 589,235 RNA-seq-derived contigs and a previously published Sanger-sequenced expressed sequence tag collection. The resulting data indicate that we now have sequence for >90% of the expressed E. esula protein-coding genes. We also assembled the gene space of E. esula by using a limited coverage (18X) genomic sequence database. In this study, the programs Velvet and Trinity produced the best gene-space assemblies based on representation of expressed and conserved eukaryotic genes. The results indicate that E. esula contains as much as 23% repetitive sequences, of which 11% are unique. Our sequence data were also sufficient for assembling a full chloroplast and partial mitochondrial genome. Further, marker analysis identified more than 150,000 high-quality variants in our E. esula L-RNA–scaffolded, whole-genome, Trinity-assembled genome. Based on these results, E. esula appears to have limited heterozygosity. This study provides a blueprint for low-cost genomic assemblies in weed species and new resources for identifying conserved and novel promoter regions among coordinately expressed genes of E. esula.

Earlier studies on the source of signals controlling correlative inhibition of root buds (underground adventitious buds located on the lateral roots) in Euphorbia esula indicated that either growing meristems (apical or axillary buds) or fully expanded leaves could prevent root buds from breaking quiescence. An investigation of the production and transport requirements of the leaf-derived signal is described. As few as three leaves remaining on budless stems greatly reduced the growth of (but not the number of growing) root buds. Also, light and CO2 fixation were necessary for the leaf effects on root bud growth, but not necessary for correlative inhibition imposed by growing axillary buds. Treatment of plants with Ametryn induced root bud growth on budless plants but not on plants with intact axillary buds. The polar auxin transport inhibitor N-1-naphthylphthalamic acid prevented transmission or the signal from growing axillary buds, but it had only a minor effect on the transmission of the leaf-derived signal. Treatment of plants with gibberellic acid (GA) induced growth of root buds under otherwise noninducing conditions to some extent in all plants. However, the greatest effects of GA were on plants with intact leaves (meristemless/budless and meristemless). GA had no significant effect on root bud quiescence under conditions that induced root bud growth.

Microarray analysis was used to follow changes in gene expression coinciding with seasonal changes in the dormancy status of crown buds of field-grown leafy spurge. Known cold-regulated genes were induced, and numerous gibberellic acid–responsive genes were down-regulated during the transition from paradormancy to endodormancy. Genes involved in photomorphogenesis were induced during endodormancy. Also, ethylene signaling responses were observed during ecodormancy rather than endodormancy. These results provide additional insights into the signals regulating expression of several genes previously associated with transition from paradormancy to growth in root buds.

Arabidopsis complementary DNA (cDNA) microarrays were hybridized with labeled cDNA from mature leaves and emerging tillers of wild oat to determine if they could identify gene expression profiles in distantly related species. More than 23% of the > 11,000 cDNAs on the array hybridized to the wild oat probe. Transcription patterns detected by hybridization to the arrays are indicators for physiological processes in the tissues tested. Coordinated expression patterns for these genes in Arabidopsis indicate common signals involved in their regulation. The results demonstrate that probing cDNA-based arrays from well-characterized species can provide valuable insight into the signal transduction processes regulating growth and development of poorly characterized species.

Canada thistle is a noxious weed that occurs in a wide range of habitats and is difficult to control because of its extensive root system and prolific seed production. Here, we focused on estimating the level of genetic diversity between populations in North Dakota as a first step in examining diversity across North America. Two types of genetic marker, intersimple sequence repeats (ISSRs) and microsatellites were used. Both marker types resulted in polymorphic alleles suitable for assessing diversity. Analysis of molecular variance (AMOVA), molecular diversity analyses, and cluster analysis were conducted. Highly significant variation was detected between populations (P < 0.01). The greatest variance recovered was between individuals within populations. Gene flow among populations in the Northern Great Plains was indicated by the presence of shared alleles between the North Dakota and Minnesota populations and in cluster formation. Multiple introductions and continued gene flow between populations has led to the continued success of Canada thistle as an invasive plant in North America.

In this review, we examine current techniques and recent advances directed toward understanding cellular mechanisms involved in controlling dormancy in vegetative propagules. Vegetative propagules (including stems, rhizomes, tubers, bulbs, stolons, creeping roots, etc.) contain axillary and adventitious buds capable of producing new stems/branches under permissive environments. Axillary and adventitious buds are distinct in that axillary buds are formed in the axil of leaves and are responsible for production of lateral shoots (branches). Adventitious buds refer to buds that arise on the plant at places (stems, roots, or leaves) other than leaf axils. Both axillary and adventitious buds generally undergo periods of dormancy. Dormancy has been described as a temporary suspension of visible growth of any plant structure containing a meristem (Lang et al. 1987). Dormancy can be subdivided into three categories: (1) ecodormancy-arrest is under the control of external environmental factors; (2) paradormancy-arrest is under the control of external physiological factors within the plant; and (3) endodormancy-arrest is under the control of internal physiological factors. One common feature in all of these processes is prevention of growth under conditions where growth should otherwise continue. There is growing evidence that lack of growth is due to blockage of cell division resulting from interactions between the signaling pathways controlling dormancy and those controlling the cell cycle.

Invasive species such as Canada thistle pose a significant threat to ecosystems. The risk of introducing invasive species has increased with human activities, and the effects of such events have economic and aesthetic impacts. Native to Europe, Canada thistle is now established throughout temperate North America. Although there is documentation of early occurrences to North America, little is known on how it has become established in diverse habitats or how it continues to spread. We examined genetic diversity within and among nearly 1,700 Canada thistle individuals from 85 North American locations with the use of seven microsatellite markers in order to address these questions. PAUP and STRUCTURE programs were used to assess genetic diversity and relationships within and between populations. Populations exhibited greater within-population diversity (> 60%) than expected for a reported clonally reproducing species. Total diversity of sampled locations in North America (0.183) was less than previously reported for European locations (0.715), but the greater mean difference between North American populations (0.264 relative to 0.246 from England) suggests strong founder effects or restriction of gene flow influencing individual populations. Furthermore, analyses identified numerous instances where individuals from geographically distant regions clustered together, indicating long-distance translocation of propogules. However, isolation by distance analysis showed significant correlation between location and population genetic distances (r = 0.1917, P = 0.006). Within populations, nearly 92% of individuals sampled harbored unique multilocus genotypes, strongly suggesting that sexual reproduction is common. Within populations, analysis of genetic structure indicated significant admixture of genotypes throughout the invasive range in North America. The recurrent distribution of seed throughout North America has led to a highly diverse gene pool and increased the adaptive success Canada thistle to a wide variety of habitats. Future technologies developed for control of Canada thistle should consider this diversity.

Weed interference with crop growth is often attributed to water, nutrient, or light competition; however, specific physiological responses to these stresses are not well described. This study's objective was to compare growth, yield, and gene expression responses of corn to nitrogen (N), low light (40% shade), and weed stresses. Corn vegetative parameters from V2 to V12 stages, yield parameters, and gene expression using transcriptome (2008) and quantitative polymerase chain reaction (qPCR) (2008/09) analyses at V8 were compared among the stresses and with nonstressed corn. N stress did not affect vegetative parameters, although grain yield was reduced by 40% compared with nonstressed plants. Shade, present until V2, reduced biomass and leaf area > 50% at V2, and recovering plants remained smaller than nonstressed plants at V12. However, grain yields of shade-stressed and nonstressed plants were similar, unless shade remained until V8. Weed stress reduced corn growth and yield in 2008 when weeds remained until V6. In 2009, weed stress until V2 reduced corn vegetative growth, but yield reductions occurred only if weed stress remained until V6 or later. Principle component analysis of differentially expressed genes indicated that shade and weed stress had more similar gene expression patterns to each other than they did to nonstressed or N-stressed tissues. However, corn grown in N-stressed conditions shared 252 differentially expressed genes with weed-stressed plants. Ontologies associated with light/photosynthesis, energy conversion, and signaling were down-regulated in response to all three stresses. Shade and weed stress clustered most tightly together, based on gene expression, but shared only three ontologies, O-METHYLTRANSFERASE activity (lignification processes), POLY(U)-BINDING activity (posttranscriptional gene regulation), and stomatal movement. Based on morphologic and genomic observations, weed stress to corn was not explained by individual effects of N or light stress. Therefore, we hypothesize that these stresses share limited signaling mechanisms.

Microarray analysis was used to identify changes in gene expression in velvetleaf that result from competition with corn. The plants were grown in field plots under adequate N (addition of 220 kg N ha−1) to minimize stress and sampled at the V6 growth stage of corn (late June). Leaf area, dry weight, and N and P concentration were similar in velvetleaf plants grown alone or with corn. Competition, however, did influence velvetleaf gene expression. Genes involved in carbon utilization, photosynthesis, red light signaling, and cell division were preferentially expressed when velvetleaf was grown in competition with corn. A less clear picture of the physiological impact of growth in monoculture was provided by the data. However, several genes involved in secondary metabolism and a gene preferentially expressed in response to phosphate availability were induced. No differences were observed in genes responsive to water stress or sequestering/transporting micronutrients.

Leafy spurge is a tenacious perennial weed of the Northern Plains. This plant maintains a perennial growth cycle by controlled production and growth of numerous underground adventitious buds. We are using molecular tools to identify signaling pathways that control underground adventitious bud growth and development in leafy spurge. Toward this end, we have used three techniques to identify genes that are differentially expressed concomitantly with the breaking of quiescence in underground buds of leafy spurge. These techniques include differential display of cDNAs, random cloning and sequencing of genes expressed in growing buds, and microarray technology. To date, we have identified more than 16 genes that are differentially expressed in underground buds of leafy spurge during dormancy break and growth initiation. A detailed expression analysis of these genes will allow them to be grouped by their responses to various signals known to play a role in control of underground bud growth. This information will be used to identify key cis-acting elements involved in the regulation of these genes. How such information on signal transduction processes may be used for developing new weed control strategies by the identification of novel target pathways and development of DNA-based herbicides is presented.

The genetic basis of weedy and invasive traits and their evolution remain poorly understood, but genomic approaches offer tremendous promise for elucidating these important features of weed biology. However, the genomic tools and resources available for weed research are currently meager compared with those available for many crops. Because genomic methodologies are becoming increasingly accessible and less expensive, the time is ripe for weed scientists to incorporate these methods into their research programs. One example is next-generation sequencing technology, which has the advantage of enhancing the sequencing output from the transcriptome of a weedy plant at a reduced cost. Successful implementation of these approaches will require collaborative efforts that focus resources on common goals and bring together expertise in weed science, molecular biology, plant physiology, and bioinformatics. We outline how these large-scale genomic programs can aid both our understanding of the biology of weedy and invasive plants and our success at managing these species in agriculture. The judicious selection of species for developing weed genomics programs is needed, and we offer up choices, but no Arabidopsis-like model species exists in the world of weeds. We outline the roadmap for creating a powerful synergy of weed science and genomics, given well-placed effort and resources.

Microarray analysis was used to identify changes in gene expression in corn leaves collected from plants at the V11–14 growth stage that resulted from competition with velvetleaf. The plants were grown in field plots under adequate N (addition of 220 kg N ha−1) and irrigation to minimize N and water stress. Consequently, only differences resulting from competition for micronutrients, light, and perhaps allelopathic stress were anticipated. Genes involved in carbon and nitrogen utilization, photosynthesis, growth and development, oxidative stress, signal transduction, responses to auxin and ethylene, and zinc transport were repressed in corn growing in competition with velvetleaf. Very few genes were induced because of competition with velvetleaf, and those that were provided little indication of the physiological response of corn. No differences were observed in genes responsive to water stress or sequestering/transporting micronutrients other than zinc, indicating that these stresses were not a major component of velvetleaf competition with corn at the developmental stage tested.

Infection by Xanthomonas axonopodis pv. manihotis (Xam) of the perennial rangeland weed leafy spurge was tested to see whether Xam might serve a potential biological control agent for this invasive weed. Although leafy spurge was susceptible to Xam infection, it recovered within 21 d after inoculation (DAI). Microarray resources available for leafy spurge allowed us to follow the physiological and signaling pathways that were altered as leafy spurge was infected and then recovered from Xam infection. The first physiological effect of Xam infection was a down-regulation of photosynthetic processes within 1 DAI. By 7 DAI, numerous processes associated with well-documented pathogenesis responses of plants were observed. Although some pathogenesis responses were still detectable at 21 DAI, other processes associated with meristem development were noted. Ontological analysis of potential signaling systems indicated jasmonic acid plays a significant role in the recovery processes.

Signals from both leaves and apical or axillary meristems of leafy spurge are known to inhibit root bud growth. To test the hypothesis that carbohydrates and growth regulators affect root bud growth, decapitated leafy spurge plants were hydroponically treated with glucose, sucrose, gibberellic acid (GA), abscisic acid (ABA), 1-naphthaleneacetic acid (NAA), 6-benzylaminopurine (BA), and a GA biosynthesis inhibitor, paclobutrazol. Both glucose and sucrose caused suppression of root bud growth at concentrations of 30 mM. The inhibitory effect of sucrose was counteracted by GA at 15 μM. In contrast, BA, ABA, NAA, and paclobutrazol inhibited root bud growth at concentrations as low as 1, 2, 1, and 16 μM, respectively. Sugar and starch levels were also determined in root buds at various times after decapitation. Buds of intact plants contained the highest level of sucrose compared with buds harvested 1, 3, and 5 d after decapitation. To determine how seasonal changes affect root bud dormancy, growth from root buds of field-grown plants was monitored for several years. Root buds of field-grown leafy spurge had the highest level of innate dormancy from October to November, which persisted until a prolonged period of freezing occurred in November or early December. Our data support the hypothesis that carbohydrates may be involved in regulating dormancy status in root buds of leafy spurge.

As a first step toward developing a genomics-based research program to study growth and development of underground adventitious shoot buds of leafy spurge, we initiated a leafy spurge expressed sequence tag (EST) database. From the approximately 2,000 clones randomly isolated from a cDNA library made from a population containing growth-induced underground adventitious shoot buds, we have obtained ESTs for 1,105 cDNAs. Approximately 29% of the leafy spurge EST database consists of expressed genes of unknown identity (hypothetical proteins), and 10% represents ribosomal proteins. The remaining 60% of the database is composed of expressed genes that show BLASTX sequence identity scores of ≥ 80 with known GenBank accessions. Clones showing sequence identity to a Histone H3, a gibberellic acid-responsive gene, Tubulin, and a light-harvesting chlorophyll a/b-binding protein were shown to be differentially expressed in underground adventitious shoot buds of leafy spurge after breaking of dormancy. RNA encoding a putative cyclin-dependent protein kinase (CDK)-activating kinase, a gene associated with cell division, and Scarecrow-like 7, a gene involved in GA signaling, were present at similar levels in dormant and growth-induced underground adventitious shoot buds. These data show how even a small EST database can be used to develop a genomics-based research program that will help us identify genes responsive to or involved in the mechanisms controlling underground adventitious shoot bud growth and development.

We have isolated both a genomic and near full length cDNA clone for a D-class cyclin gene from the perennial weed leafy spurge. Sequence analysis indicates that this gene has the highest similarity to CYCLIN D3-2 of Arabidopsis. This gene is preferentially expressed in growing shoot apices and is up-regulated in adventitious buds on resumption of growth following loss of correlative inhibition (apical dominance). CYCLIN D3-2 is also induced in nongrowing adventitious buds of plants treated with gibberellic acid or after removal of leaves—treatments known to up-regulate expression of G1 to S phase transition–specific genes such as HISTONE H3 in adventitious buds. CYCLIN D3-2 was not induced on removal of the apical and axillary buds. Expression of CYCLIN D3-2 is down-regulated in adventitious crown buds during initiation of ecodormancy in early winter. Sequence comparisons of CYCLIN D3-2 with its putative orthologue from Arabidopsis identified several conserved motifs in the promoter region and a conserved region capable of forming a stable hairpin loop in the 5′ untranslated region. Conservation of these noncoding sequences across species strongly suggests they have a regulatory function.

Genomics programs in the weed science community have not developed as rapidly as that of other crop, horticultural, forestry, and model plant systems. Development of genomic resources for selected model weeds are expected to enhance our understanding of weed biology, just as they have in other plant systems. In this report, we describe the development, characteristics, and information gained from an expressed sequence tag (EST) database for the perennial weed leafy spurge. ESTs were obtained using a normalized cDNA library prepared from a comprehensive collection of tissues. During the EST characterization process, redundancy was minimized by periodic subtractions of the normalized cDNA library. A sequencing success rate of 88% yielded 45,314 ESTs with an average read length of 671 nucleotides. Using bioinformatic analysis, the leafy spurge EST database was assembled into 23,472 unique sequences representing 19,015 unigenes (10,293 clusters and 8,722 singletons). Blast similarity searches to the GenBank nonredundant protein database identified 18,186 total matches, of which 14,205 were nonredundant. These data indicate that 77.4% of the 23,472 unique sequences and 74.7% of the 19,015 unigenes are similar to other known proteins. Further bioinformatics analysis indicated that 2,950, or 15.5%, of the unigenes have previously not been identified suggesting that some may be novel to leafy spurge. Functional classifications assigned to leafy spurge unique sequences using Munich Information Center for Protein or Gene Ontology were proportional to functional classifications for genes of arabidopsis, with the exception of unclassified or unknowns and transposable elements which were significantly reduced in leafy spurge. Although these EST resources have been developed for the purpose of constructing high-density leafy spurge microarrays, they are already providing valuable information related to sugar metabolism, cell cycle regulation, dormancy, terpenoid secondary metabolism, and flowering.