Leafy spurge, a noxious perennial weed, is a major threat to the prairie ecosystem in North America. Strategic planning to control leafy spurge requires monitoring its spatial distribution and spread. The ability to detect flowering leafy spurge at two biological control sites in southern Saskatchewan, Canada, was investigated using an unmanned aerial vehicle (UAV) system. Three flight missions were conducted on June 30, 2016, during the leafy spurge flowering period. Imagery was acquired at four flight heights and one or two acquisition times, depending on the site. The sites were reflown on June 28, 2017, to evaluate the change in flowering leafy spurge over time. Mixture tuned matched filtering (MTMF) and hue, intensity, and saturation (HIS) threshold analyses were used to determine flowering leafy spurge cover. Flight height of 30 m was optimal; the strongest relationships between UAV and ground estimates of leafy spurge cover (r2 = 0.76 to 0.90; normalized root mean square error [NRMSE] = 0.10 to 0.13) and stem density (r2 = 0.72 to 0.75) were observed. Detection was not significantly affected by the image analysis method (P > 0.05). Flowering leafy spurge cover estimates were similar using HIS (1.9% to 14.8%) and MTMF (2.1% to 10.3%) and agreed with the ground estimates (using HIS: r2 = 0.64 to 0.93, NRMSE = 0.08 to 0.25; using MTMF: r2 = 0.64 to 0.90, NRMSE = 0.10 to 0.27). The reduction in flowering leafy spurge cover between 2016 and 2017 detected using UAV images and HIS (8.1% at site 1 and 2.7% at site 2) was consistent with that based on ground digital photographs (10% at site 1 and 1.8% at site 2). UAV imagery is a useful tool for accurately detecting flowering leafy spurge and could be used for routine monitoring purposes in a biological control program.

Euphorbia esula is an exotic perennial weed that is estimated to infest 650,000 ha in North and South Dakota, Montana, and Wyoming. The estimated annual economic effect of E. esula infestations in the four-state area is about $130 million. We present the results of a survey of ranchers, local decision makers, and public land managers of grazing and nongrazing property from a five-county area in North and South Dakota, Montana, and Wyoming. A total of 565 ranchers, local decision makers, and public land managers were surveyed, which resulted in 267 completed questionnaires. The main objective of the survey was to evaluate respondent's perspectives of managerial, institutional, and social factors that may affect the rate and extent of implementation of various E. esula control strategies. The reasons for not using the various E. esula controls generally fell into environmental, financial, and educational categories. In many cases, little can be done to remove environmental constraints; however, financial constraints may be addressed through cost-share programs either offered locally or through state agencies. The other main reason for not using some controls was a lack of knowledge to work with various controls (e.g., grazing and biological controls). These obstacles could be addressed with workshops, demonstrations, or educational bulletins. These educational tools could be provided by universities or governmental agencies, as respondents indicated this was their choice for information dissemination. Disagreements among the survey groups were not substantial, and many share similar concerns in controlling the weed.

Absorption, translocation, and metabolism of AC 263,222 by leafy spurge were studied over 8 d. Based on the amount of herbicide applied and recovered from the leaf surfaces, 40% of applied AC 263,222 was absorbed by leafy spurge 2 d after treatment (DAT), with no further absorption observed by 8 DAT. Eight DAT, 19% of applied [14C]-AC 263,222 had translocated to below-ground plant parts while 4% was exuded from the roots into the sand media. AC 263,222 was not metabolized 2 DAT in the crown, root, and root buds, but 42% was metabolized in the treated leaves. Only 17% of recovered [14C] was AC 263,222 in treated leaves 8 DAT (83% metabolized), while AC 263,222 accounted for 70% of recovered [14C] in the root and root buds. HPLC analysis indicated that the balance of [14C] was associated with a single, polar metabolite. Total recovery of [14C] was 88% at 8 DAT.

Dormancy is a critical factor for the survival and persistence of weedy species. Contemporary approaches can be used to identify genes that regulate dormancy directly or indirectly to elucidate key mechanisms, signals, and pathways. Several domesticated plant species have been used as model systems to mark quantitative trait loci (QTL) that affect dormancy in seeds and vegetative propagules directly. A few weedy species have also been used to mark QTL and to determine dormancy genes using microarray analysis. Given the number of serious weeds worldwide and the role that dormancy plays in their persistence, developing fundamental knowledge on dormancy is an important step toward developing new strategies for weed management. This paper describes current research and outlines some weeds that might be candidates for dormancy investigations using molecular genetic and genomics approaches. An underlying theme in the selection of weeds for dormancy investigations is their relation to crop species and the ability to adapt existing resources to investigate dormancy in weedy plants.

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.

Real-time polymerase chain reaction (real-time PCR), also known as quantitative PCR, is used to determine relative gene expression or to quantify exact levels of mRNA in cells or tissues. Before the advent of real-time PCR, the major difficulty associated with traditional quantitative or semiquantitative PCR was to ensure that PCR reactions were quantified within the exponential phase of amplification. Real-time PCR alleviates that problem by detecting and quantifying fluorescent signals after each amplification cycle. Additionally, it does not require running gels and thus is able to produce data in 2 to 3 h. Four different types of chemistries, DNA-binding agents (SYBR Green), hydrolysis probes (TaqMan), hairpin probes (molecular beacons, scorpions), and fluorescent-labeled hybridization probes (Light Cycler), have been commonly used for real-time PCR. Among those chemistries, SYBR Green is the most economical choice. We have used real-time PCR and SYBR Green to examine the expression of a number of leafy spurge genes after growth induction and during normal seasonal growth. Because no reliable endogenous reference genes have been identified in leafy spurge, we performed PCR without an endogenous reference gene and analyzed messenger RNA (mRNA) expression based on the threshold cycle (CT) value of amplification. Excluding an endogenous reference gene from that data analysis was rather straightforward and reliable if RNA was properly prepared and quantified. Given that genomic tools, such as expressed sequence tags (ESTs), and their expression profiles are lacking for most weedy species, avoiding the use of endogenous reference genes in real-time PCR simplifies the optimization process and reduces the cost tremendously. However, we found that using a passive reference dye (ROX) to normalize non-PCR–related fluctuations in fluorescent signal is desirable.

Aphthona spp. flea beetles have established and reduced the density of leafy spurge in much of the western United States. One way to measure the long-term impact and effectiveness of a weed control program is by monitoring the changes in the seedbank over time. The change in leafy spurge stem density and seed in the seedbank were evaluated 5 yr after Aphthona spp. were released to control this weed in the Little Missouri National Grasslands in southwestern North Dakota. Leafy spurge density and seed in both loamy overflow and loamy ecological sites decreased, whereas desirable (high-seral) forbs increased 5 yr after the biological control agents were released. Leafy spurge topgrowth was reduced from an average of over 200 stems/m2 to less than 8 stems/m2 in the most densely infested sites, and leafy spurge seed was reduced from an average of 68% of the seedbank to only 14% in both ecological sites. High-seral forb seed increased by over 300% in the seedbank, which indicated the floristic quality of the sites, was returning to a preinfestation state. Species with the largest increase included western rock jasmine and fringed sage, which increased at least three-fold in both sites. Less desirable low-seral forbs and grasses accounted for about 30% of the seedbank. In a greenhouse study, native grass production was reduced nearly 50% when grown in soil from Aphthona spp. release sites compared to nonrelease sites. Switchgrass production was reduced to a greater extent (66%) than green needlegrass, little bluestem, or western wheatgrass. The cause and extent of reduced native grass production in sites where Aphthona spp. were released has yet to be determined. The decrease in leafy spurge topgrowth and seed in the soil seedbank as desirable species seed increased, should lead to the long-term recovery of the plant community.

Neither herbicides nor Aphthona spp. biological control agents provide satisfactory leafy spurge control in all environments where the weed is found. Open-release and caged field studies were conducted to determine the effect of herbicides used in combination with Aphthona spp. flea beetles on both the leafy spurge control and the biological control agent populations. Leafy spurge density was reduced more rapidly when A. nigriscutis or a mixture of A. czwalinae and A. lacertosa was combined with a fall-applied treatment of picloram plus 2,4-D than when either method was used alone. The combination treatment reduced leafy spurge density 3 to 5 yr earlier than did either the herbicides or the biocontrol agents alone and once reduced, the Aphthona flea beetles maintained acceptable control for at least 7 yr. Spring-applied herbicides eliminated the adult food source and negatively affected the flea beetle establishment, but fall-applied treatments did not reduce the flea beetle establishment or its reproduction. The combination treatment was most successful when used with established Aphthona spp. populations that were not increasing in size or reducing leafy spurge density. Land managers could realize a three- to fivefold cost savings with the combination treatment compared with herbicides alone.

Leafy spurge can be detected during flowering with either aerial photography or hyperspectral remote sensing because of the distinctive yellow-green color of the flower bracts. The spectral characteristics of flower bracts and leaves were compared with pigment concentrations to determine the physiological basis of the remote sensing signature. Compared with leaves of leafy spurge, flower bracts had lower reflectance at blue wavelengths (400 to 500 nm), greater reflectance at green, yellow, and orange wavelengths (525 to 650 nm), and approximately equal reflectances at 680 nm (red) and at near-infrared wavelengths (725 to 850 nm). Pigments from leaves and flower bracts were extracted in dimethyl sulfoxide, and the pigment concentrations were determined spectrophotometrically. Carotenoid pigments were identified using high-performance liquid chromatography. Flower bracts had 84% less chlorophyll a, 82% less chlorophyll b, and 44% less total carotenoids than leaves, thus absorptance by the flower bracts should be less and the reflectance should be greater at blue and red wavelengths. The carotenoid to chlorophyll ratio of the flower bracts was approximately 1:1, explaining the hue of the flower bracts but not the value of reflectance. The primary carotenoids were lutein, β-carotene, and β-cryptoxanthin in a 3.7:1.5:1 ratio for flower bracts and in a 4.8:1.3:1 ratio for leaves, respectively. There was 10.2 μg g−1 fresh weight of colorless phytofluene present in the flower bracts and none in the leaves. The fluorescence spectrum indicated high blue, red, and far-red emission for leaves compared with flower bracts. Fluorescent emissions from leaves may contribute to the higher apparent leaf reflectance in the blue and red wavelength regions. The spectral characteristics of leafy spurge are important for constructing a well-documented spectral library that could be used with hyperspectral remote sensing.

The combination of Aphthona spp. flea beetles and herbicides can increase leafy spurge control when compared with either method used alone, but the effect of herbicides on the biocontrol agent and conversely the effect of Aphthona on herbicides within the plant are unknown. Understanding this interaction will allow for maximum weed control with little or no negative effect on the establishment and reproduction of the biological agent. Fall application of picloram plus 2,4-D had a minimal negative effect on the A. nigriscutis population and no effect on overwintering fitness. The number of A. nigriscutis adults collected from soil cores taken from the field was similar regardless of the herbicide application date, although emergence was variable. Leafy spurge soluble and insoluble carbohydrate and soluble protein concentrations, which are indirect measurements of the herbicide's effect on nutrient availability to the insect, were similar in treated and untreated plants. Aphthona nigriscutis larval feeding did not affect the absorption or translocation of 14C–2,4-D or 14C-picloram alone or in combination in leafy spurge. The observed increase in leafy spurge control due to the combined treatment was likely an additive effect of herbicide toxicity to root tissue and A. nigriscutis larval feeding on root buds.

The impact of invasive weed management on plant community composition is highly dependent on location-specific factors. Therefore, treatment means from experiments conducted at a given set of locations will not reliably predict community response to weed management elsewhere. We developed a model that rescales treatment means to better match local conditions. The goal of this paper was to determine if this rescaling improves predictions. We used our model to predict leafy spurge stem length density and grass biomass data from field experiments. The experiments consisted of herbicide-treated plots, untreated controls, and, in some cases, grass seeding treatments. When herbicides suppressed leafy spurge, the model explained 21 to 48% more variation in grass response than did mean grass response to the same or similar herbicide treatments applied at other sites. When herbicides killed grass, the model explained 53% more variation in leafy spurge response than did mean leafy spurge response to the same herbicide treatment applied at other sites. We regressed model predictions against observed data and tested the null hypothesis that resulting slope terms were equal to 1.0. Because the null hypothesis was rejected in two of four tests, the model may systematically over- or underpredict in some situations. However, measurement error in the observed data, unintended herbicide injury, or an inaccurate allometric relationship may account for a major proportion of the systematic deviations, and these factors would not cause prediction error in some management applications. Because the model tends to be better than the means from experiments at predicting plant community composition, we conclude that the model could advance managers' ability to predict plant community responses to invasive weed management.

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.

Chemical, cultural, and biological methods have been developed to control leafy spurge in a variety of environments. Aphthona spp. biological control agents have established throughout the northern Great Plains and Rocky Mountain region and successfully controlled leafy spurge in many areas, but notable exceptions include areas with sandy soils. Leafy spurge control can be improved by combining methods such as chemical, biological, or cultural treatments, compared to a single method used alone. The effects of Aphthona spp., imazapic herbicide, and interseeded native grass species alone or in combination for leafy spurge control were evaluated at two locations in southeastern North Dakota for 5 yr. Both the Sheyenne National Grassland (SNG) and Walcott, ND, study locations had greater than 80% sand soil. Leafy spurge stem density, canopy cover, and yield were reduced for 1 to 2 yr in all treatments that included imazapic, with no difference in control between single and combination treatments. Aphthona spp. and interseeded native grasses alone or combined did not reduce leafy spurge density or cover. Aphthona spp. population remained low throughout the study at both locations. Forb yield increased during the study at the SNG but not the Walcott location. Conversely, warm-season grass yield increased at Walcott but not at the SNG. Leafy spurge stem density declined from 92 to 50 stems/m2 in 5 yr at the SNG site. The decline could not be attributed to specific treatments applied in this study and may be due to self-limitation or soil pathogens.

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.

Flea beetles (Aphthona spp.) were released in the Little Missouri National Grasslands (LMNG) in western North Dakota in 1999 to control leafy spurge. The changes in leafy spurge density and soil seedbank composition were evaluated on two ecological sites 10 yr (2009) after Aphthona spp. release to monitor the effectiveness of the insects on weed control and the associated changes in plant communities. In 2009, leafy spurge stem density averaged 2 and 9 stems m−2 (0.19 to 0.84 ft−2) in the loamy overflow and loamy sites, respectively, compared with 110 and 78 stems m−2, respectively, in 1999. Leafy spurge constituted nearly 67% of the loamy overflow seedbank in 1999, compared with 2% in 2009. In the loamy seedbank, the weed represented nearly 70% in 1999, compared with approximately 15% in 2009. As leafy spurge abundance was reduced, native species richness and seed count increased 10 yr after Aphthona spp. release. Late seral species represented 17% of the loamy overflow seedbank in 2009, an increase from 5% in 1999. However, Kentucky bluegrass, a nontarget weedy species, increased more than 250% in the loamy overflow seedbank. Late seral species were 38% of the loamy seedbank in 2009, compared with 13% in 1999. The number of native species increased from 31 in 1999 to 39 in 2009 in the loamy overflow seedbank, but only changed from 32 to 34 species in the loamy site during the same period. The reestablishment of native species has been slow, but seedbank analysis indicates the number and type of species found before the leafy spurge infestation have increased. Planting native species in selected areas may have reduced the lag time in these species return to the seedbank and reduced invasion from other nondesirable species, such as Kentucky bluegrass.

Exotic plants have the ability to modify soil seed banks in habitats they invade, but little is known about the legacy of invasion on seed banks once an exotic plant has successfully been controlled. Natural areas previously invaded by leafy spurge in the northern Great Plains typically have one of two fates following its removal: a return of native plants, or a secondary invasion of other exotic plants. It is unknown, however, if this difference in plant communities following leafy spurge control is due to seed bank differences. To answer this question, we monitored seed banks and standing vegetation for 2 yr in mixed-grass prairies that were previously invaded by leafy spurge but controlled within 5 yr of our study. We found that native plant seed banks were largely intact in areas previously invaded by leafy spurge, regardless of the current living plant community, and leafy spurge invasion history had a larger impact on cover and diversity of the vegetation than on the seed banks. Differences in plant communities following leafy spurge control do not appear to be related to the seed banks, and soil conditions may be more important in determining trajectories of these postinvasion communities.

The biological control agent Aphthona nigriscutis Foudras (Chrysomelidae) established in Fremont County, WY since 1992 on leafy spurge was released into a mixed stand a native plant Euphorbia robusta Engelm. During host range testing, E. robusta was a likely host for A. nigriscutis under laboratory conditions. In 1999, A. nigriscutis was observed feeding on both E. esula and 31 of 36 E. robusta plants present on about 2 ha (5 ac) where the visually estimated E. esula canopy cover was 50%. By August 2001, E. esula cover had declined to less than 5% and E. robusta plants had increased to 450 plants with 26 (5.8%) showing feeding damage. In 2006 Euphorbia esula ground cover was 2% and of 598 E. robusta plants originally marked, 391 could be located and four of these had damage consistent with A. nigriscutis feeding. For the 8-yr period, E. esula ground cover was inversely correlated to E. robusta density and positively correlated to A. nigriscutis feeding damage on E. robusta. This study shows that while also acceptable to A. nigriscutis in the field, feeding on E. robusta declined with declining densities of the target weed while E. robusta population densities increased. It seems that some risk in this regard is acceptable in light of the damage from the target weed and the generally high level of selectivity provided by biological control agents.

Diflufenzopyr is an auxin-transport inhibitor that has increased broadleaf weed control by some auxin herbicides. The effects of auxin herbicides when applied with diflufenzopyr for leafy spurge or Canada thistle control, herbage production, and herbicide absorption and translocation in leafy spurge were evaluated. The influence of diflufenzopyr on leafy spurge and Canada thistle control varied by herbicide. Diflufenzopyr applied with quinclorac increased both Canada thistle and leafy spurge control. Diflufenzopyr added to picloram increased leafy spurge but not Canada thistle control, whereas control of both weeds generally increased when diflufenzopyr was applied with dicamba. Canada thistle control slightly improved when diflufenzopyr was applied with clopyralid or clopyralid plus 2,4-D compared with the herbicides alone. Diflufenzopyr did not influence weed control from imazapic and reduced weed control from glyphosate. Weed control was not influenced by the ratio of diflufenzopyr to herbicide or by whether diflufenzopyr was tank mixed with or applied before the herbicides. Diflufenzopyr did not affect cool-season grass production in the greenhouse or field but did reduce sideoats grama and switchgrass production when applied alone and with dicamba in greenhouse trials. 14C-picloram and 14C-quinclorac absorption was increased when applied with diflufenzopyr in leafy spurge but nearly all absorbed herbicide remained in the treated leaf compared with the herbicides applied alone. 14C-dicamba absorption decreased from 60 to 14% when applied with diflufenzopyr and translocation to the roots decreased by a factor of 10. Overall, the increase in weed control from the addition of diflufenzopyr was much more pronounced with leafy spurge than with Canada thistle.

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.