Phytotoxic Allelochemicals From Roots and Root Exudates of Leafy Spurge (Euphorbia esula L.)

Invasive plants are a widespread problem but the mechanisms used by these plants to become invasive are often unknown. The production of phytotoxic natural products by invasive weeds is one mechanism by which these species may become successful competitors. Here we conducted a bioactivity-driven fractionation of root extracts and exudates from the invasive plant leafy spurge (Euphorbia esula L.), and structurally characterized jatrophane diterpenes and ellagic acid derivatives. Ellagic acid derivatives and one of the jatrophane diterpenes, esulone A, have been previously reported from leafy spurge, but another of the jatrophane diterpenes, kasuinine B, has not. We show that these compounds are phytotoxic but affect plants in different ways, either inducing overall plant necrosis or reducing root branching and elongation.Key Words: phytotoxicity, allelochemicals, roots, root exudates, jatrophane diterpenes, kansuinine B, ellagic acid derivatives, leafy spurge, Euphorbia esula, Arabidopsis thaliana     

Biology and host specificity ofChamaesphecia hungarica andCh. Astatiformis (Lep.: Sesiidae) two candidates for the biological control of leafy spurge,Euphorbia esula (Euphorbiaceae) in North America

Leafy spurge (Euphorbia esula (s.1.)) is an herbaceous perennial and serious weed of Eurasian origin that has been accidentally introduced into North America. The two European root-boring mothsChamaesphecia hungarica andCh. astatiformis are univoltine and overwinter as mature larvae. Both species have a lower survival rate on leafy spurge than on their field hosts, and thus are not optimal candidates for the biological control of leafy spurge. However, the rate of larval development and larval growth on the target weed and on the two field hosts is nearly the same. The experimental host range of both species is restricted to a few species in the subgenusEsula within the genusEuphorbia. The two species occupy different habitats in the steppe biome and are targeted for similar leafy spurge habitats in North America.     

Effect of Leafy Spurge (Euphorbia esula) Genotype on Feeding Damage and Reproduction of Aphthona spp.: Implications for Biological Weed Control

North American leafy spurge (Euphorbia esula L.) is genetically diverse and composed of multiple genotypes introduced from several areas of Europe and Asia. Five species of leafy spurge flea beetle (Aphthona spp.) have been introduced as biological control agents for leafy spurge, but were collected in a relatively small region of Europe. Greenhouse and field experiments were conducted to determine if observed variation in feeding preference and reproduction of Aphthona spp. on North American leafy spurge may be due in part to leafy spurge genotype. Leafy spurge genotypes were collected from Austria; Manitoba in Canada; and the states of Montana, Nebraska, North Dakota, South Dakota, and Wyoming in the United States. Leafy spurge genotype affected feeding but not egg laying by Aphthona spp. adults. Aphthona czwalinae/lacertosa fed slightly less in a free-choice test on a genotype from Manitoba (7%) compared to genotypes from Nebraska and North Dakota (14%). Aphthona flava tended to feed less on the Nebraska genotype than any other genotype evaluated. Reproduction of Aphthona spp. was greatly affected by leafy spurge genotype. For instance, A. czwalinae/lacertosa produced 72 adults per plant from a Nebraska genotype compared to 11 to 32 adults per plant from all other genotypes evaluated. Thus, some observed variation in establishment and reproduction of Aphthona spp. can be attributed to leafy spurge genotype.     

Do invasive plants structure microbial communities to accelerate decomposition in intermountain grasslands?

Invasive plants are often associated with greater productivity and soil nutrient availabilities, but whether invasive plants with dissimilar traits change decomposer communities and decomposition rates in consistent ways is little known. We compared decomposition rates and the fungal and bacterial communities associated with the litter of three problematic invaders in intermountain grasslands; cheatgrass (Bromus tectorum), spotted knapweed (Centaurea stoebe) and leafy spurge (Euphorbia esula), as well as the native bluebunch wheatgrass (Pseudoroegneria spicata). Shoot and root litter from each plant was placed in cheatgrass, spotted knapweed, and leafy spurge invasions as well as remnant native communities in a fully reciprocal design for 6 months to see whether decomposer communities were species‐specific, and whether litter decomposed fastest when placed in a community composed of its own species (referred to hereafter as home‐field advantage–HFA). Overall, litter from the two invasive forbs, spotted knapweed and leafy spurge, decomposed faster than the native and invasive grasses, regardless of the plant community of incubation. Thus, we found no evidence of HFA. T‐RFLP profiles indicated that both fungal and bacterial communities differed between roots and shoots and among plant species, and that fungal communities also differed among plant community types. Synthesis. These results show that litter from three common invaders to intermountain grasslands decomposes at different rates and cultures microbial communities that are species‐specific, widespread, and persistent through the dramatic shifts in plant communities associated with invasions.     

Microsatellite isolation from the gall midge Spurgia capitigena (Diptera: Cecidomyiidae), a biological control agent of leafy spurge

Spurgia capitigena is a gall midge that has been released as a biological control agent of leafy spurge (Euphorbia esula), a noxious rangeland weed in North America. We isolated 15 microsatellite loci from S. capitigena with from two to 27 alleles at each locus. These markers will allow us to examine the structure and levels of neutral genetic variation in native and introduced populations.     

Integration of biological control agents with other weed management technologies: Successes from the leafy spurge (Euphorbia esula) IPM program

An invasive weed can occupy a variety of environments and ecological niches and generally no single control method can be used across all areas the weed is found. Biological control agents integrated with other methods can increase and/or improve site-specific weed control, but such combinatorial approaches have not been widely utilized. The successful leafy spurge (Euphorbia esula L.) control program provides examples for future integrated weed programs that utilize biological control agents with traditional methods. Weed control methods can be used separately, such as when the leafy spurge gall midge (Spurgia esulae Gagné) reduced seed production in wooded areas while herbicides prevented further spread outside the tree line. Traditional methods also can be used directly with biological control agents. Incorporation of Aphthona spp. with herbicides has resulted in more rapid and complete leafy spurge control than either method used alone. Also, the insect population often increased rapidly following herbicide treatment, especially in areas where Aphthona spp. were established for several years but had been ineffective. Incorporation of Aphthona spp. with sheep or goat grazing has resulted in a larger decline in leafy spurge production than insects alone and in weed density than grazing alone. Controlled burns can aid establishment of biological control agents in marginally suitable environments, but timing of the fire must be coordinated to the insect’s life-cycle to ensure survival. Integration of biological control agents with revegetation programs required the agent to be the last method introduced because the cultivation and herbicide treatments necessary to establish desirable grasses and forbs were destructive to the insect. In a practical application, herbicides were combined with Aphthona spp. to help the insect establish and control leafy spurge in the habitat of the western prairie fringed orchid (Platanthera praeclara Sheviak and Bowles), an endangered species. Several experimental designs can be used to evaluate biological control agents with cultural, mechanical, and chemical control methods or with additional biological agents.     

Hierarchical Bayesian methods estimate invasive weed impacts at pertinent spatial scales

Invasive weed impact estimates are needed to determine whether or not weeds warrant costly control measures. Typically, land managers seek local weed impact estimates (e.g. ranches, parks) and policy-makers want to know how weeds are impacting entire regions. Our goal was to provide local and regional impact estimates for a ubiquitous invasive weed: leafy spurge (Euphorbia esula L.). The specific impacts we looked at related to desired species biomass production, livestock carrying capacities, and grazing land values. Our basic approach was to use an empirical model that characterizes weed biomass across the landscape in combination with another empirical model that predicts weed impact from weed biomass. Our investigation revealed that, without on-site plant biomass data, site-specific leafy spurge impacts are highly uncertain. Supplementing our general predictive model with small quantities of on-site data increased precision considerably. For the 17-state region we considered, 95% Bayesian credibility intervals indicated leafy spurge reduces cattle carrying capacities by 50–217 thousand animals a year and reduces grazing land values by 8–34 million dollars a year. Additional plant biomass data from randomly selected, leafy spurge-infested sites would shrink these fairly wide intervals.     

Seasonal shifts in dormancy status, carbohydrate metabolism, and related gene expression in crown buds of leafy spurge

Crown buds of field-grown leafy spurge (Euphorbia esula L.) were examined to determine relationships between carbohydrate metabolism and gene expression throughout para-, endo-, and eco-dormancy during the transition from summer, autumn, and winter, respectively. The data indicates that endo-dormancy plays a role in preventing new shoot growth during the transition from autumn to winter. Cold temperature was involved in breaking endo-dormancy, inducing flowering competence, and inhibiting shoot growth. An inverse relationship developed between starch and soluble sugar (mainly sucrose) content in buds during the shift from para- to endo-dormancy, which continued through eco-dormancy. Unlike starch content, soluble sugars were lowest in crown buds during para-dormancy but increased over two- to three-fold during the transition to endo-dormancy. Several genes (AGPase, HK, SPS, SuSy, and UGPase) coding for proteins involved in sugar metabolism were differentially regulated in conjunction with well-defined phases of dormancy in crown buds. Marker genes for S-phase progression, cell wall biochemistry, or responsive to auxin were also differentially regulated during transition from para-, endo-, and eco-dormancy. The results were used to develop a model showing potential signalling pathways involved in regulating seasonal dormancy status in leafy spurge crown buds.     

An efficient method for <Emphasis Type="Italic">in vitro</Emphasis> regeneration of leafy spurge (<Emphasis Type="Italic">Euphorbia esula</Emphasis> L.)

Leafy spurge (Euphorbia esula L.) is a perennial, invasive weed used as a model to study invasive plant behavior, because molecular tools (such as a deep expressed sequence tag database and deoxyribonucleic acid microarrays) have been developed for this species. However, the lack of effective in vitro regeneration and genetic transformation systems has hampered molecular approaches to study leafy spurge. In this study, we describe an efficient in vitro regeneration system. Three highly regenerative lines were selected by screening the in vitro regeneration capabilities of stem explants of 162 seedlings. The effects of various culture conditions on in vitro regeneration were then evaluated based on explant competence to form calluses and shoots. High rates of shoot regeneration can be obtained using a growth medium containing 1× woody plant basal medium and 1× Murashige and Skoog (MS) basal salts, 1× MS vitamins, 1.11 μM 6-benzylaminopurine, 1.97 μM indole-3-butyric acid, and 3% sucrose, pH 5.6–5.8. After 30 d culture, multiple shoots formed either directly from the stem or indirectly from the callus. This method is a requisite for the development of genetic transformation systems for leafy spurge and may be used to develop in vitro regeneration techniques for other species in the Euphorbiaceae.     

Energy potential of leafy spurge (Euphorbia esula)

Leafy spurge (Euphorbia esula) is a noxious, perennial weed that infests pastures, rangeland and waste areas in the northern Great Plains. The objective of this study was to determine the productive potential of this species when grown under optimum agronomic conditions. Plants were fertilized and irrigated. Oil, hydrocarbon, total protein, and dry-weight production were measured on 3 harvest dates. Calorimetric analyses were performed to determine the potential of leafy spurge as a fuel crop. The hydrocarbon content of 12 strains of leafy spurge was determined to measure genetic variability for this trait. The addition of fertilizer doubled dryweight production but did not affect percent oil or hydrocarbon content. Oil and hydrocarbon production averaged 6.8 and 0.6% on a plant dry-weight basis. Maximum production of plant biomass, protein, and hydrocarbon was obtained from a mid-July harvest. Oil content increased later in the growing season. The total protein content of leafy spurge averaged 12%. Whole-plant biomass had a caloric value of 4,407 cal/g while the oils contained 10,019 cal/g. Leafy spurge hay can produce 4 times more energy per year than wheat straw; therefore, the immediate potential of leafy spurge whole-plant biomass as a locally grown fuel crop for home-heating purposes is suggested.     

Attack behavior and host utilization ofAphthona chinchihi (Col.: Chrysomelidae), a potential biological control agent ofEuphorbia esula (Euphorbiaceae, leafy spurge) in North America

Aphthona chinchihi Chen was collected in China feeding on leafy spurge (Euphorbia esula L.). Studies were conducted on its host specificity in the laboratory, using field collected adults and their progeny.Aphthona chinchihi can effectively complement the impact of the other natural enemies of leafy spurge established from Europe in the U.S.A. and Canada. The adults feed on leaves and shoots and the larvae, which cause the main damage to the plant, feed on the hypogeous portion of the plant, seriously stressing the plant and preventing its vegetative spread. The host range ofA. chinchihi was studied with tests on adult feeding and oviposition, larval survival and host suitability, using 40 plant species or varieties distributed in 12 families. The experiments demonstrated that it has a high level of specificity. This flea beetle completed its life cycle only on leafy spurge. Also, because of its ecological valence,A. chinchihi has a very good potential as a biocontrol agent in North America.     

Is two company or a crowd: How does conspecific density affect the small-scale dispersal of a weed biocontrol agent?

To predict the growth and spread of an insect population introduced for the biological control of weeds, one must first understand the factors affecting the movement of individuals in the population. The purpose of this study was to determine how the dispersal rate of Aphthona lacertosa (Rosenhauer) (Chrysomelidae) was affected by conspecific density and by the characteristics of leafy spurge (Euphorbia esula L.: Euphorbiaceae) in patches where these beetles feed. In 2002 in Manitoba and in 2003 in Alberta, Canada, between 200 and 2500 insects were released in small patches (<10 m²) of spurge. The number and location of beetles within patches was monitored over subsequent days. In 1 m² plots within patches, spurge ramet density, the proportions of vegetative and reproductive ramets, and ramet height were measured. In both years, beetle movement within patches and emigration from patches, was not affected by conspecific density. In Manitoba in 2002, beetles aggregated non-randomly on either vegetative or reproductive ramets within plots, but plot characteristics were not related to the formation of aggregations. In Alberta in 2003, plots in which beetles aggregated had significantly higher spurge density but did not differ in either the proportion of vegetative ramets or in the amount of non-spurge vegetation. These results suggest that density-dependent dispersal does not limit the population's ability to reach densities up to 2500 beetles/m².     

Temporal dynamics of leafy spurge (Euphorbia esula) and two species of flea beetles (Aphthona spp.) used as biological control agents

The goal of this study was to evaluate the biological control program of leafy spurge (Euphorbia esula) in a large natural area, Theodore Roosevelt National Park, western North Dakota, USA. Aphthona lacertosa and Aphthona nigriscutis have been released at more than 1800 points in the 18,600-ha South Unit of the park beginning in 1989; most releases have occurred since 1994. We established permanent vegetation plots throughout the infested area of the park and determined stem counts and biomass of leafy spurge and abundance of the two flea beetle species at these plots each year from 1999 to 2001. Both biomass and stem counts declined over the 3 years of the study. Both species of flea beetle are well established within the park and have expanded into areas where they were not released. A. nigriscutis was more abundant than A. lacertosa in the grassland areas we surveyed, but in all other habitats abundances were similar. Using structural equation models, only A. lacertosa could be shown to have a significant effect on counts of mature stems of leafy spurge. A. nigriscutis numbers were positively correlated with stem counts of mature stems. Previous year’s stem counts had the greatest influence on change in stem counts over each 2-year time step examined with structural equation models.     

Organogenesis in cell cultures of leafy spurge (Euphorbiaceae) accessions from Europe and North America

Plants were regenerated from leafy spurge (Euphorbia esula L.) cell suspensions obtained from stem callus. A North Dakota accession was highly regenerable, but two accessions from Oregon and Austria formed only a few plantlets. Organogenesis occurred in media without growth regulators, under fluorescent lights (30 to 90 E m^–2 s^–1, 14 h photoperiod). Organogenesis was greatest in larger size clumps subcultured during maximum cell growth into media containing a reduced:oxidized nitrogen ratio of 33:67. Roots formed first and some clumps produced shoots. Organogenic suspension cultures also were initiated from hypocotyl and root segments of germinated seedlings, directly in liquid medium. Plantlets of the North Dakota accession formed in vitro adapted to greenhouse conditions. They were phenotypically similar to the parent plants.     

Effects of putrescine and inhibitors of putrescine biosynthesis on organogenesis inEuphorbia esula L.

Summary Exogenous putrescine (&#x2264;5 mM) had little effect on root or shoot formation in aseptically isolated hypocotyl segments of leafy spurge (Euphorbia esula L.) grown on full-strength B5 medium. Unexpectedly, putrescine inhibited root and shoot formation in hypocotyl segments grown on B5 medium diluted 10-fold. In the full-strength medium, root and shoot formation were inhibited by 0.5 mM concentrations ofdl-&#x03B1;-difluoromethylornithine (DFMO) anddl-&#x03B1;-difluoromethylarginine (DFMA). DFMO and DFMA are inhibitors of the ornithine decarboxylase and arginine decarboxylase pathways, respectively, of putrescine biosynthesis in plants. Exogenous putrescine (0.5 to 5 mM) did not reverse either the DFMO-or DFMA-induced inhibition of shoot formation. However, the DFMA-induced inhibition of root formation was partially reversed by exogenous putrescine. The auxin, indole-3-acetic acid (IAA), reduced the inhibitory effects of DFMO+DFMA (applied together) on both roots and shoots. In the first few days of culture, the endogenous levels of putrescine and spermidine, but not of spermine, increased in the presence of IAA. The levels of putrescine and spermidine in the tissues did not correlate well with either root or shoot production in the later stages of organ formation; especially in tissues treated with IAA. These results show that there were no obvious correlations between polyamine levels and organogenesis in leafy spurge hypocotyl segments, although residual putrescine or spermidine or both in the tissues at the time of excision may be indirectly involved in the early stages of root formation.     

Nature,nurture, and vegetation management: Studies with sheep and goats

Diet selection and preference by grazing animals are determined by genetic and environmental factors (i.e., nature and nurture) that interact and affect their efficacy for managing vegetation as targeted grazers. The effect of rearing environment on the consumption of leafy spurge by sheep and goats was investigated. We hypothesized that although rearing environment will affect the preference for chemically defended plants ultimately, the inherent ability to detoxify or eliminate phytotoxins will limit an animal’s preference for them. The objective of this study was to determine if sheep would consume more of the invasive weed leafy spurge (Euphorbia esula) if they were raised by goat compared to sheep raised by sheep and goat raised by goat. Sheep were raised on leafy spurge-infested pastures by either their ewe (S) or a goat doe (FS) on which they were fostered within 24 hours of birth and parturition of lamb and doe, respectively. Does that fostered lambs also raised their own goat offspring (G) such that the same doe raised the FS and G animals. The rearing environment’s effect on leafy spurge consumption was tested the following growing season by simultaneously grazing all animals on the same leafy spurge-infested rangeland and estimating percentage leafy spurge in their diet with either fecal near-infrared spectroscopy (f.NIR) or bite count. Goats consumed more leafy spurge as determined by either f.NIR (62.8%, P < 0.06) or bite count (71.9%, P < 0.01) than FS (35.2 % f.NIR, 39.3% bite count) or S (10.1 % f.NIR, 18.2% bite count). The FS consumed over twice as much leafy spurge as S and were numerically intermediate to G and S for leafy spurge consumption but not significantly different from the S sheep, most likely because one FS sheep did not eat leafy spurge during the evaluation period. Because leafy spurge is aversive to sheep but not goats, higher leafy spurge consumption by FS sheep is hypothesized to result from inoculation of their rumen microbes with microbes from the does capable of denaturing aversive phytotoxins in leafy spurge. The higher consumption of leafy spurge by G compared to FS shows that genetically determined physiological differences influence an animal’s ability to ameliorate phytotoxins and determine the upper limit of an animal’s preference for a chemically defended plant. It also indicated that in addition to the animal’s genome, the genome of an animal’s microbiome, which the mother may influence, can play an important role in diet selection.     

Post hoc assessment of an operational biocontrol program: efficacy of the flea beetle Aphthona lacertosa Rosenhauer (Chrysomelidae: Coleoptera), an introduced biocontrol agent for leafy spurge

Mixed populations of Aphthona lacertosa and Aphthona czwalinae were released at more than 50 locations in Alberta in 1997. Two and 3 years post-release, beetle populations were primarily A. lacertosa, with A. czwalinae forming less than 0.5% of the sampled populations. Beetle densities were moderate (10–70 beetles per m²) or high (>70 beetles per m²) at 14% and more than 60% of the sampled sites in 1999 and 2000, respectively. Larger beetles had greater instantaneous egg loads (r²=0.424,P=0.003). In 2000, the largest beetles were found at moderate density sites and there was a significant negative relationship between beetle size and the time taken to accumulate a degree day threshold of 1230 (for females: r²=0.678,P=0.001). Sites with the most rapid accumulation of degree days have the greatest potential for beetle population growth based on potential fecundity. Changes in leafy spurge percent cover, stem density, and canopy height from 1997 to 2000 were assessed across sites with low (<10 beetles per m²), moderate, and high beetle densities in 2000. Sites with high beetle densities had significantly greater reductions of leafy spurge within 5 m of the release point than sites with low beetle densities (P<0.017). Damage caused by the beetles at high-density sites was often visible as a halo-shaped patch of dead leafy spurge stems. The significant overall reduction of leafy spurge within release patches makes A. lacertosa a promising biocontrol agent for leafy spurge in Alberta.