Comparison of furanocoumarin concentrations of greenhouse-grownRuta chalepensis with outdoor plants later transferred to a greenhouse

Ruta chalepensis contained concentrations of furanocoumarins 25–50% of those found inR. graveolens both in the whole leaf and on its surface. On the leaf surface of plants grown all year indoors in a greenhouse, they increased steadily between November 1 and December 14 on mature upper and lower leaves. New growth upper leaves on December 14 contained less than mature upper leaves. Plants transferred from outdoors to the greenhouse showed decreased concentrations after the first two weeks, followed by recovery both in the whole leaf and on the leaf surface. Proportions of xanthotoxin and bergapten to psoralen changed during the experiment. On the leaf surfaces and in the whole upper leaves of the indoor plants, the proportions were often similar, but in the transferred plants, in most cases, psoralen was less than bergapten or xanthotoxin in the upper leaves and markedly less in the lower leaves. Implications of these findings for possible effects of environmental changes on secondary plant metabolism are discussed.     

Characterization of furanocoumarin metabolites in parsnip webworm,Depressaria pastinacella

Although metabolites of furanocoumarins have been characterized in a wide range of organisms, to date they have been identified in only a single insect species, Papilio polyxenes. Depressaria pastinacella, the parsnip webworm, like P. polyxenes a specialist on Apiaceae, routinely consumes plant tissues higher in furanocoumarin content than does P. polyxenes and is capable of faster cytochrome P-450-mediated detoxification of these compounds. In this study, we characterized metabolites of xanthotoxin, a linear furanocoumarin, and sphondin, an angular furanocoumarin, in midguts and frass of parsnip webworms. Two metabolites were isolated and identified from webworms fed artificial diet containing xanthotoxin. LC-ESI-MS analysis resulted in the determination of a MW of 266 for the compound in the frass and one of the compounds in the midgut; ¹H NMR confirmed its structure as 6-(7-hydroxy-8-methoxycoumaryl)-hydroxyacetic acid (HCHA). The second compound from the midgut had a MW of 252 and was identified by ¹H NMR and ¹³C NMR analysis as 6-(7-hydroxy-8-methoxycoumaryl)-hydroxyethanol) (HMCH). Whereas HCHA has been found in frass of Papilio polyxenes fed xanthotoxin, HMCH has not been reported previously in insects. Although the first step of metabolism of xanthotoxin in webworms as well as P. polyxenes is likely the formation of an epoxide on the furan ring, angular furanocoumarin metabolism in webworms appears to differ. The principal metabolite of sphondin was identified as demethylated sphondin (6-hydroxy-2H-furo[2,3-h]-1-benzopyran-2-one) by LC-ESI-MS and confirmed by ¹H NMR and ¹³C NMR analyses. That webworms produce metabolites of xanthotoxin in common not only with other Lepidoptera (e.g., HCHA) but with other vertebrates (e.g., HMCH) suggests a remarkable conservatism in the metabolic capabilities of cytochrome P-450s and raises the possibility that insects may share other detoxification reactions with vertebrates with respect to toxins in foodplants.     

Toxicity of linear furanocoumarins toSpodoptera exigua: Evidence for antagonistic interactions

The linear furanocoumarins psoralen, bergapten, and xanthotoxin were tested for toxicity to the beet armywormSpodoptera exigua (Hübner) under short ultraviolet (UVB) radiation. Increased dietary concentrations of each furanocoumarin significantly decreased insect larval weight, extended generation time, and induced higher mortality. Xanthotoxin was the most toxic, followed by psoralen and bergapten. Combining psoralen with bergapten, xanthotoxin, or both resulted in significantly antagonistic effects on insect mortality. The combination of bergapten and xanthotoxin, however, produced additive effects. The implications of these observations forS. exigua resistance in the wild plant accession ofApium prostratum and the enigma the findings represent for plant-insect relationships are discussed.     

Myristicin,safrole, and fagaramide as phytosynergists of xanthotoxin

The methylenedioxyphenyl-containing (MDP) inhibitors of mixed-function oxidase detoxification enzymes, myristicin, safrole, fagaramide, and isosafrole, occur with xanthotoxin or other toxic furanocoumarins in plants of the families Umbelliferae and Rutaceae. All four MDP compounds have a synergistic effect on the toxicity of xanthotoxin toHeliothis zea. Myristicin also increased the phototoxicity of xanthotoxin in the presence of UV light. The term phytosynergist is used to describe plant compounds that are present at concentrations producing no toxic effect by themselves but have a synergistic effect on cooccurring toxins.Taken in part from a thesis submitted to the Graduate College of the University of Illinois at Urbana-Champaign in partial fulfillment of the requirements for the degree of Doctor of Philosophy, June 1987.     

Metabolism and excretion of the furanocoumarin xanthotoxin by parsnip webworm,Depressaria pastinacella

The parsnip webworm,Depressaria pastinacella, feeds on plants containing high concentrations of furanocoumarins. compounds toxic to many organisms. Parsnip webworm larvae were fed radiolabeled xanthotoxin to quantify the detoxification of this furanocoumarin. They metabolized approximately 95% of the ingested xanthotoxin, indicating that metabolic detoxification is important in their tolerance to this allelochemical. Excretion of xanthotoxin and its metabolites was not restricted to the frass but also occurred by means of the silk glands. The silk glands contained half as much of the tritiated compounds as the rest of the body. Because of the feeding habits of this insect, such an excretory pathway may have implications for interactions with predators and pathogens.     

Seasonal changes of furanocoumarin concentrations in leaves ofHeracleum lanatum

Concentrations of three dermatitis-inducing furanocoumarins— xanthotoxin, bergapten and psoralen—were measured in whole leaves ofHeracleum lanatum and in extracts of the leaf surface over an entire vegetative season. The concentrations of surface furanocoumarins, localized by extraction involving brief dipping in almost-boiling water followed by HPLC quantitative analysis, increased until the middle of May and decreased until maturity. The concentration on autumn leaves (new growth) was 20–100 times as high as the ones in May, or those of similar size in April. Furanocoumarin concentrations in the whole leaf at different stages of leaf development varied, being the highest April 25, then decreasing sharply with rapid leaf enlargement. Again, in the small autumn leaves the coumarin concentration was two to three times that in April. Seasonal changes in surface furanocoumarins may be important in that these compounds are postulated to form the first defense barrier of the plant.     

Synergism between myristicin and xanthotoxin,a naturally cooccurring plant toxicant

Myristicin, a methylenedioxyphenyl (MDP)-containing phenylpropene constituent of the leaves of many plants in the family Umbelliferae, is a highly effective Synergist of the cooccurring furanocoumarin xanthotoxin. As little as 0.10 % in an artificial diet can increase the toxicity of xanthotoxin toHeliothis zea (Lepidotera: Noctuidae) fivefold. In addition to increasing the proportion of caterpillars dying at a given xanthotoxin concentration, myristicin also increases the rate at which they die and increases the time to molt of surviving larvae. That there was no increase in the deterrency of xanthotoxin in the presence of myristicin suggests that the mechanism of synergism is not behaviorial but rather is biochemical, via MDP competitive inhibition of microsomal mixed function oxidases.     

Effects of juglone on growth,photosynthesis, and respiration

The impacts of juglone on plant growth and several other physiological functions were evaluated in this study. Juglone inhibitedLemna minor growth, chlorophyll content, and net photosynthesis at treatments between 10 and 40M. Soybean leaf disks vacuum infiltrated with as little as 10M juglone had reduced photosynthesis. Oxygen evolution by chloroplasts isolated fromPisum sativum was inhibited by juglone with an I₅₀ of 2M. Micromolar treatments of juglone stimulated oxygen uptake in mitochondria isolated fromGlycine max. These data suggest perturbations of chloroplast and mitochondrial functions may contribute to plant growth reductions observed in juglone-mediated allelopathy.This study was supported by National Science Foundation grant STI-8902066.     

The effect of fertilizer additions on the solubility and plant-availability of Cd, Ni and Zn in soil

We conducted a pot experiment to investigate the effect of fertilizer additions on the solubility of Cd, Ni and Zn in soil solution and their uptake by plants. Radish (Raphanus sativus cv. Crystal Ball), oat (Avena sativa cv. Thule) and water spinach (Ipomoea aquatica cv. Kangkon) were grown in a naturally metal-rich soil. From day 7 after planting, fertilizers were added daily to each pot. Additions of fertilizer nutrients affected the pH of soil and soil solution, soluble and tissue concentrations of Ca, Mg, K, Cd, Ni and Zn differently in three plant species. The trend of soil and solution pH was in the order: water spinach < radish < oats, that resulted highest soluble and plant tissue concentrations of Cd and Zn in the water spinach followed by radish and then oats. However, Ni concentration in the soil solution increased in all pots and was not affected by pH changes. Soil solution pH increased by more than 1unit in the pots with radish and oats, indicating that mechanisms other than acidification, such as ion exchange and root exudation, may be responsible for the increased heavy metal uptake in these two plant species. Paired t-test showed significantly higher uptake of Cd and Zn in the radish plants resulting in lower concentrations of these elements in the solution. The contribution of mass flow to the supply of major cations and heavy metals varied among elements and plant species. Cadmium, Zn and K were taken up rapidly by all plant species in response to the amount supplied by mass flow. In contrast, the supply of Ni was in excess of its uptake by radish and water spinach. The uptake of all elements was positively correlated (p<0.0001) with mass flow and the transpiration rate in individual plant species. The study suggests that fertilizer cations increased the uptake of metals by improving growth conditions, but the magnitude of increase depended on plant species. This revised version was published online in November 2006 with corrections to the Cover Date.     

Allelopathic Effects of Volatile Cineoles on Two Weedy Plant Species

The volatile monoterpene analogs, 1,4-cineole and 1,8-cineole, have been identified as components of many plant essential oils, but relatively little is known about their biological activities. We compared the effects of 1,4- and 1,8-cineole on two weedy plant species by monitoring germination, mitosis, root and shoot growth, chlorophyll content, and photosynthetic efficiency. 1,4-Cineole severely inhibited growth of roots and shoots, causing cork-screw shaped morphological distortion, whereas 1,8-cineole caused a decrease in root growth and germination rates. Chlorophyll fluorescence data (yield and F _v / F _m) indicated that 1,4-cineole caused significantly higher stress (P 0.001) to photosynthesis when compared to controls. Mitotic index data showed that 1,8-cineole severely decreased (P 0.001) all stages of mitosis when compared with controls, while 1,4-cineole only caused a decrease in the prophase stage (P 0.05). Although superficially similar in structure, these two cineoles appear to have different modes of action.     

A model of S,P and N uptake by a perennial pasture

An approach is presented for simulating the uptake of S, P and N by a perennial pasture, based upon diffusion theory and plant uptake kinetics. Plant uptake was limited by either (i) diffusion to the root, (ii) root uptake capacity, or (iii) plant demand. For S and P, uptake as limited by diffusion was calculated from the nutrient depletion pattern around the root, which is dependent upon soil diffusivity for the anion, and the length of time since the root grew into the new soil. To avoid time-consuming calculations at various uptake times and distances from the root, the nutrient depletion pattern was simplified into an effective rooting radius, whereby all plant-available nutrient within the radius was made available to the plant over a 30 day period, and none from outside the radius. Mycorrhizal enhancement of P uptake was simulated by increasing the radius by a factor of between 1 and 3 when P was the primary limiting nutrient. Limitations (ii) and (iii) were included for S and P for the rare occasions when nutrient concentrations were high either in a single layer, or in the profile overall. For N, uptake was limited by uptake capacity, adjusted for soil N concentration, root length density and soil moisture content.Because of a lack of data on diffusion coefficients close to the plant root, a calibration factor was applied to the effective rooting radius, which is described in the second paper in the series.     

Juglone Disrupts Root Plasma Membrane H+-ATPase Activity and Impairs Water Uptake, Root Respiration, and Growth in Soybean (Glycine max) and Corn (Zea mays)

Juglone is phytotoxic, but the mechanisms of growth inhibition have not been fully explained. Previous studies have proposed that disruption of electron transport functions in mitochondria and chloroplasts contribute to observed growth reduction in species exposed to juglone. In studies reported here, corn and soybean seedlings grown in nutrient solution amended with 10, 50, or 100 microM juglone showed significant decreases in root and shoot dry weights and lengths with increasing concentrations. However, no significant differences in leaf chlorophyll fluorescence or CO2-dependent leaf oxygen evolution were observed, even in seedlings that were visibly affected. Disruption of root oxygen uptake was positively correlated with increasing concentrations of juglone, suggesting that juglone may reach mitochondria in root cells. Water uptake and acid efflux also decreased for corn and soybean seedlings treated with juglone, suggesting that juglone may affect metabolism of root cells by disrupting root plasma membrane function. Therefore, the effect of juglone on H+-ATPase activity in corn and soybean root microsomes was tested. Juglone treatments from 10 to 1000 microM significantly reduced H+-ATPase activity compared to controls. This inhibition of H+-ATPase activity and observed reduction of water uptake offers a logical explanation for previously documented phytotoxicity of juglone. Impairment of this enzyme's activity could affect plant growth in a number of ways because proton-pumping in root cells drives essential plant processes such as solute uptake and, hence, water uptake.     

Allelochemicals fromPilocarpus goudotianus leaves

The effect on germination, shoot, and root growth by bergapten, xanthotoxin, imperatorin, xanthyletin, xanthoxyletin, luvangetin, donatin and alloxanthoxyletol fromPilocarpus goudotianus leaves, onLactuca sativa var. nigra seedlings has been evaluated. A structure-activity correlation is discussed based on the bioassay results. Furanocoumarins appear to be the most active compounds in comparison with pyrano- and simple coumarins. The presence of an oxygenated function at C-8 decreases the germination effect in furano- and pyranocoumarins, while C-5 substituents do not cause significant changes on the activity.Part 2 in the series:Natural Product Models as Allelochemicals. For Part 1 see: Macíaset al. (1992).     

Induction of cytochrome P450-mediated detoxification of xanthotoxin in the black swallowtail

Xanthotoxin is a phototoxic allomone found in many of the host plants of the black swallowtail,Papilio polyxenes (Lepidoptera: Papilionidae). When added to the diet of final instar larvae, xanthotoxin can induce the cytochrome P450 monooxygenase (P450) activity in midgut microsomes by which it is detoxified. Induction is dose-dependent, increasing sevenfold when larvae feed on parsley treated topically with xanthotoxin at 0.5 or 1.0% fresh weight. Although xanthotoxin exerts much of its toxic effects when photoactivated by ultraviolet light, induction of P450 activity did not differ in the presence or absence of ultraviolet light. Despite a 4.7-fold induction of xanthotoxin-metabolizing P450 activity, total P450 content measured in the same microsomal samples did not increase significantly. These data indicate that multiple forms of P450 exist in the black swallowtail midgut and that they are differentially induced by xanthotoxin.     

Penicillium bilaji and phosphorus fertilization effects on the growth,development, yield and common root rot severity of spring wheat

A strain ofPenicillium bilaji Chalabuda (PB) has recently been commercially released as a seed inoculant to increase phosphorus (P) uptake by wheat (Triticum aestivum L.). The purpose of this study was to compare the effects of drill applied P (15 kg P ha^–1) with PB seed inoculation on early growth, development, P uptake, and grain yield of Stoa spring wheat at four sites in North Dakota.Fertilization with P consistently enhanced early season growth, main stem development, tillering and P uptake. Seed inoculation with PB had little or no effect on these traits. Phosphorus fertilization tended to increase common root rot severity (CRR, incited byCochliobolus sativus (Ito & Kurib) Drechs.), while PB inoculation had no effect. Grain yields were significantly increased by P fertilization at one location. Inoculation with PB also increased grain yield at this location. The reason why PB inoculation increased yield at this location is not evident, as plant growth and P uptake were not enhanced earlier in the season. Averaged across all four sites, PB inoculation increased wheat yields 66 kg ha^–1, which is similar to averaged yield responses reported from the Prairie Provinces of Canada.     

The Allelochemical Sorgoleone Inhibits Root H+-ATPase and Water Uptake

Sorghum plants inhibit the growth of some adjacent species. Root exudates from grain sorghum (Sorghum bicolor), consisting primarily of the quinone sorgoleone, are phytotoxic to several plant species, yet the mechanisms of growth inhibition remain to be fully explained. Disruption of electron transport functions in isolated mitochondria and chloroplasts has been reported as one explanation for growth inhibition. In the studies reported here, however, soybean seedlings grown in nutrient solution with 10, 50, or 100 microM sorgoleone showed no disruption of photosynthesis, as measured by leaf fluorescence and oxygen evolution, yet their mean leaf surface area was less when grown in 100 microM sorgoleone. Furthermore, in the presence of these same concentrations of sorgoleone, decreased nutrient solution use by soybean seedlings and decreased H+-ATPase activity in corn root microsomal membranes were observed. This suggests that impairment of essential plant processes, such as solute and water uptake, driven by proton-pumping across the root cell plasmalemma should also be considered as a mechanism contributing to observed plant growth inhibition by sorgoleone.     

Defense of parsnip webworm against phototoxic furanocoumarins: Role of antioxidant enzymes

The parsnip webworm,Depressaria pastinacella (Lepidoptera: Oecophoridae), feeds on plants rich in furanocoumarins, phototoxic allomones. Final-instar larvae possess high levels of activities of antioxidant enzymes (Superoxide dismutase, catalase, glutathione reductase), which detoxify oxygen radicals generated from the furanocoumarins of their host plants. When added to an artificial diet, three linear furanocoumarins (xanthotoxin, bergapten, imperatorin) do not increase levels of the antioxidant enzymes. However, on diets containing both xanthotoxin and piperonyl butoxide, a cytochrome P-450 inhibitor, food utilization indices of the insect are reduced and superoxide dismutase activity is enhanced. These data suggest that cytochrome P-450s act as a primary detoxification system of ingested furanocoumarin, and antioxidant enzymes as a backup system to detoxify oxygen radicals generated by unmetabolized furanocoumarins.     

Analysis,isolation and insecticidal activity of linear furanocoumarins and other coumarin derivatives fromPeucedanum (Apiaceae: Apioideae)

Peucedanum arenarium Waldst. & Kit.,P. austriacum (Jacq.) Koch,P. coriaceum Reichenb.,P. longifolium Waldst. & Kit,P. officinale L.,P. oreoselinum (L.) Moench,P. ostruthium L., andP. palustre (L.) Moench accumulate different structural types of coumarins including simple coumarins, linear furanocoumarins, linear dihydropyranocoumarins, angular dihydrofuranocoumarins and angular dihydropyranocoumarins. Linear furanocoumarins, known for various biological activities, include some well-known antifeedants, such as bergapten, isopimpinellin, and xanthotoxin. The aim of this investigation was to screen the diverse coumarins fromPeucedanum for insecticidal activity. LC was used to analyze and isolate coumarins for the bioassays. A growth inhibition bioassay with 17 derivatives, comprising all structural types fromPeucedanum, carried out withSpodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) as test organism, indicated the majority of the linear furanocoumarins and the angular dihydrofuranocoumarin athamantin as active compounds. Oxygenation of the prenyl residue of linear furanocoumarins decreased activity. Further formation of an ester with angelic acid even resulted in complete inactivity. Five active linear furanocoumarins, bergapten, isopimpinellin, xanthotoxin, isoimperatorin, and imperatorin, and two linear furanocoumarins with a substituted furan ring, peucedanin and 8-methoxypeucedanin, were compared in a dietary utilization bioassay. Relative growth rate (RGR) and relative consumption rate (RCR) divided the tested coumarins in three groups of similar activity. Isopimpinellin and peucedanin slightly decreased RGR and RCR of the treated larvae, and xanthotoxin, isoimperatorin, and 8-methoxypeucedanin heavily decreased RGR and RCR. Bergapten and imperatorin differed by the lowest RGR values and rather high RCR values. The effects caused by these two coumarins indicate specific postingestive toxicity. The results obtained in this study add to the reputation of coumarins to be an effective chemical defense, postulating that chemical diversity is a necessary trait for well-defended plants.     

Fate of photosensitizing furanocoumarins in tolerant and sensitive insects

The fate of [¹⁴C]xanthotoxin (8-methoxypsoralen) was studied in larvae of insect species that are tolerant (Papilio polyxenes Stoll) or sensitive (Spodoptera frugiperda J.E. Smith) to the phototoxic effects of photosensitizing psoralens. Both insects metabolize xanthotoxin by oxidative cleavage of the furan ring, but the detoxification occurs at a much more rapid rate inP. polyxenes in which >95% of an oral 5 g/g xanthotoxin dose is metabolized within 1.5 hr after treatment. The detoxification of psoralens byP. polyxenes appears to occur primarily in the midgut tissue prior to absorption, with the result that the intact phototoxin does not reach appreciable levels in body tissues. Studies with an angular furanocoumarin indicated that isopsoralens are metabolized byP. polyxenes at a somewhat slower rate than observed for psoralens; however, a reduced rate of metabolic detoxification of isopsoralens probably does not explain the fact that psoralen tolerance inP. polyxenes does not extend to the isopsoralen series.     

Plant–herbivore–carnivore Interactions in Cotton, <Emphasis Type="Italic">Gossypium hirsutum</Emphasis>: Linking Belowground and Aboveground

Most studies on plant–herbivore interactions focus on either root or shoot herbivory in isolation, but above- and belowground herbivores may interact on a shared host plant. Cotton (Gossypium spp.) produces gossypol and a variety of other gossypol-like terpenoids that exhibit toxicity to a wide range of herbivores and pathogens. Cotton plants also can emit herbivore-induced volatile compounds at the site of damage and systemically on all tissues above the site of damage. As these volatile compounds attract natural enemy species of the herbivore, they are thought to represent an indirect plant defense. Our study quantified gossypol and gossypol-like compounds in cotton plants with foliage feeding (Heliocoverpa zea), root feeding (Meloidogyne incognita), or their combination. Cotton plants with these treatments were studied also with respect to induced local and systemic volatile production and the attraction of the parasitic wasp Microplitis croceipes to those plants. We also evaluated whether foliage or root feeding affected foliar nitrogen levels in cotton. After 48 hr of leaf feeding and 5 wk of root feeding, local and systemic induction of volatiles (known to attract parasitoids such as M. croceipes) occurred with herbivore damage to leaves, and it increased in levels when root herbivory was added. Nevertheless, M. croceipes were equally attracted to plants with both leaf and root damage and leaf damage only. In contrast to previous studies in cotton, production of gossypol and gossypol-like compounds was not induced in leaf and root tissue following foliage or root herbivory, or their combination. We conclude that root feeding by M. incognita has little influence on direct and indirect defenses of Gossypium hirsutum against insect herbivory.