Mechanism of damage-induced alkaloid production in wild tobacco

Greenhouse-grown tobacco plants of the speciesNicotiana sylvestris (Solanaceae) subjected to leaf damage show a fourfold increase in the alkaloid content of their undamaged leaves. This increase in nicotine and nornicotine concentrations begins 19 hr after the end of the damage regime, reaches a maximum at nine days, and wanes to control levels 14 days after the start of leaf damage. The increase in leaf alkaloid content in damaged plants is largely due to a 10-fold increase in the alkaloid concentration of the xylem fluid entering leaves, which, in turn, suggests that increased synthesis of alkaloids is occurring in the roots. This research distinguishes between positive and negative cues affecting the change in xylem fluid alkaloid concentrations. A negative cue, such as auxin, when lost or diminished as a result of leaf damage could signal the alkaloidal response. Indeed, exogenous applications of auxin to damaged leaves inhibit the alkaloidal response. However, attempts to block endogenous auxin transport by steam girdling or applying an auxin transport inhibitor fail to mimic the effect of leaf damage on leaf alkaloid concentrations. The damage cue appears to be a positive cue that is related to the timing and the amount of leaf damage rather than to the amount of leaf mass lost. Moreover, when performed proximally to leaf damage, steam girdling truncates the alkaloidal response. This induced alkaloidal response appears to be triggered by a phloem-borne cue that allows the plant to distinguish between different types of leaf damage. The physiological and ecological consequences of the mechanism of this damage-induced alkaloidal response are further explored.     

Elevational variation of quinolizidine alkaloid contents in a lupine (Lupinus argenteus) of the Rocky Mountains

Quinolizidine alkaloid contents of leaves and seeds ofLupinus argenteus (Fabaceae) collected from seven different localities near Gothic, Colorado were determined by capillary GLC. Differences in alkaloid levels between sites are substantial and alkaloid quantity decreases as elevation increases. Leaves at the lowest elevation, for example, contain six times the alkaloid levels of leaves at the highest elevation. Seeds from plants of low-and high-elevation sites were grown under identical conditions in the green-house. Alkaloid levels of leaves of seedlings were significantly higher in those seedlings derived from populations of low elevations than those of high elevations, indicating that the observed differences in the field are at least partly genetic and not environmental. To determine whether predation rates were responsible for these genetic differences, data on seed predation rates and observations on herbivory were collected.     

Damage-induced alkaloids in tobacco: Pot-bound plants are not inducible

Field-grown wild tobacco plants (Nicotiana sylvestris) were subjected to a defoliation regime designed to mimic the rate and amount of leaf mass removed by one tobacco hornworm per plant. Undamaged leaves on these plants undergo a dramatic (457% for leaf position 5, 410% for leaf position 8) increase in total leaf alkaloids compared to same-age and positioned control leaves on undamaged control plants. However, potted greenhouse-grown plants fail to exhibit the same damage-induced increase in alkaloid content. The greenhouse environment differs from the field environment in factors known to influence leaf alkaloid content, particularly soil N, P, K, near-UV radiation, and relative humidity. However, altering these environmental factors does not make potted plants able to increase their leaf alkaloid levels in response to defoliation. Transplanting plants into larger pots with more soil does allow the plants to respond to defoliation. Thirty days after transplanting, the plants are again unresponsive to damage, probably as a result of becoming pot-bound. This result suggests a mechanism for the induction response, specifically that leaf damage triggers synthesis of these alkaloids in the roots, and offers a potentially valuable experimental tool for the study of induced-plant defenses in tobacco and other plants that synthesize alkaloids in their root tissues.     

Role of Alkaloids in Resistance of Yellow Lupin to Red-Legged Earth Mite Halotydeus destructor

Eleven varieties of Lupinus luteus were tested in choice and no-choice experiments for their resistance to feeding by the red-legged earth mite (Halotydeus destructor). Three were found to show resistance, and the alkaloid fraction from these varieties strongly deterred mites from feeding. The alkaloid components of the extracts, lupinine, a number of acyl derivatives of lupinine, and sparteine were tested for deterrent activity. Sparteine was the most potent, with significant activity at 0.001% concentration. Quantitation of the levels of nonpolar alkaloids in the cotyledons and leaves of the 11 varieties was by gas chromatography–mass spectrometry. An inverse correlation was found between the concentrations of these alkaloids and the damage caused by the mites; varieties with levels <100 g/g (fresh wt) showed a high damage index. The involvement of sparteine and the bipiperidine alkaloid ammodendrine in the deterrence to mite feeding is discussed.     

Influence of alkaloid concentration on acceptability of tall larkspur (Delphinium spp.) to cattle and sheep

Tall larkspur (Delphinium spp.) is a serious toxic plant problem on western U.S. ranges. The major toxins in tall larkspur are methyllycaconitine (MLA) and 14-deacetylnudicauline (14-DAN); the sum of both is termed the toxic alkaloid concentration. Toxic alkaloids comprise about 20–50% of the total alkaloid concentration in tall larkspur. Toxic and total alkaloid concentration generally declines with maturity, whereas cattle and sheep consumption of larkspur typically increases with plant maturity. We hypothesized that cattle and sheep consumption of tall larkspur was negatively related to higher concentrations of total or toxic alkaloid. We compared consumption of several collections of dried, ground larkspur and fresh larkspur in a series of trials. In another trial, a crude alkaloid fraction was extracted with ethanol, added to alfalfa hay, and consumption compared to untreated alfalfa hay, alcohol-treated hay, and the essentially alkaloid-free plant residue. In all cases we correlated amounts eaten with total and toxic alkaloid concentration. A grazing trial was also conducted to relate larkspur consumption over time to alkaloid concentrations. Total alkaloid concentrations in dried, whole-plant collections ranged from 9.3 to 38.8 mg/g of dry weight, whereas toxic alkaloid concentrations varied from 0.0 to 7.1 mg/g. In one pen trial, cattle preferred a larkspur collection (P<0.01) that contained no toxic alkaloids but had a high total alkaloid concentration (39 mg/g). There was no correlation (P>0.05), however, between concentrations of total or toxic alkaloids and amount of dry plant consumed in this or any other trial. Conversely, sheep consumption tended to be negatively influenced by total and toxic alkaloid concentration (P0.08). In the trials with extract, cattle preferred the alcohol-treated hay and rejected the alkaloid-free residue (P<0.01), whereas the alkaloid-treated hay was of intermediate acceptability. Cattle preferred the alkaloid-treated hay over the alkaloid-free residue, indicating that alkaloids did not deter consumption. Conversely, the alkaloid-treated hay was less preferred than either untreated or alcohol-treated hay, suggesting a negative effect on acceptability. There was no correlation between alkaloid concentration and amount of treated feed eaten. In field trials, the amount of composited, fresh leaves or flowers eaten by cattle was influenced by plant part (P=0.04), but was not related (P>0.05) to alkaloid concentration. Cattle preferred leaves over flowers when offered individual plants differing in phenological stage and/or amount of shade, but alkaloid concentration was not related to consumption. We conclude that knowledge of the concentration of total or toxic alkaloid in tall larkspur will give little or no indication of plant acceptability to cattle. Even though accurate predictions can be made about the potential toxicity of larkspur based on the concentration of toxic alkaloids, predictions about consumption must be based primarily on plant phenology.     

Systemic induction of feeding deterrents in cotton plants by feeding ofSpodoptera SPP. Larvae

Cotton,Gossypium hirsutum L., has been shown to exhibit systemic induced resistance to arthropods under certain conditions. We conducted experiments to determine the effects of previous feeding ofSpodoptera exigua Hübner andSpodoptera littoralis (Boisd.) larvae on feeding behavior, growth, and survival of larvae subsequently feeding on cotton. In one feeding choice test,S. exigua larvae preferred young leaves from undamaged control plants to undamaged young leaves from a previously damaged plant. Feeding deterrence was noticeable after only 6 hr of initial feeding damage by larvae, and there was almost complete deterrence after 30 and 54 hr of continuous feeding. In a second feeding choice test,S. littoralis larvae fed more on mature leaves from undamaged control plants than on undamaged mature leaves from previously damaged plants. In no-choice tests, third instars ofS. littoralis fed undamaged young leaves from damaged plants did not gain weight and died by the seventh day, whereas larvae fed young leaves from undamaged control plants gained weight and pupated within 11 days. Sixth instars ofS. littoralis fed either old damaged leaves, old undamaged leaves, or young undamaged leaves all from previously damaged plants gained weight slowly and took more than 12 days to pupate, whereas larvae fed young leaves from undamaged plants gained weight rapidly and pupated within five days of the beginning of the experiment.     

Detrimental effects ofCinchona leaf alkaloids on larvae of the polyphagous insectSpodoptera exigua

YoungCinchona ledgeriana plants contain two types of alkaloid: indole alkaloids in the leaves and quinoline alkaloids in the root. FromCinchona leaves, a crude alkaloid extract was made, containing the cinchophylline type of indole alkaloids and a small amount of 5-methoxytryptamine. The leaf alkaloid extract exerted a strong detrimental effect on the growth of larvae of the polyphagous beet armyworm,Spodoptera exigua (Lepidoptera). Feeding of larvae on an artificial diet containing the leaf alkaloids at the same concentrations as those found in the plant resulted in significant growth reduction, retardation in development, and mortality of the larvae. Cinchophyllines are composed of 5-methoxytryptamine coupled to a corynantheal unit. When incorporated into the artificial diet, 5-methoxytryptamine alone had no effect on the 5.exigua larvae. Corynantheal, however, had a strong detrimental effect on growth of the larvae, its effect being comparable to that of the leaf alkaloid extract. In contrast to the indole-type leaf alkaloids, the quinolinetypeCinchona root alkaloids did not affect growth and development of the larvae. These results suggest that the indole-type alkaloids, which inCinchona plants are present at the highest concentrations in the young, vulnerable leaflets, are involved in the chemical defense of the plant against herbivorous insects.     

Importance of quinolizidine alkaloids in the relationship between larvae ofUresiphita reversalis (Lepidoptera: Pyralidae) and a host plant,Genista monspessulana

Larvae ofUresiphita reversalis feed almost exclusively on legumes in the tribe Genisteae, which characteristically contain a variety of quinolizidine alkaloids. The larvae are aposematic, and onGenista monspessulana, a major host in California, they feed on the youngest leaves, at the periphery of the plant. These leaves, which were preferred over older foliage in choice tests, contained four to five times the level of alkaloid found in older leaves. The major alkaloids detected in these plants were dehydroaphylline andN-methylcytisine, together accounting for 74% of the total. Preliminary analyses showed the alkaloid profile of exuviae from larvae feeding on these plants was very similar to that of the plants. Two alkaloids, sparteine and cytisine, which are known components of some hosts ofU. reversalis, were phagostimulants for fifth-instar larvae when added to sucrose-impregnated glass-fiber disks. In addition, when sparteine was added to foliage ofG. monspessulana, effectively doubling the percent dry weight of alkaloid, the growth rate of late-instar larvae was positively affected. Cytisine added to plants had no discernible effect on growth of larvae. Alkaloid levels in larvae and in their frass were proportional to levels in the plants on which they fed. Although the majority of alkaloid was excreted, that which was sequestered by the insect was found entirely in the integument, possibly confering some protection from predators.     

Foliar and floral pyrethrins ofChrysanthemum cinerariaefolium are not induced by leaf damage

Pyrethrins are a class of potent insecticides produced byChrysanthemum cinerariaefolium. Simulated herbivory does not affect concentrations of pyrethrins in damaged and undamaged expanding and fully expanded leaves, or flowers of greenhouse or field-grown plants.     

Developmental stage of herbivorePseudaletia separata affects production of herbivore-induced synomone by corn plants

The female parasitic waspCotesia kariyai discriminated between the volatiles of corn leaves infested by younger host larvaePseudaletia separata (first to fourth instar) and uninfested leaves in a Y-tube olfactometer; the wasps were attracted to the infested leaves. In contrast, when corn plants were infested by the later stages (fifth and sixth instar) of the armyworm, the wasps did not distinguish between infested corn leaves and uninfested corn leaves in the olfactometer. Mechanically damaged leaves were no more attractive than undamaged leaves, and host larvae or their feces were not attractive to the parasitoid. Through chemical analysis, the herbivore-induced plant volatiles were identified in the headspace of infested corn leaves. The herbivore-induced volatiles (HIVs) constituted a larger proportion of the headspace of corn leaves infested by early instar armyworms than of corn leaves infested by late instar armyworms. Application of third-instar larval regurgitant onto artificially damaged sites of leaves resulted in emission of parasitoid attractants from the leaf, whereas leaves treated with sixth-instar regurgitant did not. The function of this herbivore-stage related specificity of herbivore-induced synomones is discussed in a tritrophic context.     

Differential induction of tomato foliar proteins by arthropod herbivores

The effects of mechanical and chemical damage and three types of biotic damage on the activities of four foliar proteins of the tomato plant (Lycopersicon esculentum Mill var. Castlemart) were assayed. Proteinase inhibitor, polyphenol oxidase, peroxidase, and lipoxygenase activities were assayed in damaged leaflets and compared with activities in undamaged leaflets. These proteins are putative plant defenses in tomato. Differential induction of these proteins by the various damage-treatments was demonstrated, such that different subsets of the four proteins were induced by different types of damage. This study clearly demonstrates the ability of plants to respond differentially to different types of damage. Possible mechanisms for this differential induction and the implications of differential induction for plant defense are discussed.     

Differences in Sterol Composition between Male and Female Gonads of Dominant Limpet Species

The present study demonstrated here for the first time that there are statistically significant differences in sterol composition between male and female gonads of the dominant limpets Cellana grata and Cellana toreuma, which are intertidal gastropods. Among 11 different sterols identified in this study, unusually high levels (11.2–19.8% of total sterols) of the Δ⁸-sterols 5α-cholest-8-en-3β-ol (zymostenol) and 5α-cholesta-8,24-dien-3β-ol (zymosterol), which have never been reported in aquatic invertebrate gonads, were present in only the male gonads.     

Effect of nitrogen and water treatment on leaf chemistry in horsenettle (Solanum carolinense), and relationship to herbivory by flea beetles (Epitrix spp.) and tobacco hornworm (Manduca sexta)

We studied the interaction between plants (horsenettle; Solanum carolinense) and herbivorous insects (flea beetles; Epitrix spp., and tobacco hornworm; Manduca sexta) by focusing on three questions: (1) Does variation in nitrogen availability affect leaf chemistry as predicted by the carbon-nutrient balance (CNB) hypothesis? (2) Does variation in plant treatment and leaf chemistry affect insect feeding? (3) Is there an interaction between the insect herbivores that is mediated by variation in leaf chemistry? For three successive years (1998-2001), we grew a set of clones of 10 maternal plants under two nitrogen treatments and two water treatments. For each plant in the summer of 2000, we assayed herbivory by hornworms in both indoor (detached leaf) and outdoor (attached leaf) assays, as well as ambient flea beetle damage. Estimates of leaf material consumed were made via analysis of digitized leaf images. We also assayed leaves for total protein, phenolic, and glycoalkaloid content, and for trypsin inhibitor, polyphenol oxidase, and peroxidase activity. Despite strong effects of nitrogen treatment on growth and reproduction, only total protein responded as predicted by CNB. Leaf phenolic levels were increased by nitrogen treatment, polyphenol oxidase activity was decreased, and other leaf parameters were unaffected. Neither hornworm nor flea beetle herbivory could be related to plant treatment or genotype or to variation in any of the six leaf chemical parameters. A negative relationship between flea beetle and hornworm herbivory was found, but was not apparently mediated by any of the measured leaf chemicals. Because leaf resistance was maintained in low nitrogen plants at the apparent expense of growth and reproduction, our results support the concept of a fitness cost of defense, as predicted by the optimal defense hypothesis.     

Comparison of larval and adult P-450 activity levels for alkaloid metabolism in desertDrosophila

The cytochrome P-450 monooxygenase system has been implicated in plant utilization by at least three species ofDrosophila (D. nigrospiracula, D. mettleri, andD. mojavensis) that are endemic to the Sonoran Desert of the southwestern United States and northwestern Mexico. Basal and induced levels of total cytochrome P-450 were determined for third-instar and decapitated 2- to 5-day post eclosion adults of the three desert species. Total P-450 levels, both basal and induced for all species assayed, were significantly higher for adults than for larvae by up to 20-fold. On a per organism basis, the levels of in vitro metabolism of the cactus alkaloid, carnegine, and patterns of response to induction by cactus tissue for adult desertDrosophila approximated those of larvae. Induction by phenobarbital, however, resulted in levels of in vitro carnegine metabolism that were up to 5.6-fold higher in adults than in larvae.     

Predicting toxicity of tall larkspur (Delphinium barbeyi): measurement of the variation in alkaloid concentration among plants and among years

Tall larkspur (Delphinium barbeyi) is the principal mountain larkspur responsible for the majority of cattle deaths on mountain rangelands in western Colorado and central and southern Utah in the United States. Ten plants in each of two tall larkspur populations in the mountains near Ferron and Salina, Utah, were marked, and single stalks were harvested periodically through the growing season for 4 yr. Toxic alkaloid concentration [alkaloids containing the N-(methylsuccimimido)-anthranilik ester group] was determined by Fourier transform infrared (FTIR) spectroscopy. Individual larkspur plants varied in alkaloid concentrations, especially in early growth (14–38 mg/g). As the concentration declined over the growing season, variation among plants also declined. There were yearly differences in alkaloid concentration among individual plants (P < 0.01) and populations (P < 0.001), even after accounting for differences in phenological growth between years. Variables such as precipitation, temperature, days since snow melt, growing degree days (sum of mean temperature each day from snow melt), and plant height and weight were all considered in a Mallows Cp multiple regression selection procedure to predict alkaloid concentration. The mixed model procedure in SAS adjusted the regression equation for locations and years. Growing degree days was the best single predictor of alkaloid levels: ln y = (3.581 – 0.00423 GDD), R ² = 0.85. Internal validation of this equation within individual years and locations from which the equation was developed, produced correlations between observed versus predicted values ranging from r = 0.73 to 0.93. External validations on nine other larkspur populations produced correlations ranging from r = 0.76 to 0.99. This predictive equation can provide a tool for ranchers and land managers to make management decisions of when to graze cattle in larkspur areas.     

Inhibition and induction of barnacle settlement by natural products present in octocorals

Barnacle settlement inhibitors and inducers are present in the gorgonianLeptogorgia virgulata and the pennatulaceanRenilla reniformis. The inhibitors are low-molecular-weight compounds (<20,000 daltons) that were detected in soft tissue homogenates and dialysates of homogenate and in ambient gorgonian water. Settlement was almost completely inhibited at a dialysate concentration of 1.0 g wet weight equivalents/liter. The inhibitors probably function in chemical defense against predation and fouling, and could prove useful in ship fouling control. The settlement inducers are high-molecular-weight substances (>20,000 daltons) that adsorb to surfaces.     

Antagonism between jasmonate- and salicylate-mediated induced plant resistance: effects of concentration and timing of elicitation on defense-related proteins,herbivore, and pathogen performance in tomato

The jasmonate (JA) and salicylate (SA) signaling pathways in plants provide resistance to herbivorous insects and pathogens. It is known that these pathways interact, sometimes resulting in antagonism between the pathways. We tested how the timing and concentration of elicitation of each pathway influenced the interaction between the jasmonate and salicylate pathways measured in terms of five biochemical responses and biological resistance to caterpillars and bacteria. The salicylate pathway had a stronger effect on the jasmonate pathway than did the reverse. The negative signal interaction was generated by two distinct paths in the plant. A negative interaction in the biochemical expression of the two pathways was most consistent in the simultaneous elicitation experiments compared to when the elicitors were temporally separated by two days. Herbivore bioassays with Spodoptera exigua also consistently reflected an interaction between the two pathways in the simultaneous elicitation experiments. The negative signal interaction reducing biological resistance to the herbivore was also demonstrated in some temporally separated treatment combinations where attenuation of the biochemical response was not evident. Concentration of the elicitors had an effect on the pathway interaction with consistent biochemical and biological antagonism in the high concentration experiments and inconsistent antagonism in the low concentration experiments. The bacterial pathogen, Pseudomonas syringae pv. tomato (Pst), consistently showed reduced lesion development on plants with SA responses activated and, in some experiments, on JA-elicited plants. Resistance to Pst was not reduced or enhanced in dual-elicited plants. Thus, signal interaction is most consistent when elicitors are applied at the same time or when applied at high doses. Signal interaction affected the herbivore S. exigua, but not the pathogen Pst.     

Elevational Trends in Defense Chemistry, Vegetation, and Reproduction in Sanguinaria canadensis

Evaluation of biotic interactions along geographic gradients reveals that pressure on plant populations by herbivores and pathogens increases as latitude decreases, and is accompanied by a parallel increase in the number and toxicity of alkaloid-bearing plants. We compared rhizome alkaloid content with plant reproductive and vegetative characters in Sanguinaria canadensis (Papaveraceae) along an elevational gradient over two growing seasons to ascertain 1) if alkaloid production in bloodroot varies among populations and systematically with elevation, and 2) if there exists a correlation between isoquinoline alkaloid, vegetative and reproductive production. In general, alkaloid content in bloodroot rhizomes declines with elevation, increases with rhizome water content, varies by site, and fluctuates seasonally with plant growth and reproduction. Alkaloid content was positively correlated with vegetative and reproductive effort with few exceptions. Analysis of total protopine and benzophenanthridine alkaloid concentrations revealed generally similar patterns as those of individual alkaloid concentrations, although significant differences did appear between individual alkaloid concentrations.     

Systemic,genotype-specific induction of two herbivore-deterrent iridoid glycosides in Plantago lanceolata L. in response to fungal infection by Diaporthe adunca (Rob.) Niessel

Iridoid glycosides are a group of terpenoid secondary plant compounds known to deter generalist insect herbivores. In ribwort plantain (Plantago lanceolata), the iridoid glycosides aucubin and catalpol can be induced following damage by insect herbivores. In this study, we investigated whether the same compounds can be induced following infection by the fungal pathogen Diaporthe adunca, the causal agent of a stalk disease in P. lanceolata. Significant induction of aucubin and catalpol was observed in two of the three plant genotypes used in this study following inoculation with the pathogen. In one of the genotypes, induction occurred within 6 hr after inoculation, and no decay was observed within 8 days. The highest level of induction was observed in reproductive tissues (spikes and stalks) where infection took place. In these tissues, iridoid glycoside levels in infected plants were, on average, 97% and 37% higher than the constitutive levels in the corresponding control plants, respectively. Significant induction was also observed in leaves (24%) and roots (17%). In addition to significant genotypic variation in the level of induction, we found genetic variation for the tissue-specific pattern of induction, further broadening the scope for evolutionary fine-tuning of induced responses. Recent studies have revealed a negative association between iridoid glycoside levels in P. lanceolata genotypes and the amount of growth and reproduction of D. adunca that these genotypes support. However, for the three genotypes used in the present study, differences in resistance were not related to their constitutive or induced levels of iridoid glycosides, suggesting that additional resistance mechanisms are important in this host-pathogen system. We conclude that iridoid glycosides in P. lanceolata can be induced both by arthropods and pathogenic micro-organisms. Pathogen infection could, therefore, potentially enhance resistance to generalist insect herbivores in this species.     

The role of manganese and nitrogen nutrition in the susceptibility of wheat plants to take-all in Western Australia

Five field experiments are described which measured the effect of take-all on grain yield of wheat when 5 levels of manganese fertilizer were applied in a factorial combination with 5 different types of nitrogen fertilizer.Ammonium nitrogen fertilizer, either as ammonium sulphate or ammonium chloride, lowered the severity of take-all. By contrast, sodium nitrate had no effect on the incidence and severity of take-all. Ammonium chloride and ammonium sulphate were equally effective at controlling take-all, suggesting that the chloride or sulphate ion had little or no effect on the disease.Manganese sulphate decreased take-all severity at two trial sites. Where manganese was deficient, an application of manganese lowered the severity of take-all, had no effect on the incidence and increased the dry matter and grain yields of the wheat plants. There were no beneficial effects of applied manganese if the wheat plants were adequately supplied with soil manganese.The results suggest that take-all is more severe where plants are deficient in either manganese or nitrogen. The work also suggests that manganese deficiency is not necessarily the reason why the wheat plants grown on the acid soils of south-west Western Australia are prone to take-all.