Elicitor-enhanced syringin production in suspension cultures of Saussurea medusa

Syringin production and related secondary metabolism enzyme activities in suspension cultures of Saussurea medusa treated with different elicitors (yeast extract, chitosan and Ag⁺) were investigated. All elicitors enhanced syringin production, and the optimal feeding protocol was the combined addition of 1.5% (v/v) yeast extract, 0.2 g l⁻¹ chitosan and 75 μM Ag⁺ at the 15th day of the cell culture. The highest syringin production reached 741.9 mg l⁻¹, which was 3.6−fold that of the control. The glucose−6-phosphate dehydrogenase (EC 1.1.1.49), phenylalanine ammonia lyase (EC 4.3.1.5) and peroxidase (EC 1.11.1.7) activities increased significantly after elicitor treatment. The maximum enzyme activities were obtained when the treatment time was 6 h.     

Host-Pathogen Interactions : XXI. Extraction of a Heat-Labile Elicitor of Phytoalexin Accumulation from Frozen Soybean Stems

An extract of frozen and thawed soybean (Glycine max L. Merr. cv. Wayne) stems is active, in wounded soybean cotyledons, as a heat-labile elicitor of phytoalexins. The elicitor activity of the extract is destroyed by heating to 95°C for 10 minutes. The fraction that contains heat-labile elicitor activity releases heat-stable elicitor-active molecules from purified soybean cell walls. Heat-labile elicitor activity voids a Bio-Gel P-6 column and can be absorbed onto and eluted from a DEAE Sephadex ion exchange column. Using the cotyledon phytoalexin elicitor assay, maximum heatlabile elicitor activity was obtained when soybean stems were extracted with acetate buffer at pH 6.0. Addition of 1 millimolar CaCl₂ increased apparent heat-labile elicitor activity. The heat-labile elicitor stimulated maximum phytoalexin accumulation when applied to cotyledons immediately after the cotyledons were cut. Partially purified stem extracts lost heat-labile elicitor activity during storage for several days at 3°C. The possible role of a heat-labile elicitor in stimulation of phytoalexin accumulation by both abiotic and biotic elicitors is discussed.     

Host-Pathogen Interactions : XXV. Endopolygalacturonic Acid Lyase from Erwinia carotovora Elicits Phytoalexin Accumulation by Releasing Plant Cell Wall Fragments

Heat-labile elicitors of phytoalexin accumulation in soybeans (Glycine max L. Merr. cv Wayne) were detected in culture filtrates of Erwinia carotovora grown on a defined medium containing citrus pectin as the sole carbon source. The heat-labile elicitors were highly purified by cation-exchange chromatography on a CM-Sephadex (C-50) column, followed by agarose-affinity chromatography on a Bio-Gel A-0.5m gel filtration column. The heat-labile elicitor activity co-purified with two α-1,4-endopolygalacturonic acid lyases (EC 4·2·2·2). Endopolygalacturonic acid lyase activity appeared to be necessary for elicitor activity because heat-inactivated enzyme preparations did not elicit phytoalexins. The purified endopolygalacturonic acid lyases elicited pterocarpan phytoalexins at microbial-inhibitory concentrations in the soybean-cotyledon bioassay when applied at a concentration of 55 nanograms per milliliter (1 × 10⁻⁹ molar). One of these lyases released heat-stable elicitors from soybean cell walls, citrus pectin, and sodium polypectate. The heat-stable elicitor-active material solubilized from soybean cell walls by the lyase was composed of at least 90% (w/v) uronosyl residues. These results demonstrate that endopolygalacturonic acid lyase elicits phytoalexin accumulation by releasing fragments from pectic polysaccharides in plant cell walls.     

Genetic transformation of Indian isolate of Lemna minor mediated by Agrobacterium tumefaciens and recovery of transgenic plants

Transgenic plants of an Indian isolate of Lemna minor have been developed for the first time using Agrobacterium tumefaciens and hard nodular cell masses ‘nodular calli’ developed on the BAP - pretreated daughter frond explants in B₅ medium containing sucrose (1.0 %) with 2,4-D (5.0 μM) and 2-iP (50.0 μM) or 2,4-D (50.0 μM) and TDZ (5.0 μM) under light conditions. These calli were co-cultured with A. tumefaciens strain EHA105 harboring a binary vector that contained genes for β-glucuronidase with intron and neomycin phosphortransferase. Transformed cells selected on kanamycin selection medium were regenerated into fronds whose transgenic nature was confirmed by histochemical assay for GUS activity, PCR analysis and Southern hybridization. The frequency of transformation obtained was 3.8 % and a period of 11–13 weeks was required from initiation of cultures from explants to fully grown transgenic fronds. The pretreatment of daughter fronds with BAP, use of non-ionic surfactant, presence of acetosyringone in co-cultivation medium, co-culture duration of 3 d and 16 h photoperiod during culture were found crucial for callus induction, frond regeneration and transformation of L. minor. This transformation system can be used for the production of pharmaceutically important protein and in bioremediation.     

A new approach for achievement of inulin accumulation in suspension cultures of Jerusalem artichoke (Helianthus tuberosus) using biotic elicitors

A promising protocol for achievement the accumulation rate of inulin compound in a suspension culture of Jerusalem artichoke (Helianthus tuberosus) was established. The effect of incorporated of cell cultures in combining with two type of biotic elicitors Aspergillus niger extract and Methyl-Jasmonate incorporation feeding medium on leaf cell growth patterns and production of inulin was investigated. The maximum value of cell growth parameters and highest content of inulinase activity (0.395 u/ml) were resulted from elicitation of augmented MS-medium with A. niger extract at the level of 0.2% in combination with Methyl-Jasmonate (150 μM) as compared with other concentrations after 2 weeks of cultivation. The chemical analyses of the different cell lines were spectro-photometerically performed. This study clearly indicates that combining of A. niger and Methyl-Jasmonate elicitors plays a critical role on inulin process and its accumulation in Jerusalem artichoke cell cultures.     

Induction of defense responses in cultured parsley cells by plant cell wall fragments

Cell suspension cultures of parsley (Petroselinum crispum) accumulated coumarin phytoalexins and exhibited increased β-1,3-glucanase activity when treated with either a purified α-1,4-d-endopolygalacturonic acid lyase from Erwinia carotovora or oligogalacturonides solubilized from parsley cell walls by endopolygalacturonic acid lyase. Coumarin accumulation induced by the plant cell wall elicitor was preceded by increases in the activities of phenylalanine ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL) and S-adenosyl-l-methionine:xanthotoxol O-methyltransferase (XMT). The time courses for the changes in these three enzyme activities were similar to those observed in cell cultures treated with a fungal glucan elicitor. The plant cell wall elicitor was found to act synergistically with the fungal glucan elicitor in the induction of coumarin phytoalexins. As much as a 10-fold stimulation in coumarin accumulation above the calculated additive response was observed in cell cultures treated with combinations of plant and fungal elicitors. The synergistic effect was also observed for the induction of PAL, 4CL, and XMT activities. These results demonstrate that plant cell wall elicitors induce at least two distinct biochemical responses in parsley cells and further support the role of oligogalacturonides as important regulators of plant defense.     

Host-Pathogen Interactions: IX. Quantitative Assays of Elicitor Activity and Characterization of the Elicitor Present in the Extracellular Medium of Cultures of Phytophthora megasperma var. sojae

Resistance of soybean (Glycine max L.) seedlings to Phytophthora megasperma var. sojae (Pms) is in part due to the accumulation in infected tissue of a compound which is toxic to Pms. The accumulation of this compound, a phytoalexin called glyceollin, is triggered by infection, but it can also be triggered by molecules, “elicitors,” present in cultures of Pms. The ability of the Pms elicitor to stimulate phytoalexin accumulation in soybean tissues has been used as the basis for biological assays of elicitor activity. Two bioassays were developed and characterized in this study of the Pms elicitor. These bioassays use the cotyledons and the hypocotyls of soybean seedlings. The cotyledon assay was used to characterize the extracellular Pms elicitor. This elicitor was isolated from Pms cultures and purified by ion exchange and molecular sieving chromatography. The extracellular Pms elicitor was determined to be a predominantly 3-linked glucan, which is similar in composition and structure to a polysaccharide component of Pms mycelial walls.     

Elicitor enhanced production of protoberberine alkaloids from in vitro cell suspension cultures of <Emphasis Type="Italic">Tinospora cordifolia</Emphasis> (Willd.) Miers ex Hook. F. &amp; Thoms

The present research investigates the effect of Piriformospora indica, an endophytic fungus, on production of protoberberine alkaloids in in vitro cell suspension cultures of Tinospora cordifolia. Although T. cordifolia produces a number of protoberberine alkaloids, the simultaneous production of jatrorrhizine and palmatine in cell suspension cultures of T. cordifolia was observed for the first time with the use of P. indica as biotic elicitor. The cells in suspension cultures were elicitated with P. indica on 14th day of culture initiation and the production of the alkaloids on 16th day was monitored. The autoclaved as well as filter sterilized cultures of P. indica were used in addition to the use of fungal cell extract. The elicitor effect of P. indica was analyzed and compared with other abiotic elicitor (methyl jasmonate) and biotic elicitors (chitin and chitosan). The culture filtrate of P. indica in the filter sterilized (5.0% v/v) form gave better response with enhanced 4.2-fold production of jatrorrhizine (10.72 mg/g DW) and 4.0-fold production of palmatine (4.39 mg/g DW). The production of these compounds was at par with that achieved in methyl jasmonate (at 250 µM) treated cell suspension cultures.     

Elicitation of camptothecin production in cell cultures ofCamptotheca acuminata

Campothecin production was increased with elicitors, methyl jasmonate, jasmonic acid, yeast extract elicitor, and ferulic acid in suspension cultures ofCamptotheca acuminata. jasmonic acid was found to be the most efficient elicitor. Camptothecin production increased 11 times by using the optimum dosing concentration of jasmonic acid which was 50 μM. The kinetics of camptothecin accumulation in response to the treatment with jasmonic acid showed that the camptothecin accumulation reached the maximum value at 4 days after jasmonic acid dosing and then a rapid decrease in camptothecin accumulation was observed.     

Assessment of pharmacological activities of two medicinal plant of Bangladesh: Launaea sarmentosa and Aegialitis rotundifolia roxb in the management of pain,pyrexia and inflammation

Background

The current study aims at evaluating the analgesic, anti-pyretic and anti-inflammatory properties of methanolic extract of the stem, bark and leaves of Launaea sarmentosa and Aegialitis rotundifolia roxb.

Results

The AELS and AEAR extract presented a significant (***p < 0.001) dose dependent increase in reaction time in writhing method and showed inhibition of 63.1% and 57.1% respectively at the doses of 400 mg/kg body weight while standard drug showed (P < 0.001) inhibition of 69.23%. In tail immersion method, AELS and AEAR showed maximum time of tail retention at 30 min in hot water i.e. 6.93 sec and 6.54 sec respectively at highest doses of 400 mg/kg body weight than lower dose while standard pentazocine showed reaction time of 7.62 sec. The AELS and AEAR extract also exhibited promising anti-inflammatory effect as demonstrated by statistically significant inhibition of paw volume by 32.48% and 26.75% respectively at the dose of 400 mg/kg body weight while the value at the dose of 200 mg/kg body weight were linear to higher dose at the 3^rd hour of study. On the other hand, Standard indomethacin inhibited 40.13% of inflammation (***P < 0.001). In Cotton-pellet granuloma method, AELS and AEAR extract at the dose of 400 mg/kg body weight exhibited inhibition of inflammation of 34.7% and 29.1% respectively while standard drug showed (P < 0.001) inhibition of 63.22%. Intraperitoneal administration of AELS and AEAR showed dose dependent decrease in body temperature in brewer’s yeast induced hyperthermia in rats at both doses. However, AELS significantly decreased body temperature (***p < 0.001) at 400 mg/kg compared to control.

Conclusions

Present work propose that the methanolic extract of Launaea sarmentosa and Aegialitis rotundifolia roxb possesses dose dependent pharmacological action which supports its therapeutic use in folk medicine possibly mediated through the inhibition or blocking of release of prostaglandin and/or actions of vasoactive substances such as histamine, serotonin and kinins.     

Significant enhancement of oleanolic acid accumulation by biotic elicitors in cell suspension cultures of Calendula officinalis L.

The effects of elicitors on cell growth and oleanolic acid (OA) accumulation in shaken cell suspension cultures of Calendula officinalis were investigated. Elicitors were added individually at various concentrations to 5-day-old cell cultures and their effects monitored at 24 h intervals for 4 days. Different effects on OA accumulation were observed depending on the day of treatment. Jasmonic acid was the most efficient elicitor. After 72 h of treatment with 100 μM JA, the intracellular content of OA reached its maximum value (0.84 mg g⁻¹ DW), which was 9.4-fold greater than that recorded in an untreated control cultures. The addition of chitosan at 50 mg l⁻¹ produced a 5-fold enhancement of OA accumulation (0.37 mg g⁻¹ DW) after 48 h of treatment. Treatment with yeast extract at 200 mg l⁻¹ for 96 h or with pectin at 2 mg l⁻¹ for 48 h produced identical cellular levels of OA (0.22 mg g⁻¹ DW). Lastly, 48 h elicitation with homogenate of the fungus Trichoderma viride produced a 1.8-fold increase in oleanolic acid content (0.12 mg g⁻¹ DW). In addition to significantly stimulating OA accumulation and its secretion into the culture medium, the elicitors also caused slight inhibition of cell growth.     

Coronatine,a more powerful elicitor for inducing taxane biosynthesis in Taxus media cell cultures than methyl jasmonate

Coronatine is a toxin produced by the pathogen Pseudomonas syringae. This compound has received much attention recently for its potential to act as a plant growth regulator and elicitor of plant secondary metabolism. To gain more insight into the mechanism by which elicitors can affect the biosynthesis of paclitaxel (Px) and related taxanes, the effect of coronatine (Cor) and methyl jasmonate (MeJA) on Taxus media cell cultures has been studied. For this study, a two-stage cell culture was established, in which cells were first cultured for 14 days in a medium optimised for growth, after which the cells were transferred to medium optimised for secondary metabolite production. The two elicitors were added to the medium at the beginning of the second stage. Total taxane production in the cell suspension was significantly enhanced by both elicitors, increasing from a maximum level of 8.14 mg/L in control conditions to 21.48 mg/L (day 12) with MeJA and 77.46 mg/L (day 16) with Cor. Expression analysis indicated that the txs, t13oh, t2oh, t7oh, dbat, pam, bata and dbtnbt genes were variably induced by the presence of the elicitors. Genes encoding enzymes involved in the formation of the polihydroxylated hypothetical intermediate (TXS, T13OH, T2OH, T7OH) and the phenylalanoil CoA chain (PAM) were stronger induced than those encoding enzymes catalysing the last steps of the Px biosynthetic pathway (DBAT, BAPT and DBTNBT). Notably, although taxane accumulation differed qualitatively and quantitatively following MeJA- or Cor-elicitation, gene expression induction patterns were similar, inferring that both elicitors may involve distinct but yet uncharacterised regulatory mechanisms.     

Improved isoflavonoid production in Pueraria candollei hairy root cultures using elicitation

The effect of abiotic and biotic elicitors (methyl jasmonate, chitosan, salicylic acid, Agrobacterium, and yeast extract) at various concentrations on total isoflavonoid accumulation was studied in the hairy root cultures of Pueraria candollei. All elicitors stimulated isoflavonoid production. Yeast extract (0.5 mg/ml) was the most efficient giving total isoflavonoids at 60 ± 1 mg/g dry wt, which was 4.5-fold higher than control hairy roots on day 3 of elicitation.     

Factors affecting efficient in vitro micropropagation of Muscari muscarimi Medikus using twin bulb scale

Endemic Muscari muscarimi Medikus is the most fragrant plant among Muscari species and has a high ornamental potential. The natural populations of M. muscarimi, are severely affected by increased environmental pollution and urbanization. There is a need to develop a micropropagation method that should serve effectively for commercial propagation and conservation. Therefore, the study targeted to set up a strategy for efficient in vitro bulblet regeneration system of M. muscarimi using twin scale bulb explants on 1.0 × MS medium containing 4.44, 8.88, 17.76 μM BAP (6-Benzylaminopurine) plus 2.685, 5.37, 10.74 μM NAA (α-Naphthalene acetic acid). Maximum number of 19 daughter axillary bulblets and 16 daughter adventitious bulblets per twin bulb scale explant was regenerated on 1.0 × MS medium containing 17.76 μM BAP plus 10.74 μM NAA and 17.76 μM BAP plus 2.685 μM NAA respectively. The daughter bulblets regenerated on twin bulb scales on 8 out of 9 regeneration treatment could be easily rooted on 1.0 × MS medium containing 4.9 μM IBA (Indole-3-butyric acid). The daughter bulblets regenerated on 9th treatment (1.0 × MS medium containing 17.76 μM BAP plus 10.74 μM NAA) were transferred to 1.0 × MS medium containing 30 g/l sucrose to break negative carry over effect of this dose of BAP–NAA, where they grew 2–3 roots of variable length. Daughter bulblet diameter was increased by culturing them on 1.0 × MS medium containing 4.44 μM BAP plus 5.37 μM NAA. The results verified that both age and the source of explants had significant effect on regeneration. In another set of experiments, twin scales were obtained from in vitro regenerated daughter bulblets, although they induced bulblets, yet their bulblet regeneration percentage, mean number of bulblets per explant and their diameter were significantly reduced. In vitro regenerated bulblets were acclimatized in growth chamber under ambient conditions of temperature and humidity on peat moss, where they flowered. The study provides important information about selection of suitable micropropagation medium, strategies to improve bulblet diameter and rooting of M. muscarimi which offers a scope for commercial propagation.Abbreviations: MS medium, Murashige Skoog medium; BAP, 6-Benzylaminopurine; NAA, α-Naphthalene acetic acid; IBA, Indole-3-butyric acid     

Protection of human erythrocyte using Crinumasiaticum extract and lycorine from oxidative damage induced by 2-amidinopropane

The intention of this investigation was to evaluate the free radical scavenging activity and erythrocyte protective activity of ethanolic extract of Crinumasiaticum (L) and lycorine. The ethanolic extract of C. asiaticum (L) and lycorine were found to have different levels of antioxidant properties in the test models. Both ethanolic extract of C. asiaticum (L) (0.5–2.5 mg/ml) and lycorine (0.010 mg–0.050 mg/ml) increases the percentage of lipid peroxidation inhibition (26.25 ± 0.23% and 19.25 ± 0.23%) and enhances the free radical scavenging activity (20.92 ± 0.22% and 20.52 ± 0.22%), scavenging of hydrogen peroxide (25.67 ± 0.17% and 23.07 ± 0.3%) superoxide anion scavenging activity (27.69 ± 0.16% and 16.09 ± 0.7%) at concentration of 2.5 and 0.050 mg of C. asiaticum (L) and lycorine, respectively. But compared with tocopherol (P < 0.05) less activity was observed by C. asiaticum (L) and lycorine. The ethanolic extract of C. asiaticum (L) and lycorine were normalized to reduce the level of glutathione and also to sustain the status of protein in erythrocytes during the peroxyl radical [2,2-azobis (2-amidinopropane) dihydrochloride (AAPH)] induced oxidative damage in ex vivo model. The present results of the investigations demonstrated that protective nature of the C. asiaticum (L) and lycorine will be considered as a significant natural antioxidant source.     

Rapid Activation of Phenylpropanoid Metabolism in Elicitor-Treated Hybrid Poplar (Populus trichocarpa Torr. & Gray x Populus deltoides Marsh) Suspension-Cultured Cells

Elicitor induction of phenylpropanoid metabolism was investigated in suspension-cultured cells of the fast-growing poplar hybrid (Populus trichocarpa Torr. & Gray × Populus deltoides Marsh) H11-11. Treatment of cells with polygalacturonic acid lyase or two fungal elicitors resulted in rapid and transient increases in extractable l-phenylalanine ammonia lyase and 4-coumarate:coenzyme A ligase enzyme activities. The substrate specificity of the inducible 4-coumarate:coenzyme A ligase enzyme activity appeared to differ from substrate specificity of 4-coumarate:coenzyme A ligase enzyme activity in untreated control cells. Large and transient increases in the accumulation of l-phenylalanine ammonia-lyase and 4-coumarate:coenzyme A ligase mRNAs preceded the increases in enzyme activities and were detectable by 30 minutes after the start of elicitor treatment. Chalcone synthase, cinnamyl alcohol dehydrogenase, and coniferin β-glucosidase enzyme activities were unaffected by the elicitors, but a large and transient increase in β-glucosidase activity capable of hydrolyzing 4-nitrophenyl-β-glucoside was observed. Subsequent to increases in l-phenylalanine ammonialyase and 4-coumarate:coenzyme A ligase enzyme activities, cell wall-bound thioglycolic acid-extractable compounds accumulated in elicitor-treated cultures, and these cells exhibited strong staining with phloroglucinol, suggesting the accumulation of wall-bound phenolic compounds.     

Optimized production of isoflavones in cell cultures of Psoralea corylifolia L. Using elicitation and precursor feeding

The effect of biotic elicitors (yeast extract, chitosan), signaling molecule (salicylic acid), and polyamines (putrescine and spermidine) was studied with respect to isoflavones accumulation in cell suspension cultures of corylifolia L. Untreated cell suspension (control) accumulated 1.66% dry wt of daidzein and 0.165% dry wt of genistein. In precursor feeding experiment, phenylalanine at 0.5 mM concentration led to 1.3 fold higher production of daidzein (1.99% dry wt) and genistein (0.22% dry wt). In biotic elicitors, yeast extract (100 mg/L) was found to be the most efficient elicitor to induce higher production levels of daidzein (2.21% dry wt) and genistein (0.293% dry wt) in suspension cultures. Salicylic acid (signaling molecule) at 1 mM concentration stimulated the maximum accumulation of daidzein (3.4% dry wt) and genistein (0.41% dry wt) 2 days after elicitation. In case of polyamines, spermidine (100 mM) resulted in highest accumulation of daidzein (3.2% dry wt) and genistein (0.475% dry wt) after 7 days of addition, which was 2.4 fold of that in control. This is the first report on kinetics of isoflavone production in response to elicitation in cell suspension of P. corylifolia.     

Elicitation as yield enhancement strategy for glycyrrhizin production by cell cultures of <Emphasis Type="Italic">Abrus precatorius</Emphasis> Linn.

Glycyrrhizin is an important phytoconstituent of licorice which is widely used in the pharmaceutical and food industry. As the roots and leaves of Abrus precatorius also contain glycyrrhizin, it can be used as an alternative source of glycyrrhizin. In spite of extensive research work undertaken with cultures of Glycyrrhiza glabra, the glycyrrhizin production remains elusive. Successful production of glycyrrhizin in cell cultures of A. precatorius is being reported for the first time in our study. Cell cultures of A. precatorius L. were treated with the elicitors prepared from the fungi (Aspergillus niger and Rhizopus stolonifer), yeast extract, salicylic acid, ascorbic acid, and eugenol to induce and enhance the synthesis of glycyrrhizin. In the present study, an integrated yield enhancement strategy, developed by the addition of selected elicitor (A. niger and ascorbic acid) at optimized concentrations, resulted in 24.6 g/l dry cell weight biomass and 53.62 mg/l glycyrrhizin, which was 5.22 times higher in productivity in comparison to control cultures.     

Glycopeptide elicitors of stress responses in tomato cells: N-linked glycans are essential for activity but act as suppressors of the same activity when released from the glycopeptides

Induction of ethylene, an early symptom of the stress response in tomato (Lycopersicon esculentum [L.] Mill) cells, was used as a bioassay to purify elicitor activity from yeast extract. The purified elicitor preparation consisted of small glycopeptides (mean relative molecular weight of approximately 2500) and induced ethylene biosynthesis and phenylalanine ammonia-lyase activity half-maximally at 15 nanograms per milliliter. Elicitor activity was partially abolished by pronase and almost completely by endo-β-N-acetylglucosaminidase H, α-mannosidase, or periodate. The oligosaccharides released upon treatment with endo-β-N-acetylglucosaminidase H competitively inhibited the elicitor activity of the glycopeptides. This suppressor activity was abolished by periodate oxidation and α-mannosidase treatment. The suppressors were chromatographically separated into four active fractions with sizes corresponding to 7 to 10 monosaccharides. They consisted predominantly of mannose and contained also N-acetylglucosamine and glucose. The suppressors had no effect on the response of the tomato cells to a different elicitor, derived from cell walls of Phytophthora megasperma f. sp. glycinea. This strongly suggests that different recognition sites exist for different elicitors in tomato cells, and that the oligosaccharide suppressors act specifically on the perception of just one elicitor. The hypothesis is put forward that the suppressors bind to one of the elicitor recognition sites nonproductively, i.e. without producing a signal, thereby preventing induction of the stress responses by the corresponding elicitor.