Jens Kvist Nielsen

HOST PLANT DISCRIMINATION WITHIN CRUCIFERAE: FEEDING RESPONSES OF FOUR LEAF BEETLES (COLEOPTERA: CHRYSOMELIDAE) TO GLUCOSINOLATES, CUCURBITACINS AND CARDENOLIDES

Feeding responses of four Chrysomelidae to six less acceptable plants and to compounds from them were investigated by means of leaf disc tests. Significant differences were found between responses of different species, and plants containing potent feeding inhibitors were always rejected Cucurbitacins are potent feeding inhibitors to Phyllotreta nemorum, and this species does not eat Iberis species containing these compounds. Cardenolides are potent feeding inhibitors to P undulata, P tetrastigma and Phaedon cochleartae, and these three species do not eat the cardenolide containing Cheiranthus and Erysimum

Seasonal variation in leaf glucosinolates and insect resistance in two types of Barbarea vulgaris ssp. arcuata.

Leaves from natural populations of Barbarea vulgaris ssp. arcuata (Brassicaceae) in Denmark were examined for glucosinolate content and resistance to the crucifer specialist flea beetle Phyllotreta nemorum. Two types of the plant (P- and G-type) could be recognized. Leaves of the G-type contained the glucosinolates (only side chains mentioned): (S)-2-hydroxy-2-phenylethyl- (2S), indol-3-ylmethyl- (4) and in trace amount (R)-2-hydroxy-2-phenylethyl- (2R), 2-phenylethyl- (1) and 4-methoxyindol-3-ylmethyl- (5). Leaves of the P-type were dominated by 2R and 4, and had only trace amounts of 1, 2S, and 5 but contained in addition the previously unknown (R)-2-hydroxy-2-(4-hydroxyphenyl)ethyl- (3R). The epimer, (S)-2-hydroxy-2-(4-hydroxyphenyl)ethyl- (3S) was found in populations believed to be hybrids, and in B. orthoceras. 2S, 2R, desulfo 2S,-2R, -3S and -3R were isolated and identified by NMR and MS. Acylated glucosinolates or allylglucosinolate were not detected in leaves. The glucosinolate content in August was variable, 3-46 micromol/g dry wt, but was low in most populations, 3-15 micromol/g dry wt. In general, the glucosinolate content increased during the autumn, to 35-75 micromol/g dry wt in November. The G-type was resistant to neonate larvae of Phyllotreta nemorum in August and September (survival in 3-day bioassay typically 0%), and gradually lost the resistance in October and November (survival in 3-day bioassay 40-90%), and there was no correlation between glucosinolate content and resistance. Neither did glucosinolates explain the difference in resistance between the P-type (always susceptible) and the G-type (resistant in the summer season).

A Saponin Correlated with Variable Resistance of Barbarea vulgaris to the Diamondback Moth Plutella xylostella

Two types of Barbarea vulgaris var. arcuata, the G-type and the P-type, differed in resistance to larvae of the diamondback moth (DBM) Plutella xylostella. Rosette plants of the G-type were fully resistant to the DBM when grown in a greenhouse or collected in the summer season, but leaves collected during the late fall were less resistant, as previously found for flea beetle resistance. The P-type was always susceptible. Extracts of resistant leaflets inhibited larval growth in a bioassay, and a growth-inhibiting fraction was isolated by activity-guided fractionation. A triterpenoid saponin (1) was isolated from this fraction and identified as 3-O-β-cellobiosyloleanolic acid from spectroscopic data and analysis of hydrolysis products. The decrease in resistance of the G-type in the fall was correlated with a decrease in the level of 1, from 0.6–0.9 to <0.2 μmol/g dry wt. Compound 1 was not detected in the susceptible P-type. We conclude that 1 is correlated with the variable resistance of B. vulgaris foliage to the DBM.

Flavonoids from cabbage are feeding stimulants for diamondback moth larvae additional to glucosinolates: Chemoreception and behaviour

In caterpillars two styloconic contact chemoreceptors on the maxillary galea are assumed to contain the main taste receptors involved in host plant selection. The diamondback moth, Plutella xylostella L. is a specialist feeder of plants in the Brassicaceae, a plant family characterized by the biosynthesis of glucosinolates. We used pea (Pisum sativum L., Leguminosae) as a neutral non‐host for a dual‐choice leaf disc assay to quantify feeding stimulation by glucosinolates and flavonoids. Increasing concentrations of sinigrin resulted in significant preferences for sinigrin‐treated leaf discs, with a threshold between 1 and 3 μM. Millimolar concentrations of four of the five flavonol triglucosides likewise elicited a significant preference for flavonoid‐treated leaf discs. A mixture of four flavonoids and sinigrin was significantly preferred over sinigrin‐treated leaf discs alone. Vigorous unicellular electrophysiological responses of medial maxillary styloconic taste sensilla were observed in response to five glucosinolates (glucocapparin, sinigrin, glucobrassicin, glucoiberin, and gluconasturtiin). This medial taste neuron responded in a dose‐dependent manner to a concentration series of sinigrin, with a threshold of response of ca. 1 μM. The lateral sensillum styloconicum contained a neuron sensitive to sucrose, glucose, and fructose. However, no responses in the two types of maxillary styloconic sensilla to the phagostimulatory flavonoids could be detected, suggesting that other taste organs mediate chemoreception of flavonoids. We conclude that diamondback moth larvae employ a combination of biosynthetically distinct categories of feeding stimulants which allows for a higher degree of discriminatory ability than when this would be based on glucosinolates alone.

Identification of Defense Compounds in Barbarea vulgaris against the Herbivore Phyllotreta nemorum by an Ecometabolomic Approach

Winter cress (Barbarea vulgaris) is resistant to a range of insect species. Some B. vulgaris genotypes are resistant, whereas others are susceptible, to herbivory by flea beetle larvae (Phyllotreta nemorum). Metabolites involved in resistance to herbivory by flea beetles were identified using an ecometabolomic approach. An F2 population representing the whole range from full susceptibility to full resistance to flea beetle larvae was generated by a cross between a susceptible and a resistant B. vulgaris plant. This F2 offspring was evaluated with a bioassay measuring the ability of susceptible flea beetle larvae to survive on each plant. Metabolites that correlated negatively with larvae survival were identified through correlation, cluster, and principal component analyses. Two main clusters of metabolites that correlate negatively with larvae survival were identified. Principal component analysis grouped resistant and susceptible plants as well as correlated metabolites. Known saponins, such as hederagenin cellobioside and oleanolic acid cellobioside, as well as two other saponins correlated significantly with plant resistance. This study shows the potential of metabolomics to identify bioactive compounds involved in plant defense.

Glucosinolates, flea beetle resistance, and leaf pubescence as taxonomic characters in the genus Barbarea (Brassicaceae)

Glucosinolate content of leaves and roots, diversity in leaf pubescence, and resistance to two near-isogenic lines of the flea beetle Phyllotreta nemorum with or without an R-gene, were determined for 27 accessions of 7 Barbarea taxa, i.e. B. stricta, B. orthoceras, B. intermedia, B. verna, B. vulgaris var. vulgaris, the G-type of B. vulgaris var. arcuata and the P-type of B. vulgaris var. arcuata. Four variable glucosinolate biosynthetic characters were deduced. For (formally) homophenylalanine-derived glucosinolates: (1). Presence or absence of 2-hydroxylation, and if present, R- or S-configuration of 2-hydroxylation; (2). presence or absence of p-hydroxylation; and for tryptophan-derived glucosinolates: (3). presence or absence of N-methoxyglucobrassicin; and (4). presence or absence of 1,4-dimethoxyglucobrassicin. Three phenotypes of leaf-pubescence were observed; (1). glabrous to glabrate leaves; (2). glabrous to glabrate leaves with hairs along the edge; (3). pubescent leaves. The hairs were characterized as simple by scanning electron microscopy. Full resistance to a flea beetle line (ST) was found in B. vulgaris var. vulgaris and in the G-type of var. arcuata; partial resistance was found in B. verna and B. intermedia, while the remaining taxa were fully susceptible to the ST line. All investigated Barbarea taxa were susceptible to larvae from another line containing an R-gene, indicating a similar flea beetle resistance mechanism in the three resistant species. Most Barbarea taxa could be characterized by a particular combination of the investigated characters. The most aberrant was the P-type of B. vulgaris var. arcuata, and the taxonomic status of this type should be reconsidered.

Responses of the flea beetles Phyllotreta nemorum and P. cruciferae to metabolically engineered Arabidopsis thaliana with an altered glucosinolate profile

Summary. Insects feeding on Cruciferae recognize their host plants at least partially by means of specific responses to glucosinolates. However, the effects of variations in glucosinolate levels on the acceptability of plants for specialized insects are not well understood. A survey of the literature demonstrated positive, no, as well as negative correlations between plant acceptability and glucosinolate levels. The present study took advantage of the presence of transgenic Arabidopsis thaliana plants with increased glucosinolate levels. Transgenic A. thaliana contain the CYP79A1 gene from Sorghum bicolor. This gene encodes an enzyme which converts L-tyrosine into p-hydroxyphenylacetaldoxime in the biosynthesis of cyanogenic glycosides in S. bicolor. In transgenic A. thaliana plants, endogenous enzymes convert p-hydroxyphenylacetaldoxime into p-hydroxybenzylglucosinolate (sinalbin), which is not found naturally in this plant. The introduction of CYP79A1 resulted in a four-fold increase in total glucosinolate levels in transgenic A. thaliana plants. Although these changes in glucosinolate levels were rather dramatic, they did not have any effects on the acceptability of A. thaliana for the two flea beetle species, Phyllotreta nemorum and P. cruciferae. The flea beetles did not discriminate between transgenic and wildtype plants. Furthermore, they did not discriminate between leaf discs of wildtype plants where different concentrations of p-hydroxybenzylglucosinolate had been applied topically on the leaf surface. Feeding in P. nemorum was stimulated by extremely high levels of allylglucosinolate while this compound had no effect on P. cruciferae. It is concluded that the effect of glucosinolates on adapted insects depends on the chemical or physical environment in which the glucosinolates are found.

UDP-Glycosyltransferases from the UGT73C Subfamily in Barbarea vulgaris Catalyze Sapogenin 3-O-Glucosylation in Saponin-Mediated Insect Resistance

Triterpenoid saponins are bioactive metabolites that have evolved recurrently in plants, presumably for defense. Their biosynthesis is poorly understood, as is the relationship between bioactivity and structure. Barbarea vulgaris is the only crucifer known to produce saponins. Hederagenin and oleanolic acid cellobioside make some B. vulgaris plants resistant to important insect pests, while other, susceptible plants produce different saponins. Resistance could be caused by glucosylation of the sapogenins. We identified four family 1 glycosyltransferases (UGTs) that catalyze 3-O-glucosylation of the sapogenins oleanolic acid and hederagenin. Among these, UGT73C10 and UGT73C11 show highest activity, substrate specificity and regiospecificity, and are under positive selection, while UGT73C12 and UGT73C13 show lower substrate specificity and regiospecificity and are under purifying selection. The expression of UGT73C10 and UGT73C11 in different B. vulgaris organs correlates with saponin abundance. Monoglucosylated hederagenin and oleanolic acid were produced in vitro and tested for effects on P. nemorum. 3-O-β-d-Glc hederagenin strongly deterred feeding, while 3-O-β-d-Glc oleanolic acid only had a minor effect, showing that hydroxylation of C23 is important for resistance to this herbivore. The closest homolog in Arabidopsis thaliana, UGT73C5, only showed weak activity toward sapogenins. This indicates that UGT73C10 and UGT73C11 have neofunctionalized to specifically glucosylate sapogenins at the C3 position and demonstrates that C3 monoglucosylation activates resistance. As the UGTs from both the resistant and susceptible types of B. vulgaris glucosylate sapogenins and are not located in the known quantitative trait loci for resistance, the difference between the susceptible and resistant plant types is determined at an earlier stage in saponin biosynthesis.

HOST PLANT SELECTION OF THE HORSERADISH FLEA BEETLE PHYLLOTRETA ARMORACIAE (COLEOPTERA: CHRYSOMELIDAE): IDENTIFICATION OF TWO FLAVONOL GLYCOSIDES STIMULATING FEEDING IN COMBINATION WITH GLUCOSINOLATES

Two flavonol glycosides have been isolated from horseradish leaves and identified as kaempferol 3–0‐xylosylgalactoside and quercetin 3–0‐xylosylgalactoside. The kaempferol glycoside stimulates feeding in the monophagous flea beetle Phyllotreta armoraciae as do glucosinolates. Combinations of allylglucosinolate and the kaempferol glycoside are, however, more stimulatory than any of the compounds alone. The role of flavonol glycosides in host plant selection of P. armoraciae is discussed. This is the first record of a crucifer feeding insect being stimulated to feed by an allelochemic which is not a glucosinolate.

Cucurbitacin E and I in Iberis amara: Feeding inhibitors for Phyllotreta nemorum

Abstract Cucurbitacin E and cucurbitacin I have been isolated from green parts of Iberis amara and identified by TLC, UV and MS. It is shown that cucurbitacins act as feeding inhibitors for the flea beetle Phyllotreta nemorum. The most potent feeding inhibitors in green parts of I. amara towards P. nemorum are cucurbitacin E and I, and the concentrations of these compounds in the plant are found to be high enough to prevent feeding of the flea beetle.