Herbivore-Induced Changes in Tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>) Primary Metabolism: A Whole Plant Perspective

Induced changes in primary metabolism are important plant responses to herbivory, providing energy and metabolic precursors for defense compounds. Metabolic shifts also can lead to reallocation of leaf resources to storage tissues, thus increasing a plant’s tolerance. We characterized whole-plant metabolic responses of tomato (Solanum lycopersicum) 24 h after leaf herbivory by two caterpillars (the generalist Helicoverpa zea and the specialist Manduca sexta) by using GC-MS. We measured 56 primary metabolites across the leaves, stems, roots, and apex, comparing herbivore-attacked plants to undamaged plants and mechanically damaged plants. Induced metabolic change, in terms of magnitude and number of individual concentration changes, was stronger in the apex and root tissues than in undamaged leaflets of damaged leaves, indicating rapid and significant whole-plant responses to damage. Helicoverpa zea altered many more metabolites than M. sexta across most tissues, suggesting an enhanced plant response to H. zea herbivory. Helicoverpa zea herbivory strongly affected concentrations of defense-related metabolites (simple phenolics and precursor amino acids), while M. sexta altered metabolites associated with carbon and nitrogen transport. We conclude that herbivory induces many systemic primary metabolic changes in tomato, and that changes often are specific to a single tissue or type of herbivore. The potential implications of primary metabolic changes are discussed in relation to resistance and tolerance.     

Reassessment of the role of gut alkalinity and detergency in insect herbivory

Previously it was reported that significant amounts of the tomato phenolic, chlorogenic acid, were oxidized in the digestive system of generalist feedersSpodoplera exigua andHelicoverpa zea. The covalent binding of the oxidized phenolic (i.e., quinone) to dietary protein exerts a strong antinutritive effect against larvae. In this study, we examined the fate of ingested chlorogenic acid in larvalManduca sexta, a leaf-feeding specialist of solanaceous plants. Significant amounts of chlorogenic acid were bound to excreted protein byM. sexta when larvae fed on tomato foliage. However, in the case ofM. sexta we suggest that the strong alkalinity and detergency of the midgut may minimize the antinutritive effects of oxidized phenolics. The solubility of tomato leaf protein is significantly greater at pH 9.7, representative of the midgut ofM. sexta, than at pH 8.0, representative of the midguts ofH. zea and S. exigua. We suggest that this increase in solubility would compensate for any loss in bioavailability of essential amino acids caused by the covalent binding of chlorogenic acid to amino acids. Furthermore, lysolecithin, a surfactant likely to contribute to the detergent properties of the midgut fluid, was shown to enhance protein solubility as well as inhibit polyphenol oxidase activity. The adaptive significance of gut alkalinity and detergency is discussed.Approved by the Director of the Arkansas Agricultural Experiment Station.     

Identification and Cloning of a Pheromone-Binding Protein from the Oriental Beetle, Exomala orientalis

We have identified and cloned a pheromone-binding protein (EoriPBP) from the Japanese and American populations of the Oriental beetle, Exomala orientalis (Coleoptera: Scarabaeidae). The protein showed more than 90% amino acid identity to the previously identified pheromone-binding proteins from Popillia japonica (PjapPBP) and Anomala osakana (AosaPBP), as well as to one of the odorant-binding proteins from Phyllopertha diversa (PdivOBP1). EoriPBP has 116 amino acids, with a calculated molecular mass of 12,981 Da, pI of 4.3, and six highly conserved cysteine residues. 5-RACE amplifications led to the characterization of a signal peptide with 19 amino acids. The signal peptide showed high amino acid identity to the signal peptide for AosaPBP. Comparison of the amino acid sequences of the PBPs involved in the detection of similar ligands, i.e., monounsaturated lactones and ketone, suggests that the most variable residues among the PBPs from E. orientalis, P. japonica, and A. osakana are probably the most discriminating residues. As with the pheromone-binding protein from Bombyx mori, the residues at positions 61, 64, 71, and 82 in EoriPBP, PajpPBP, and AosaPBP, which are either valine, leucine, isoleucine, or methionine, are likely to be specificity determinants.     

Structure-activity relationships for analogs of (+)-(E)-endo-Β-bergamoten-12-oic acid,an oviposition stimulant ofHelicoverpa zea (Boddie)

-Bergamotenoic acid, a compound previously shown to stimulate oviposition inH. zea, was converted into a set of bicyclic analogs and tested with a set of acyclic side chain analogs to ascertain the molecular structure that maximizes insect behavioral response. While changes in the bicyclic ring elicited no variation in response, alteration in the side chain structure of-bergamotenoic acid resulted in significant changes in moth preference. Free rotation about the C-C bond proximal to the carboxylic acid group appears to be an important structural factor, since saturation of the side chain double bond significantly increased activity. The carboxylic acid group seems to be required for strong oviposition stimulation, since analogs lacking the carboxylic acid group exhibited no significant oviposition activity. Oviposition preference ofH. zea was also influenced by the length of the hydrocarbon chain to which the carboxylic acid is attached. While hexanoic acid was found inactive, the ovipositional preference for the heptanoic and octanoic acids was greatest for the one 8-carbon tested. This and other work suggest that carboxylic acids of specific chain lengths influence the oviposition behavior of bothHelicoverpa andHeliothis species and may be associated with host-plant selection. The potential use of this information in designing integrated pest management strategies for control ofH. zea is discussed.     

Terminal steps in pheromone biosynthesis byHeliothis virescens andH. zea

In vivo application to the sex pheromone gland ofHeliothis Virescens andH. Zea of large quantities of alcohols normally present in small amounts resulted in the preferential conversion of the alcohols to the corresponding pheromonal aldehydes. Amounts of the minor component aldehydes were increased up to 15-fold by selectively applying large quantities of the alcohol precursors. Using this technique, we have inducedH. virescens to convert bombykol, the sex pheromone of the silkworm, to the corresponding aldehyde, bombykal, and have induced femaleH. zea to produce the same sex pheromone components used byH. virescens by applying tetradecanol and (Z)-9-tetradecenol to the surface of the gland. Further, treatedH. zea females were attractive toH. virescens males and caused males to attempt interspecific copulation repeatedly. We have also found that the enyzme involved in this conversion is dependent on the presence of molecular oxygen, indicating that a nonspecific alcohol oxidase is responsible for the terminal biosynthetic step in pheromone production by bothH. virescens andH. zea.Mention of a commercial product or proprietary does not constitute endorsement by either the University of Guelph or U.S.D.A.     

Do Caterpillars Secrete “Oral Secretions”?

The oral secretions or regurgitant of caterpillars contain potent elicitors of plant induced responses. These elicitors are recognized by host plants to differentiate between simple mechanical injury and the presence of herbivores. In some cases, this level of recognition is highly specific. Despite the in-depth chemical characterization of these elicitors, little is known about the amounts delivered in regurgitant during feeding. In this study, we use a fluorescent dye to label regurgitant in order to visualize caterpillar regurgitation during feeding. The procedure is highly sensitive and allows us to visualize nanoliter amounts of regurgitant. We examined the propensity of larval Helicoverpa zea, Heliothis virescens, Spodoptera exigua, Spodoptera frugiperda, and Manduca sexta to regurgitate on various host plants. These species were selected because they have been among the most intensely studied in terms of elicitors. Our results indicate that most larvae did not regurgitate following a brief feeding bout (∼10 min) during which they ate ca. 0.40 cm² of leaf. When larvae did regurgitate, it was typically less than 10 nl. This is several orders of magnitude less than is typically used in most studies on oral secretions. The frequency of regurgitation appears to vary depending upon the host plant. Larval H. zea are less likely to regurgitate when feeding on tomato leaves compared to corn mid-whorl tissue. Our results have importance in understanding the role of oral secretions in plant recognition of herbivory. Because caterpillars did not routinely regurgitate during feeding, it is likely that they avoid the elicitation of some plant defensive responses during most feeding bouts.     

Inhibition ofCampoletis sonorensis parasitism ofHeliothis zea and of parasitoid development by 2-tridecanone-mediated insect resistance of wild tomato

Field populations ofHeliothis spp. were sampled for levels of naturally occurring larval parasitism on six tomato lines varying in levels of 2-tridecanone-mediated resistance toManduca sexta (L.) andLeptinotarsa decemlineata (Say). Second and third instars were parasitized byCampoletis sonorensis (Cameron) (Hymenoptera: Ichneumonidae) andCotesia (=Apantales)marginiventris (Cresson) (Hymenoptera: Braconidae) in 1984 through 1986 and byMicropletis croceipes (Cresson) (Hymenoptera: Braconidae) in 1986. Differences in parasitism by individual and multiple species among host plants were not demonstrated. However, levels of parasitism were low and variable among replicates. Total larval parasitism averaged across all plant lines was less than 6% in 1984 and 1986 and approximately 11% in 1985. In laboratory cage studies,C. sonorensis parasitized fewerH. zea larvae on tomato foliage with high levels of 2-tridecanone than on foliage with low levels. RearingH. zea on diet containing 2-tridecanone and 2-undecanone did not alter incidence of parasitism byC, sonorensis; nor did rearing parasitizedH. zea larvae on chemically treated host diets precondition the parasitoid to higher or lower mortality when transferred to foliage as a substrate for cocoon spinning, regardless of the foliage genotype. However, parasitoid survival during cocoon spinning on foliage varied significantly among plant lines in a manner corresponding to the level of 2-tridecanone-mediated resistance of the foliage. Parasitoid mortality was greatest on highly resistant foliage and lowest on susceptible foliage.     

Interaction of nuclear polyhedrosis virus with catechols: Potential incompatibility for host-plant resistance against noctuid larvae

Two major orthodihydroxy phenolics ofLycopersicon esculentum, rutin and chlorogenic acid, have previously been identified as potential sources of host-plant resistance against the tomato fruitwormHeliothis zea. We report here the possible incompatibility of these chemically based resistance factors with viral control ofH, zea. We have found that both rutin and chlorogenic acid significantly inhibited the infectivity of nuclear polyhedrosis viruses. Chlorogenic acid, when added to tissue culture medium containing TN-368 ovarian cells, inhibited the infectivity of a multiply embedded virus (AcMNPV) by over 86%. Rutin or chlorogenic acid, when fed toH. zea, inhibited the infectivity of a singly embedded nuclear polyhedrosis virus (HzSNPV), with the greatest degree of inhibition occurring at low doses of viral inoculum. Additionally, the ingestion of these phytochemicals significantly prolonged the survival time of virally infectedH. zea larvae. These results suggest that the effectiveness of nuclear polyhedrosis viruses in controllingH. zea populations may be adversely affected by varieties ofL. esculentum with significant levels (eg. 3.5 mol/g wet weight) of rutin or chlorogenic acid.     

Comparison of anacardic acid biosynthetic capability between insect-resistant and-susceptible geraniums

The garden geranium (Pelargonium xhortorum) has been shown to secrete anacardic acids in the form of a viscous sticky exudate from tall glandular trichomes, and this exudate provides a sticky trap defense against small pest species. The anacardic acids from genetically related pest-resistant and -susceptible plants have been characterized, and resistance has been shown to depend upon the presence of5 unsaturated anacardic acids. In this study, the biosynthesis of these anacardic acids was comparatively investigated by incubating [¹⁴C]methyl palmìtate, margarate, stearate, oleate and linoleate on floral buds of resistant and susceptible plants. In addition, the incorporation of [¹⁴C]valine, -isoleucine, and -leucine into anacardic acids was also studied. Nineteen anacardic acids were quantitated utilizing an improved HPLC technique. Fatty acids and, to a much lesser extent, amino acids were incorporated into anacardic acids. There are at least two pathways of biosynthesis operating: direct elongation, and -oxidation with reincorporation of the [¹⁴C]acetate, the latter being more prevalent in the resistant plant. The amino acids were processed into branched chain anacardic acids, isoleucine being the precursor of the anteiso compounds, and valine the iso branched ones. The major difference between resistant and susceptible plants was the ability of resistant plants, but not the susceptible plants, to synthesize5 unsaturated anacardic acids. Both types of plants were capable of directly incorporating¹⁴C-labeled fatty acid methy esters into anacardic acids regardless of the plant's normal anacardic acid composition, thus bypassing the plant's tightly controlled regulation of the chemical structures of anacardic acids. No evidence was found for5 desaturation of saturated anacardic acids. A revised biosynthesis scheme is presented.     

Herbivore-Induced Defenses in Tomato Plants Enhance the Lethality of the Entomopathogenic Bacterium, <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> var. <Emphasis Type="Italic">kurstaki</Emphasis>

Plants can influence the effectiveness of microbial insecticides through numerous mechanisms. One of these mechanisms is the oxidation of plant phenolics by plant enzymes, such as polyphenol oxidases (PPO) and peroxidases (POD). These reactions generate a variety of products and intermediates that play important roles in resistance against herbivores. Oxidation of the catecholic phenolic compound chlorogenic acid by PPO enhances the lethality of the insect-killing bacterial pathogen, Bacillus thuringiensis var. kurstaki (Bt) to the polyphagous caterpillar, Helicoverpa zea. Since herbivore feeding damage often triggers the induction of higher activities of oxidative enzymes in plant tissues, here we hypothesized that the induction of plant defenses would enhance the lethality of Bt on those plants. We found that the lethality of a commercial formulation of Bt (Dipel® PRO DF) on tomato plants was higher if it was applied to plants that were induced by H. zea feeding or induced by the phytohormone jasmonic acid. Higher proportions of H. zea larvae killed by Bt were strongly correlated with higher levels of PPO activity in the leaflet tissue. Higher POD activity was only weakly associated with higher levels of Bt-induced mortality. While plant-mediated variation in entomopathogen lethality is well known, our findings demonstrate that plants can induce defensive responses that work in concert with a microbial insecticide/entomopathogen to protect against insect herbivores.     

Alleviation of α-tomatine-induced toxicity to the parasitoid,Hyposoter exiguae,by phytosterols in the diet of the host,Heliothis zea

The solitary, endoparasitic ichneumonid,Hyposoter exiguae (Viereck) is a parasitoid of the tomato fruitworm,Heliothis zea (Boddie). However, the parasitoid is deleteriously affected by the tomato glycoalkaloid, -tomatine, ingested from hosts (H. zea) fed on artificial diets or semipurified extracts of tomato plants that contained -tomatine. -Tomatine causes prolonged larval development; disruption or prevention of pupal eclosion; deformation of antennal, abdominal, and genital structures; and reduction in adult weight and longevity of the parasitoid. These toxic effects are exacerbated when the dietary dose of -tomatine is increased from 12 mol to 20 mol/g dry wt of diet. However, the toxicity of -tomatine is attenuated in parasitoids reared from hosts fed on artificial diets that contain equimolar or supramolar amounts of 3-OH-sterols admixed with -tomatine. Further, the toxicity of extracts from the foliage of different cultivars of tomatoes toH. exiguae is contingent upon the composite levels of -tomatine and total phytosterol (free and esterified). Cultivars with a low total sterol-tomatine ratio are more toxic toH. exiguae. The possible mode of action of -tomatine toxicity toH. exiguae by disruption of sterol metabolism is discussed.     

Growth inhibitors in tomato (Lycopersicon) to tomato fruitworm (Heliothis zea)

Several compounds that retard the larval growth of the tomato fruitworm,Heliothis zea (Boddie) have been isolated and identified from tomato leaves,Lycopersicon esculentum Mill. The major allelochemics are -tomatine (I), chlorogenic acid (II), rutin (III), and a new caffeyl derivative of an aldaric acid (IV). The isolation, analyses, and toxicity of these compounds toH. zea are presented.Reference to a company and/or product named by the department is only for purposes of information and does not imply approval or recommendation of the product to the exclusion of others which may also be suitable     

Mortality and inhibition ofHelicoverpa zea Egg parasitism rates byTrichogramma in relation to trichome/methyl ketone-mediated insect resistance ofLycopersicon hirsutum f. glabratum,accession PI 134417

The glandular trichome/methyl ketone-mediated insect resistance of the wild tomato,Lycopersicon hirsutum f. glabratum C.H. Mull, accession PI 134417, toManduca sexta (L.) andLeptinotarsa decemlineata (Say) was shown to affect adverselyTrichogramma pretiosum Riley. AdultT. pretiosum were killed by direct contact with PI 134417 foliage and by exposure to its volatiles. This effect was greatly reduced or eliminated by removing the exudate of the glandular trichomes from the foliage. 2-Tridecanone, a principal consituent of the foliar glandular trichomes of PI 134417, was toxic to adultT. pretiosum at concentrations similar to those associated with PI 134417 foliage. Incubation of parasitizedHelicoverpa (=Heliothis) zea (Boddie) eggs on PI 134417 foliage or 2-tridecanone-treated filter paper significantly reduced the proportion of eggs producing adult parasitoids. Similarly, incubation of parasitizedH. zea eggs on filter paper treated with 2-undecanone, another constituent of the glandular trichomes of PI 134417, caused an increase in the percentage of host eggs containing dead parasitoid pupae.     

Kairomones and their use for management of entomophagous insects: II. Mechanisms causing increase in rate of parasitization byTrichogramma spp.

When the effect of the kairomone, tricosane, on parasitization byTrichogramma achaeae Nagaraja and Nagarkatti of eggs ofHeliothis zea (Boddie) was studied in petri dish tests, the greatest percentage parasitization ( = 64%) was obtained if the entire filter paper was treated. Treatment of smaller areas (about the eggs) resulted in decreased parasitism. In the greenhouse, highest parasitization ( = 71%) byT. pretiosum (Riley) ofH. zea eggs placed on pea seedlings grown in pie pans was obtained if the whole pan was treated; lowest parasitism ( = 29%) occurred when the pans were untreated. Parasitization was intermediate ( = 52%) in other pans treated only at selected spots. Dissections ofH. zea eggs collected from kairomonetreated and untreated field plots revealed that eggs ofTrichogramma spp. were more efficiently distributed (less superparasitism) among host eggs in treated plots. These kairomones increase parasitization ofTrichogramma spp. by releasing and continuously reinforcing an intensified searching behavior rather than by attracting and guiding the parasite directly to the host.Hymenoptera: Trichogrammatidae.In cooperation with the University of Georgia College of Agriculture Experiment Station, Coastal Plain Station, Tifton, Georgia.Mention of a proprietary product in this paper does not constitute an endorsement of this product by the USDA.     

Constraints to herbivore-induced systemic responses: bidirectional signaling along orthostichies in Nicotiana attenuata

We investigated the impact of leaf vascular connections on systemically transmitted herbivore-induced gene expression in Nicotiana attenuata. Although systemic signaling is clearly associated with the plant vascular system, few studies consider vascular architecture when measuring systemically induced defenses. N. attenuata is a plant with dispersed phyllotaxis approximating 3/8 in the rosette stage of growth. We mimicked Manduca sexta herbivory by introducing larval regurgitant to wounds produced with a standardized continuous mechanical wounding and investigated mRNA accumulation of genes. Herbivory in N. attenuata induces the expression of genes coding for a proteinase inhibitor protein (PI), threonine deaminase (TD, EC 4.3.1.19), a luminal-binding protein (BiP), and an -dioxygenase (-DOX). We measured the systemic response of sink leaves when orthostichous (growing at an angular distance of 0 degrees) source leaves were treated, and vice versa, and compared it to the systemic response of leaves growing at the maximum angular distance of 180 degrees. Vascular architecture clearly controlled the intensity of systemic mRNA accumulation within the 4-hr time frame of the experiment. In addition, we found signal translocation to be bidirectional, travelling from source to sink as well as from sink to source leaves, which argues against a phloem-based assimilate-linked signal identity.     

Identification and bioassay of kairomones forHelicoverpa zea

Hexane extracts of leaves of 307 accessions from 73 host plant species ofHelicoverpa zea were analyzed by gas chromatography (GC) and used forH. zea oviposition and neonate larvae orientation bioassays. The gas chromatographic (GC) retention times of compounds statistically associated with behavioral activity were identified by correlation of GC peak area with oviposition and larval orientation preferences. Although taxonomically diverse in their origin, compounds for study were purified from extracts of species of the genusLycopersicon, due to their relative abundance. The structures of eight long-chain alkanes associated with oviposition preference were assigned by mass spectrometry, and the structures of five similarly associated organic acids and a terpenoid alkene were identified by¹H and¹³C nuclear magnetic resonance spectroscopy. The structures of a number of other phytochemicals from the plant leaves were identified for comparative purposes, including a previously unknown terpene, 7-epizingiberene. Bioassays were performed on the isolated acids and on the alkane wax fractions of severalLycopersicon species, and significant differences were found in oviposition stimulation for both classes of compounds. Of the hundreds of compounds found in the extracts, none were observed to act as oviposition deterrents. The results of these bioassays may be useful in explaining the broad host range ofH. zea, as well as the process and evolution of host plant selection for oviposition.     

Kairomones and their use for management of entomophagous insects

Volatile chemicals emanating from an excretion (apparently meconium) and abdominal tips of femaleHeliothis zea (Boddie) moths mediated increased rates of parasitization ofH. zea eggs byTrichogramma pretiosum Riley. A blend of synthetic chemicals, consisting of hexadecanal, (Z)-7-hexadecenal, (Z)-9-hexadecenal, and (Z)-11-hexadecenal, which has been identified as the sex pheromone of and from the abdominal tip of femaleH. zea moths, also increased rates of parasitization ofH. zea eggs byT. pretiosum in greenhouse experiments. In addition, parasitization ofH. zea eggs by wildTrichogramma spp., in field plots of cotton,Gossypium hirsutum L., treated with a similar blend of chemicals, in Conrel fibers, was more than double that in untreated plots.Hymenoptera: Trichogrammatidae.Lepidoptera: Noctuidae.In cooperation with the University of Georgea College of Agriculture Experiment Stations, Coastal Plain Station, Tifton, Georgia 31793.Mention of a proprietary product does not constitute endorsement by the USDA.     

Sex pheromone specificity inHeliothis

Electric grid traps baited withHeliothis subflexa (Guenée),H. virescens (F.), orH. zea (Boddie) females captured conspecific males with few exceptions.Heliothis subflexa females reduced the attraction ofH. virescens andH. zea males when used as bait simultaneously with females of either of these two species. Backcrosses were made withH. virescens males and female hybrids from a cross betweenH. subflexa females andH. virescens males. The backcross (BC) females andH. virescens females attracted approximately equal numbers ofH. virescens males in field traps. BC males released in field cages were attracted toH. virescens females and to the synthetic pheromone ofH. virescens. When laboratory-reared maleH. virescens, BC males,H. subflexa males, and F₁ hybrid males were exposed to the synthetic pheromone ofH. virescens in Plexiglas wind tunnels,H. virescens males and BC males responded to the pheromone, butH. subflexa and F₁ hybrid males did not. The peak activity of bothH. subflexa andH. zea males occurred approx. 4 hr after sunset. MaleH. zea were active throughout most of the night; maleH. virescens were most active approx. 6 hr after sunset.This paper reports the results of research only. Mention of a pesticide or of a commercial or proprietary product in this paper does not constitute a recommendation for use by the U.S. Department of Agriculture nor does it imply registration under FIFRA as amended.Lepidoptera: Noctuidae.