Secondary plant metabolites, those believed not be primary for sustaining the life of an organism, have been an important and growing area of research in recent years. The areas of interest have focused on interactions involving chemicals metabolized by plants, insect herbivores, parasitoids of herbivores, and plant pathogens. This focus is attributed to the expanding awareness and need to prevail upon biological controls to keep plant pests in check in man made environments.

It has been shown that chemical pathways are induced in plants in response to attack by insect herbivores and pathogens. Attack by insect herbivores activates the octadecanoid pathway via lipoxygenation of linolenic acid (LOX) which in turn produces jasmonic acid. Jasmonic acid serves as a signal for expression of a number compounds such as proteinase inhibitor (PINII), polyphenol oxidase and steroid glycoalkaloids that appear to contribute to plant resistance against many insect attackers and some pathogens. Fungal, bacterial and viral pathogen infections induces a different biochemical pathway that produces salicylic acid which in turn signals the formation of pathogenesis-related (PR or P4) proteins, and phytoalexins. This pathway has been shown to confer resistance to many pathogens and some insects. These pathways lead to systemic responses by the plant respectively called induced resistance (IR) and systemic acquired resistance (SAR). These induced plant defenses can cause reciprocal-induced resistance as well as signaling conflicts, which may compromise resistance to other pests.

Secondary compounds produced by plants in response to insect herbivory additionally include volatiles that attract parasitic and predatory insects. These volatiles appear to have evolved secondarily from plant defense responses, and have been shown to be clear, specific and emitted at a time when insects are foraging, important features for attracting specific parasitoids and predators.

Research in these areas could potentially lead to inducement in plants of SAR and IR in a vaccine form, as well as development of semiochemical products that could be placed in a field to attract parasitoids and predators of the crops common insect herbivores. Gene modification might allow plants to produce these chemicals without inducement. There are pros and cons to these possibilities.
 
 

Much research has been focused in recent years on the interactions that occur between insect herbivores, plant pathogens and their hosts. This research has revealed many interesting and valuable responses that are used by plants to limit damage that can occur from insect feeding and pathogen infection. Additionally, tritropic interactions have been exposed that show the ability of many plants to bring on additional forces to help them combat herbivore attack, in the form of herbivore parasitoids. This paper views this research as a body to fully utilize the information in formulating future development of products, practices and gene modification that can assist man in controlling herbivore and pathogen attack on plants of economic value.
 
 

Specific chemical pathways have been shown to be induced in plants as a result of insect herbivore feeding or attack by pathogens (1). The consequences of wounding from insect herbivory and infection can be different, although both can trigger oxidative reaction in the plant that contribute to induced responses and symptoms. Insect herbivory can trigger the octadecanoid pathway via lipoxygenation of linolenic acid (LOX) which in turn produces jasmonic acid. Jasmonic acid (JA) serves as a signal for expression of a number compounds such as proteinase inhibitor (PINII), polyphenol oxidase and steriod glycoalkaloids. These appear to contribute to plant resistance

against many insect attackers and some pathogens. The consequence of pathogen infection can induce the production of salycylic acid and in turn the expression of SAR genes, production of pathogenisis-related (PR or P4) protein, and phytoalexins. These two very different plant responses have been coined respectively induced resistance (IR) and systemic acquired resistance (SAR).

The first step in the development of SAR is the recognition of pathogen infection by a plant. Once the plant reacts to the pathogen, signals are released that trigger resistance in adjacent as well as distant tissues. Currently there is no common denominator that can be used to group inducing pathogens, and not all plant/ pathogen interactions lead to SAR inducement. The markers that correlate with SAR initiation are cDNAs that are expressed in distant tissues in SAR induced plants. Nine families of genes have been induced in uninfected leaves of induced plants. Along with being reliable markers, many of these genes apparently have an active role in resistance. Several of the SAR genes encode antimicrobial activities and the expression of certain SAR genes in plants imparts a pathogen tolerance indicating a direct role in maintaining SAR. Conceivably, each taxonomic group of plants may have evolved its own set of SAR genes in response to evolutionary pressure from a specific spectrum of pathogens (2).

Induced resistance denotes a change in a plant that causes a decrease in any measure of herbivore performance. The response is ubiquitous in plants, having been reported in over 100 plant species from approximately 30 families (5). Plant responses are in the form of secondary metabolite production largely induced by volicitin, a component of the oral secretions of feeding insect herbivores. These secondary metabolites are deleterious to the arthropods, and aid the plant in defense against attack.

Although there are two main pathways that have been shown, there is not a strict dichotomy of signaling by insects or pathogens. In general the expression of PINII can be associated with wounding and feeding by insect herbivores, and P4 expression with fungal and bacterial infection. However there is overlap in response pathways. Aphid Macrosiphum euphorbiae feeding on tomato plants Lycopersicon esculentum, induced P4 but not PINII, and infection by Pseudomonas syringae pv. Tomato (Pst)induced both P4 and PINII(1). It is tempting to simplify the signaling responses according to insect vs. pathogen, but this is not the reality of what is indicated by the literature.

Stumbling blocks to simple, clear cut answers in regards to signaling pathways in plants and their interactions with herbivores and pathogens are numerous. Tomato plants fed on by noctuid Helicoverpa zea larvae caused leaf-systemic increases in resistance to both H. zea feeding and Pst. , and infection of leaflets by Pst increased leaf-systemic resistance for both H. zea and for Pst whereas infection of leaflets by Phytophthora infestans had no effect on resistance of leaflets to H. zea. Additionally it was shown that introduction of benzothiadiazole (BTH), a salicylate mimic, and inducer of SAR into plants compromised their ability to express PINII and induced resistance to Pst but improved suitability of leaflets for H. zea (4).

The specificity of IR to various organisms has been observed in tomato plants. Specificity of the range of organisms affected by a given induced response showed that restricted feeding by Helicoverpa zea increased the resistance of tomato plants to four different organisms, an aphid species, mite species, noctuid species and a phytopathogen (5). Thus, causing a generalized resistance against diverse organisms. Prior aphid feeding resulted in better sources of food for a noctuid species, indicating that there is some specificity in the elicitation of chemical responses in tomato to different herbivore species feeding.

There is evidence from in vivo studies that the SAR and IR pathways can interfere with one another. Tomato plants grown in the field treated with BTH, JA or BTH/JA, to stimulate SAR and IR, showed an inverse mobilization of plant defenses. SAR induced plants exhibited greater suitability to caterpillar Spodoptera exigua larvae than IR induced or control plants. BTH/JA treated plants exhibited a greater abundance of Pst lesions than those treated with just BTH, indicating that JA partially reversed the protective effect of BTH against Pst (8). This negative cross-talk between responses of the SAR and IR pathways was demonstrated again by reducing phenylpropanoid biosynthesis which in turn reduces the expression of SAR. Tomato plants with reduced SAR exhibited resistance to Heliothis virescens larval feeding. Likewise, larval resistance was reduced in plants with enhanced SAR (6).

Additional evidence shows that JA-induced responses although benefiting plants under insect attack, are costly and result in reduced seed yield in plants not under insect pressure (7). Evidence of the cost of induced SAR responses are not available, but similar processes most likely will apply to plant responses to pathogen attack. This coupled with the evidence above indicate that attempts to artificially stimulate, or genetically modify plants to express SAR or IR on demand may not result in the expected and desired outcome. The evidence available points toward a number of possible negative scenarios such as, plants resistant to pathogens, but very susceptible to herbivore attack, and the reverse situation, and plants wasting resources protection themselves from non-existent threats. Additionally, the specificity in responses indicates that there are multiple systems of response in tomato, and likely other plant species and more studies are necessary to get a better understanding of the coordination by plants of multiple defense systems against multiple biotic threats.

The octadecanoid pathway, in addition to inducing secondary metabolites that can be deleterious to herbivores and pathogens, releases volatiles of two types, green leafy and herbivore-induced. Green leafy are released from storage or synthesized from stored intermediates. These constitutive and green leafy volatiles are released immediately, whether damage is caused mechanically or herbivore induced, and levels drop when damage stops. The release of different volatiles (mostly terpenoids and indole) only

occurs several hours after the damage starts. Insect herbivore feeding stimulates the de novo production and release of these herbivore-induced volatiles (8). This response is systemic, as undamaged leaves of injured plants also emit herbivore-induced volatiles. Chemical labeling experiments established that the systemic volatiles are synthesized at the site of release, suggesting that a mobile chemical messenger is transported from the damage location to distal, undamaged leaves to trigger synthesis and volatile release. Cotton plants release volatiles from undamaged leaves after 2 to 3 days of continuous larval feeding. All systemically released compounds are induced by caterpillar damage and are not released in significant amounts by undamaged plants. Additionally, plants damaged with a razor did not release systemic compounds (9). In response to insect feeding on the leaves, cotton plants emitted green leafy volatiles and various volatiles produced de novo. Plants treated with insect regurgitant also produced both types of volatiles, whereas undamaged control plants produced much lower levels of volatiles (3).

The production of volatiles has been shown to attract herbivore parasitoids. Tomato plants in vivo treated with jasmonic acid, stimulating an IR response, contained twice as many parasitoid larvae Hyposoter exiguae (found developing on naturally occurring lepidopteran pest, Spodoptera exigua), as untreated plants (10). In response to insect feeding on cotton plant leaves, elevated levels of volatiles are released. Many of these volatiles are biosynthesized de novo following insect damage (3).

The release of herbivore-induced volatiles from insect injured plants allows them to enlist the aid of natural enemies, guiding them with chemical cues to the herbivore. Turlings et al (8) presents and confirms aspects of plant chemical cues that appear most relevant for functioning as an alert system to attract parasitiods. The signal must be clear enough that the natural enemy can perceive, and distinguish it from background noise. It should be specific, so it can be counted on as a reliable indicator of a suitable host or prey, and it should be emitted at a time when the natural enemy is foraging.

The evidence presented strongly supports the view that herbivore-induced emissions of volatiles serve to attract natural enemies of the herbivores. Clarity and timing appear to be perfectly attuned with the parasitoids’ biology. Clarity indicated quantitatively, shows that plants fed on by insects emit far more terpinoids than plants mechanically damaged. One corn seedling can emit several micrograms of a defense-related volatile per hour. This is a much greater amount than normally seen in insect pheromone communication, where a few nanograms of sex pheromone per hour can be detected from a distance by potential mates (8).

Signals are emitted soon after damage from a herbivore begins, and are emitted strongest diurnally, the time the parasitoid is actively foraging. Chemical labeling studies have established that the compounds released in much greater quantities during the day and specifically in response to insect damage are synthesized de novo and are not stored in the plant, therefore showing to be the volatiles that are herbivore-induced.

Observed nonspecific nature of many of the signals can be explained from the plants perspective; as long as the correct natural enemies are attracted, the plants do not suffer is other parasitoids and predators "mistakenly" trace the odors as well. Specificity of chemical signals, in some cases, has been demonstrated, based on quantifiable differences. Tobacco, cotton, and corn plants each produced distinct volatile blends in response to damage by two closely related herbivore species, Heliothis virescens and Helicoverpa zea. The specialist parasitic wasp Cardiochiles ngriceps exploits these differences and identifies infestation by its host H. virescens (11). This sophisticated plant/ herbivore/ parasitoid interaction reinforces the belief that herbivore-induced plant signals are important for the foraging success of parasitoids and for the plants defense.

Specificity on the part of the wasp has also been demonstrated in the ability to find a suitable host when that host feeds on multiple plant species. The wasps have overcome this obstacle by developing the ability to learn chemical cues associated with the presence of a host. The chemicals to which a female wasp is exposed during interactions with her host familiarize her with particular host location cues. A successful host experience increases the wasp’s responsiveness to host-associated chemicals. For example, female wasps that have had an ovipositing experience on a plant/host complex are more likely to have appropriate landing responses on host-damaged plants than those that have not had an ovipositing experience on that particular plant/host complex (12).

Induced resistance and release of parasitoid attracting volatiles has been shown to negatively affect parasitic wasps (10). Hyposoter exiguae, an endoparasitic wasp, reared in vitro on Spodoptera exigua larvae fed on induced foliage show reduced performance on caterpillar hosts grown on induced compared to those grown on control foliage. S. exigua larvae feeding on induced plants have reduced growth rates compared to larvae feeding on control plants. All in all, production of volatiles that influence attraction of natural enemies may be vital to the plant’s defense system, overcoming the negative influences of induction on wasp performance.

Release of volatiles by insect damaged and chemically induced plants can also serve as a means by which insect herbivores locate possible host plants. Female Colorado potato beetle adults were attracted to potato plants 24 hours after damage by larval feeding, or after foliage was treated with regurgitant from Colorado potato beetle or cabbage looper larvae, or after foliage was treated with methyl jasmonate to induce IR (13). These findings indicate an important role of potato plant, and potentially other plant species odorants that are induced by herbivory in Colorado potato beetle, and other insects, host finding behavior. Conversely, it has been shown that volatiles released in response to high-density herbivore attack can repel other like insects. For example, wheat seedlings without herbivore damage attract aphids, whereas odors released from wheat seedling with a high density of aphids repel other aphids (7).

The production of terpenoids such as those in herbivore-induced volatiles produced by plants is metabolically costly. Terpinoids are more expensive to manufacture per gram that most other primary and secondary metabolites due to the need for extensive chemical reduction in their production. When herbivory levels are low, induced plants produced fewer seeds than their non-induced counterparts. With intermediate herbivory, chemically induced plants experienced less feeding on the foliage and have a higher fitness level than non-induced, insect-damaged plants (12). This is a fine line to walk for the plant in terms of accurately interpreting and reacting to external information and activating defense responses appropriately so as not to unnecessarily waste energy producing defenses that could cause lowered fitness and production levels.

It is clear from all of the above-presented evidence that plant volatiles interact with many tropic levels. As in the case of IR and SAR responses, there are many varying reactions, of what at first glance appear to be predictable responses, but after further investigation are not. It would be tempting to take advantage of the knowledge that these herbivore-induced volatiles attract parasitoids, and genetically modify plants to produce these compounds all the time, or on demand. This would likely, as has been shown, lower the economic production level of the plant. It would also be tempting to create a pheromone lure that would attract the parasitoids to a field at the onset of a predicted herbivore epidemic, but it must first be know if the volatile might have the reverse effect of attracting the herbivore, creating a larger problem.

Recent work suggests that herbivore-induced volatile release, as well as other plant defense responses are triggered by an active component or components associated with the feeding herbivore which allows the plant to respond differently to mechanical wounding and damage caused by chewing insects. In cotton, induced volatiles that are synthesized in response to wounding are released in greater quantities as a result of caterpillar feeding than as a result of mechanical damage alone (3). Two compounds introduced by insect feeding have been identified that elicit herbivore-induced volatile release. Beta- glucosidase, present in the regurgitant of Pieris brassicae caterpillars, triggers the same volatile release as feeding by the larvae on cabbage plants. The enzyme activity does not appear to be plant derived, as its activity is retained when caterpillars are fed on a beta-glucosidase free diet. It appears that the enzyme acts to cleave sugars coupled to organic compounds that they become more volatile and are released (13). The second compound, volicitin, was isolated from insect oral secretions and identified as a key component in plant recognition of damage from insect herbivory (14).

Volicitin was isolated from the regurgitant of Spodoptera exigua larvae and identified as N-(17-hydroxylinolenoyl)-L-glutamine. Its potency as an elicitor of maize volatiles that attract parasitoids was confirmed by incubating maize seedling in very low concentration of pure natural volicitin, releasing relatively large amount of terpenoids. These seedlings were highly attractive to the parasitoid Microplitis croceipes (15).

Chemical analysis has established that the fatty acid portion of volicitin is plant derived, although volicitin has not been found in plant tissues. The biogenetic origin of volicitin shows that the linolenic acid portion (an essential fatty acid in the diet of Lepidoptera) is plant derived, and the caterpillar synthesizes this elicitor by adding a hydroxyl group and glutamine of insect origin. Thus although the precursor of volicitin is obtained from plants, the bioactive product has only been found in the caterpillar. This strongly suggests that these molecules play an important yet still unknown role in metabolism or some other process critical to the life of the herbivorous insects.(12). Ironically, these insect-catalyzed modifications to linolenic acid are critical for the biological activity that triggers the release of plant volatiles, which in turn attract natural enemies of the caterpillar(16).

The mechanisms that regulate the synthesis and release of plant volatiles are poorly understood. Since different herbivore feeding can induce different blends of volatiles, it would logically follow that there are a number of elicitor compounds yet to be identified in insect oral secretions. The actual mechanisms involved in the triggering of biosynthesis and release of volatiles is of yet unknown. Do they have anything to do with actual triggering of the octadecanoid signaling pathway, or the production of jasmonic acid, or activation of an oxidative burst, all of which are associated with the wounding of plant tissue. Additionally we do not know how elicitors are involved with the evidence of systemic volatile release. Does the herbivore-induced elicitor act as a mobile messenger, triggering whole plant volatile synthesis? Furthermore, why do herbivores produce compounds that activate plant chemical defenses? what function, if any, do these compounds serve in herbivore metabolism? These are all good questions yet to be answered as related to the role that herbivore induced volatile elicitors play in the plant/ herbivore/ parasitoid tritropic relationship.
 
 

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