Direct Defense Responses Research Articles

Dynamic distress calls: volatile info chemicals induce and regulate defense responses during herbivory.

Plants are continuously threatened by a plethora of biotic stresses caused by microbes, pathogens, and pests, which often act as the major constraint in crop productivity. To overcome such attacks, plants have evolved with an array of constitutive and induced defense mechanisms- morphological, biochemical, and molecular. Volatile organic compounds (VOCs) are a class of specialized metabolites that are naturally emitted by plants and play an important role in plant communication and signaling. During herbivory and mechanical damage, plants also emit an exclusive blend of volatiles often referred to as herbivore-induced plant volatiles (HIPVs). The composition of this unique aroma bouquet is dependent upon the plant species, developmental stage, environment, and herbivore species. HIPVs emitted from infested and non-infested plant parts can prime plant defense responses by various mechanisms such as redox, systemic and jasmonate signaling, activation of mitogen-activated protein (MAP) kinases, and transcription factors; mediate histone modifications; and can also modulate the interactions with natural enemies via direct and indirect mechanisms. These specific volatile cues mediate allelopathic interactions leading to altered transcription of defense-related genes, viz., proteinase inhibitors, amylase inhibitors in neighboring plants, and enhanced levels of defense-related secondary metabolites like terpenoids and phenolic compounds. These factors act as deterrents to feeding insects, attract parasitoids, and provoke behavioral changes in plants and their neighboring species. This review presents an overview of the plasticity identified in HIPVs and their role as regulators of plant defense in Solanaceous plants. The selective emission of green leaf volatiles (GLVs) including hexanal and its derivatives, terpenes, methyl salicylate, and methyl jasmonate (MeJa) inducing direct and indirect defense responses during an attack from phloem-sucking and leaf-chewing pests is discussed. Furthermore, we also focus on the recent developments in the field of metabolic engineering focused on modulation of the volatile bouquet to improve plant defenses.

A Cognitive Behavioral Defense Mechanism of Plants Against Herbivory

Plants interactions with animals, insects, and pathogens is the need of ecosystem. Herbivory is the most critical part of food chain. In an ecosystem, the relationship between prey and predators and mutualism are inevitable breathing junctions. Plants are eaten by herbivores, but plants in return have evolved in such a way which affect herbivores and other organisms that come in contact with them at every level from basic biological interaction to genetics. In other words, plants have adapted strategies to defend themselves against herbivory.Mainly, there are two defense strategies, 1) direct defense responses and 2) indirect defense responses. The former one includes production of such enzymes which reduce the digestibility of consumed food,reallocation of resources, morphological adaptations, and the production of specialized bioactive molecules such as alkaloids, phenolics, terpenoids, glucosinolates.The later one includes the production of volatiles and extrafloral nectars to attract pollinators and other microorganisms to reduce enemy pressure and for their own benefit, pollination. All these strategies are to intoxicate or repel the insect herbivores and to reduce the herbivory.

Effects of Salicylic Acid Concentration and Post-Treatment Time on the Direct and Systemic Chemical Defense Responses in Maize (Zea mays L.) Following Exogenous Foliar Application.

Salicylic acid (SA) plays a critical role in allergic reactions of plants to pathogens and acquired systemic resistance. Thus far, although some research has been conducted on the direct effects of different concentrations of SA on the chemical defense response of treated plant parts (leaves) after at multiple post-treatments times, few research has reported on the systematic effects of non-treated parts (roots). Therefore, we examined direct and systemic effects of SA concentration and time following foliar application on chemical defense responses in maize variety 5422 with two fully expanded leaves. In the experiments, maize leaves were treated with different SA concentrations of 0.1, 0.5, 1.0, 2.5, 5.0 mM, and then, the presence of defense chemicals and enzymes in treated leaves and non-treated roots was measured at different time points of 3, 12, 24, 48, 72 h following SA foliar application. The results showed that direct and systemic effects of SA treatment to the leaf on chemical defense responses were related to SA concentration and time of measurement after spraying SA. In treated leaves, total phenolics content increased directly by 28.65% at the time point of 12 h following foliar application of 0.5 mM SA. DIMBOA (2,4-dihydroxy-7-methoxy-2H, 1, 4-benzoxazin-3 (4H)-one) content was directly enhanced by 80.56~551.05% after 3~72 h following 0.5~5.0 mM SA treatments. Polyphenol oxidase and superoxide dismutase activities were directly enhanced after 12~72 h following 0.5~5.0 mM SA treatments, whereas peroxidase and catalase activities were increased after 3~24 h following application of 1.0~5.0 mM SA. In non-treated roots, DIMBOA content and polyphenol oxidase activity were enhanced systematically after 3~48 h following 1.0~5.0 mM SA foliar treatments. Superoxide dismutase activities were enhanced after 3~24 h following 0.5~2.5 mM SA applications, but total phenolics content, peroxidase and catalase activity decreased in some particular concentrations or at the different times of measurement in the SA treatment. It can be concluded that SA foliar application at 1.0 and 2.5 mM produces strong chemical defense responses in maize, with the optimal induction time being 24 h following the foliar application.

Volatiles and hormones mediated root-knot nematode induced wheat defense response to foliar herbivore aphid

Plant root-leaf communication signals are critical for plant defense. Numerous studies show that belowground organisms can alter systemically resistance traits in aboveground parts against herbivores. However, there are limited studies on root-knot nematode-aphid interaction. Moreover, the impact of nematode's initial density and infection time on plant defense is poorly understood. Here we aim to examine the induced defense responses by root-knot nematode Meloidogyne incognita against aboveground feeding aphid Sitobion avenae in wheat. Further, we investigated the influence of the nematode infection density as well as the length of infection in these interactions. We tested the direct and indirect defense responses triggered by M. incognita against S. avenae as well as how the responses affect the preference of Harmonia axyridis. Plant volatiles and hormones were determined to explore plant defense mechanisms that mediate aboveground-belowground defense. The photosynthetic rate was tested to examine plant tolerance strategy. We found that, both low and high densities M. incognita root infection at 7 days post inoculation (dpi) reduced the feeding of the aphid S. avenae. Behavioral assay showed that H. axyridis preferred plants co-damaged by both M. incognita and S. avenae at 7 dpi. M. incognita infection induced the changes of jasmonic acid, salicylic acid and volatile content, which mediated plant response to S. avenae. Furthermore, photosynthetic rate in wheat increased at 5 dpi under 300 M. incognita or 1000 M. incognita infection. These results suggest that plant roots induced multiple defense strategies against foliar herbivores as damages increased. Our study provides evidence of a complex dynamic response of wheat aboveground defense against aphids in response to belowground nematode damage on a temporal scale.

Comparative analyses of responses to exogenous and endogenous antiherbivore elicitors enable a forward genetics approach to identify maize gene candidates mediating sensitivity to herbivore-associated molecular patterns.

Crop damage by herbivorous insects remains a significant contributor to annual yield reductions. Following attack, maize (Zea mays) responds to herbivore-associated molecular patterns (HAMPs) and damage-associated molecular patterns (DAMPs), activating dynamic direct and indirect antiherbivore defense responses. To define underlying signaling processes, comparative analyses between plant elicitor peptide (Pep) DAMPs and fatty acid-amino acid conjugate (FAC) HAMPs were conducted. RNA sequencing analysis of early transcriptional changes following Pep and FAC treatments revealed quantitative differences in the strength of response yet a high degree of qualitative similarity, providing evidence for shared signaling pathways. In further comparisons of FAC and Pep responses across diverse maize inbred lines, we identified Mo17 as part of a small subset of lines displaying selective FAC insensitivity. Genetic mapping for FAC sensitivity using the intermated B73 × Mo17 population identified a single locus on chromosome 4 associated with FAC sensitivity. Pursuit of multiple fine-mapping approaches further narrowed the locus to 19 candidate genes. The top candidate gene identified, termed FAC SENSITIVITY ASSOCIATED (ZmFACS), encodes a leucine-rich repeat receptor-like kinase (LRR-RLK) that belongs to the same family as a rice (Oryza sativa) receptor gene previously associated with the activation of induced responses to diverse Lepidoptera. Consistent with reduced sensitivity, ZmFACS expression was significantly lower in Mo17 as compared to B73. Transient heterologous expression of ZmFACS in Nicotiana benthamiana resulted in a significantly increased FAC-elicited response. Together, our results provide useful resources for studying early elicitor-induced antiherbivore responses in maize and approaches to discover gene candidates underlying HAMP sensitivity in grain crops.

Extracellular DNA as an elicitor of broad-spectrum resistance in Arabidopsis thaliana

Like in mammals, the plant immune system has evolved to perceive damage. Damaged-associated molecular patterns (DAMPs) are endogenous signals generated in wounded or infected tissue after pathogen or insect attack. Although extracellular DNA (eDNA) is a DAMP signal that induces immune responses, plant responses after eDNA perception remain largely unknown. Here, we report that signaling defenses but not direct defense responses are induced after eDNA applications enhancing broad-range plant protection. A screening of defense signaling and hormone biosynthesis marker genes revealed that OXI1, CML37 and MPK3 are relevant eDNA-Induced Resistance markers (eDNA-IR). Additionally, we observed that eDNA from several Arabidopsis ecotypes and other phylogenetically distant plants such as citrus, bean and, more surprisingly, a monocotyledonous plant such as maize upregulates eDNA-IR marker genes. Using 3,3′-Diaminobenzidine (DAB) and aniline blue staining methods, we observed that H2O2 but not callose was strongly accumulated following self-eDNA treatments. Finally, eDNA resulted in effective induced resistance in Arabidopsis against the pathogens Hyaloperonospora arabidopsidis, Pseudomonas syringae, and Botrytis cinerea and against aphid infestation, reducing the number of nymphs and moving forms. Hence, the unspecificity of DNA origin and the wide range of insects to which eDNA can protect opens many questions about the mechanisms behind eDNA-IR.

Plant Lectins as Insecticidal Agents Against Cotton Leafworm Spodoptera littoralisand Their Potential Applications in Crop Protection

Background: The production of insecticidal proteins is one of the greatest vital direct defense responses in plants against the attack by phytophagous insects. One particular class of the entomotoxic proteins present in many plant species is lectins that is used against a wide range of important insects. Aim: The insecticidal activity of two lectins from seeds of white kidney bean (Phaseolus vulgaris) and soybean (Glycine max) on the growth, development and survival of the cotton leafworm, Spodoptera littoralis Boisd. (Lepidoptera: Noctuidae) was studied. Methods: Lectin products were isolated and purified using ammonium sulfate precipitation. Insecticidal activity, growth-inhibitory and physiological effects on the differential haemocyte count of lectin products were evaluated against S. littoralis. In vivo effects of lectin products on some S. littoralis enzymes were also determined. Results: Lectin contents in 250 g seeds were 700 and 500 mg in soybean and white kidney bean, respectively. The total protein concentrations were 160-163 and 118-120 mg/mL in soybean and white kidney bean, respectively. The insecticidal activity of lectin products exerted a strong effect on the larval weight gain and the reduction of the weight reached to 75.25%. Furthermore, the other developmental stages of pupation and adult formation were also affected. An elongated larval duration was noticed since larval duration was 24.8 days compared with control (12.0 days). Complete pupation (100%) was recorded in the control; however, a significant decrease in pupation was found to be 54.0% in treated larvae. Lectin products increased the average number of 123.9 haemocytes (123.9) compared to the control (94.4 haemocytes/field). In addition, lectin products inhibited the activity of total proteases, α-amylase, GST and total esterases. Conclusion: It can be concluded that the lectins have high potential in pest control strategies based on their activity towards the cotton leafworm, S. littoralis. These findings suggest that lectin products are interesting tools for integrated pest management in important agriculture crops.

The Induced Resistance Lexicon: Do's and Don'ts.

To be protected from biological threats, plants have evolved an immune system comprising constitutive and inducible defenses. For example, upon perception of certain stimuli, plants can develop a conditioned state of enhanced defensive capacity against upcoming pathogens and pests, resulting in a phenotype called 'induced resistance' (IR). To tackle the confusing lexicon currently used in the IR field, we propose a widely applicable code of practice concerning the terminology and description of IR phenotypes using two main phenotypical aspects: local versus systemic resistance, and direct versus primed defense responses. Our general framework aims to improve uniformity and consistency in future scientific communication, which should help to avoid further misinterpretations and facilitate the accessibility and impact of this research field.

Zucchini Plants Alter Gene Expression and Emission of (E)-β-Caryophyllene Following Aphis gossypii Infestation.

Zucchini (Cucurbita pepo L.) is widely cultivated in temperate regions. One of the major production challenges is the damage caused by Aphis gossypii (Homoptera: Aphididae), a polyphagous aphid, which can negatively affect its host plant, both directly by feeding and indirectly by vectoring viruses. To gain insights into the transcriptome events that occur during the zucchini–aphid interaction and to understand the early-to-late defense response through gene expression profiles, we performed RNA-sequencing (RNA-Seq) on zucchini leaves challenged by A. gossypii (24, 48, and 96 h post-infestation; hpi). Data analysis indicated a complex and dynamic pattern of gene expression and a transient transcriptional reconfiguration that involved more than 700 differentially expressed genes (DEGs), including a large number of defense-related genes. The down-regulation of key genes of plant immunity, such as leucine-rich repeat (LRR) protein kinases, transcription factors, and genes associated with direct (i.e., protease inhibitors, cysteine peptidases, etc.) and indirect (i.e., terpene synthase) defense responses, suggests the aphid ability to manipulate plant immune responses. We also investigated the emission of volatile organic compounds (VOCs) from infested plants and observed a reduced emission of (E)-β-caryophyllene at 48 hpi, likely the result of aphid effectors, which reflects the down-regulation of two genes involved in the biosynthesis of terpenoids. We showed that (E)-β-caryophyllene emission was modified by the duration of plant infestation and by aphid density and that this molecule highly attracts Aphidius colemani, a parasitic wasp of A. gossypii. With our results we contributed to the identification of genes involved in cucurbit plant interactions with phloem feeders. Our findings may also help pave the way toward developing tolerant zucchini varieties and to identify molecules for sustainable management of harmful insect populations.

Direct and indirect plant defenses induced by (Z)-3-hexenol in tomato against whitefly attack

Plants have evolved induced defense mechanisms against insect herbivore attacks. The application of plant elicitors can enhance the biological control of crop pests by inducing plant defense responses, and (Z)-3-hexenol (z3HOL) is recognized as a herbivore-induced plant volatile that plays an important role in the chemical communication between plants and pests. We investigated whether z3HOL can activate the defense mechanism of tomato (Solanum lycopersicum) against the whitefly Bemisia tabaci, one of the major insect pests of cultivated tomato. Exposure to z3HOL vapors in the glass jars decreased weight gain and oviposition in B. tabaci and negatively influenced the feeding behavior of B. tabaci by shortening the total feeding period and phloem ingestion and increasing the frequency of stylet puncture. Furthermore, exposure to z3HOL induced jasmonic acid- and salicylic acid-mediated defense responses and enhanced B. tabaci-induced volatile emissions in tomato. The elevated release of tomato volatiles increased the attraction of the parasitoid Encarsia formosa and improved its parasitism on B. tabaci. Our results indicate that z3HOL can serve as an elicitor that triggers direct and indirect defense responses against B. tabaci by modulating signaling pathways in tomato. These findings will not only help us to gain a better understanding of how z3HOL influences the crop community, but also provide new insights into the potential application of z3HOL in integrated pest management systems.

Induction of defensive proteins in Solanaceae by salivary glucose oxidase of Helicoverpa zea caterpillars and consequences for larval performance

Salivary glucose oxidase (GOX) is one of the most abundant salivary proteins in generalist caterpillar Helicoverpa zea. GOX has been hypothesized to benefit H. zea by modulating direct defense responses of plants. Although the function of this protein has been studied, its role remains unclear. The study aims to test the hypothesis that GOX induces similar defensive responses among Solanaceous plants, and has similar consequences for larval performance of H. zea. Using six different plants in Solanaceae, including tomato (Solanum lycopersicum cv. Better Boy and S. lycopersicum var. cerasiforme), bell pepper (Capsicum annuum cv. Revolution), habanero pepper (Capsicum chinense), tomatillo (Physalis philadelphica cv. Tamayo), and tobacco (N. benthamiana), we tested the impact of GOX on induction of two common defense proteins, trypsin protease inhibitors (TPI) and polyphenol oxidases (PPO), and on relative growth rate of H. zea larvae. We found that GOX specifically induced TPI activity in tomato and habanero pepper, and the level of defense protein depended on leaf location. In addition, prior application of GOX did not increase the performance of H. zea in any plant tested. Changes in performance in tomato and habanero pepper matched the induction of TPI. In summary, our findings indicate that GOX induces similar defense responses in some Solanacean plants, but largely depends on species/genotype of plant, and that the presence of GOX did not benefit larval H. zea by modulating direct defense responses of plants. Other mechanisms must be involved in driving the evolution of this salivary protein.

Morphophysiological basis of plant defense mechanisms against insect pests

Plants have been interacting with insects for several hundred million years, leading to the development of an intricate and dynamic defense system against insect herbivores. This defense system is further categorized as direct and indirect defense responses based on their expression. Specific or direct defensive responses are mediated by various morphological, biochemical and molecular mechanisms to counteract the effects of herbivore attack while indirect defense is mediated by releasing volatile mixtures to attract natural enemies of plant eating insect herbivores. The structural defense includes both morphological and anatomical traits viz., thorns, spines etc., whereas biochemical mechanisms of defense against the herbivores are wide-ranging, highly dynamic and includes production of different secondary metabolites-terpenes, phenolics and N-containing compounds. The molecular defense mechanisms include intracellular wound signalling viz., systemin signalling and jasmonate regulation of defense responsive genes. The defensive compounds are developed either constitutively or in response to plant damage, affecting insect herbivores’ feeding, development, and survival. In addition, plants also release volatile organic compounds which attract the herbivores’ natural enemies. These strategies either act independently or in conjunction with each other.

Molasses Grass Induces Direct and Indirect Defense Responses in Neighbouring Maize Plants.

Plants have evolved intricate defence strategies against herbivore attack which can include activation of defence in response to stress-related volatile organic compounds (VOCs) emitted by neighbouring plants. VOCs released by intact molasses grass (Melinis minutiflora), have been shown to repel stemborer, Chilo partellus (Swinhoe), from maize and enhance parasitism by Cotesia sesamiae (Cameron). In this study, we tested whether the molasses grass VOCs have a role in plant-plant communication by exposing different maize cultivars to molasses grass for a 3-week induction period and then observing insect responses to the exposed plants. In bioassays, C. partellus preferred non-exposed maize landrace plants for egg deposition to those exposed to molasses grass. Conversely, C. sesamiae parasitoid wasps preferred volatiles from molasses grass exposed maize landraces compared to volatiles from unexposed control plants. Interestingly, the molasses grass induced defence responses were not observed on hybrid maize varieties tested, suggesting that the effect was not simply due to absorption and re-emission of VOCs. Chemical and electrophysiological analyses revealed strong induction of bioactive compounds such as (R)-linalool, (E)-4,8-dimethyl-1,3,7-nonatriene and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene from maize landraces exposed to molasses grass volatiles. Our results suggest that constitutively emitted molasses grass VOCs can induce direct and indirect defence responses in neighbouring maize landraces. Plants activating defences by VOC exposure alone could realize enhanced levels of resistance and fitness compared to those that launch defence responses upon herbivore attack. Opportunities for exploiting plant-plant signalling to develop ecologically sustainable crop protection strategies against devastating insect pests such as stemborer C. partellus are discussed.

Transcriptome and Metabolome Reprogramming in Tomato Plants by Trichoderma harzianum strain T22 Primes and Enhances Defense Responses Against Aphids.

Beneficial fungi in the genus Trichoderma are among the most widespread biocontrol agents of plant pathogens. Their role in triggering plant defenses against pathogens has been intensely investigated, while, in contrast, very limited information is available on induced barriers active against insects. The growing experimental evidence on this latter topic looks promising, and paves the way toward the development of Trichoderma strains and/or consortia active against multiple targets. However, the predictability and reproducibility of the effects that these beneficial fungi is still somewhat limited by the lack of an in-depth understanding of the molecular mechanisms underlying the specificity of their interaction with different crop varieties, and on how the environmental factors modulate this interaction. To fill this research gap, here we studied the transcriptome changes in tomato plants (cultivar “Dwarf San Marzano”) induced by Trichoderma harzianum (strain T22) colonization and subsequent infestation by the aphid Macrosiphum euphorbiae. A wide transcriptome reprogramming, related to metabolic processes, regulation of gene expression and defense responses, was induced both by separate experimental treatments, which showed a synergistic interaction when concurrently applied. The most evident expression changes of defense genes were associated with the multitrophic interaction Trichoderma-tomato-aphid. Early and late genes involved in direct defense against insects were induced (i.e., peroxidase, GST, kinases and polyphenol oxidase, miraculin, chitinase), along with indirect defense genes, such as sesquiterpene synthase and geranylgeranyl phosphate synthase. Targeted and untargeted semi-polar metabolome analysis revealed a wide metabolome alteration showing an increased accumulation of isoprenoids in Trichoderma treated plants. The wide array of transcriptomic and metabolomics changes nicely fit with the higher mortality of aphids when feeding on Trichoderma treated plants, herein reported, and with the previously observed attractiveness of these latter toward the aphid parasitoid Aphidius ervi. Moreover, Trichoderma treated plants showed the over-expression of transcripts coding for several families of defense-related transcription factors (bZIP, MYB, NAC, AP2-ERF, WRKY), suggesting that the fungus contributes to the priming of plant responses against pest insects. Collectively, our data indicate that Trichoderma treatment of tomato plants induces transcriptomic and metabolomic changes, which underpin both direct and indirect defense responses.

Tetranins: new putative spider mite elicitors of host plant defense.

The two-spotted spider mite (Tetranychus urticae) is a plant-sucking arthropod herbivore that feeds on a wide array of cultivated plants. In contrast to the well-characterized classical chewing herbivore salivary elicitors that promote plant defense responses, little is known about sucking herbivores' elicitors. To characterize the sucking herbivore elicitors, we explored putative salivary gland proteins of spider mites by using an Agrobacterium-mediated transient expression system or protein infiltration in damaged bean leaves. Two candidate elicitors (designated as tetranin1 (Tet1) and tetranin2 (Tet2)) triggered early leaf responses (cytosolic calcium influx and membrane depolarization) and increased the transcript abundances of defense genes in the leaves, eventually resulting in reduced survivability of T. urticae on the host leaves as well as induction of indirect plant defenses by attracting predatory mites. Tet1 and/or Tet2 also induced jasmonate, salicylate and abscisic acid biosynthesis. Notably, Tet2-induced signaling cascades were also activated via the generation of reactive oxygen species. The signaling cascades of these two structurally dissimilar elicitors are mostly overlapping but partially distinct and thus they would coordinate the direct and indirect defense responses in host plants under spider mite attack in both shared and distinct manners.

Herbivore-specific induction of indirect and direct defensive responses in leaves and roots.

Herbivory can induce both general and specific responses in plants that modify direct and indirect defence against subsequent herbivory. The type of induction (local versus systemic induction, single versus multiple defence induction) likely depends both on herbivore identity and relationships among different responses. We examined the effects of two above-ground chewing herbivores (caterpillar, weevil) and one sucking herbivore (aphid) on indirect defence responses in leaves and direct defence responses in both leaves and roots of tallow tree, Triadica sebifera. We also included foliar applications of methyl jasmonate (MeJA) and salicylic acid (SA). We found that chewing herbivores and MeJA increased above-ground defence chemicals but SA only increased below-ground total flavonoids. Herbivory or MeJA increased above-ground indirect defence response (extrafloral nectar) but SA decreased it. Principal component analysis showed there was a trade-off between increasing total root phenolics and tannins (MeJA, chewing) versus latex and total root flavonoids (aphid, SA). For individual flavonoids, there was evidence for systemic induction (quercetin), trade-offs between compounds (quercetin versus kaempferitrin) and trade-offs between above-ground versus below-ground production (isoquercetin). Our results suggest that direct and indirect defence responses in leaves and roots depend on herbivore host range and specificity along with feeding mode. We detected relationships among some defence response types, while others were independent. Including multiple types of insects to examine defence inductions in leaves and roots may better elucidate the complexity and specificity of defence responses of plants.

The terpene synthase gene family in Gossypium hirsutum harbors a linalool synthase GhTPS12 implicated in direct defence responses against herbivores.

Herbivore-induced terpenes have been reported to function as ecological signals in plant-insect interactions. Here, we showed that insect-induced cotton volatile blends contained 16 terpenoid compounds with a relatively high level of linalool. The high diversity of terpene production is derived from a large terpene synthase (TPS) gene family. The TPS gene family of Gossypium hirsutum and Gossypium raimondii consist of 46 and 41 members, respectively. Twelve TPS genes (GhTPS4-15) could be isolated, and protein expression in Escherichia coli revealed catalytic activity for eight GhTPS. The upregulation of the majority of these eight genes additionally supports the function of these genes in herbivore-induced volatile biosynthesis. Furthermore, transgenic Nicotiana tabacum plants overexpressing GhTPS12 were generated, which produced relatively large amounts of (3S)-linalool. In choice tests, female adults of Helicoverpa armigera laid fewer eggs on transgenic plants compared with non-transformed controls. Meanwhile, Myzus persicae preferred feeding on wild-type leaves over leaves of transgenic plants. Our findings demonstrate that transcript accumulation of multiple TPS genes is mainly responsible for the production and diversity of herbivore-induced volatile terpenes in cotton. Also, these genes might play roles in plant defence, in particular, direct defence responses against herbivores.

Methyl jasmonate induction of cotton: a field test of the 'attract and reward' strategy of conservation biological control.

Natural or synthetic elicitors can affect plant physiology by stimulating direct and indirect defence responses to herbivores. For example, increased production of plant secondary metabolites, a direct response, can negatively affect herbivore survival, development and fecundity. Indirect responses include increased emission of plant volatiles that influence herbivore and natural enemy behaviour, and production of extrafloral nectar that serves as a food source for natural enemies after their arrival on induced plants. Therefore, the use of elicitors has potential for the study of basic aspects of tritrophic interactions, as well as application in biorational pest control, i.e. an 'attract and reward' strategy. We conducted a field study to investigate the effects of methyl jasmonate, an elicitor of plant defence responses, on three trophic levels: the plant, herbivores and natural enemies. We made exogenous applications of methyl jasmonate to transgenic cotton and measured volatile emission, extrafloral nectar production and plant performance (yield). We also assessed insect abundance, insect performance, and parasitism and predation of brown stink bug, Euschistus servus, eggs in methyl jasmonate-treated and untreated control plots. Application of methyl jasmonate increased emission of volatiles, in particular, (+)-limonene and (3E)-4,8-dimethyl-1,3,7-nonatriene, and production of extrafloral nectar, but not yield, compared with the control treatment. Despite increased volatile and extrafloral nectar production, methyl jasmonate application did not affect plant bug performance, or mortality of E. servus egg masses, and only marginally influenced insect abundance. Mortality of E. servus eggs varied over the course of the study. Overall, methyl jasmonate treatment affected cotton plant-induced responses, but not the insects that inhabit the plants. Our results were probably influenced by reduced natural enemy colonization of cotton from adjacent non-crop habitats, and subsequent low within-field population recruitment. Much remains to be learned about the effects of exogenous application of plant-produced 'enhancers' on the behaviour of natural enemies before crop physiology can be manipulated to enhance pest control.

Early plant defence against insect attack: involvement of reactive oxygen species in plant responses to insect egg deposition.

Pinus sylvestris responds to insect egg deposition by ROS accumulation linked with reduced activity of the ROS scavenger catalase. Egg mortality in needles with hypersensitive response (HR)-like symptoms is enhanced. Aggressive reactive oxygen species (ROS) play an important role in plant defence against biotic stressors, including herbivorous insects. Plants may even generate ROS in response to insect eggs, thus effectively fighting against future larval herbivory. However, so far nothing is known on how ROS-mediated plant defence against insect eggs is enzymatically regulated. Neither do we know how insects cope with egg-induced plant ROS. We addressed these gaps of knowledge by studying the activities of ROS-related enzymes in Pinus sylvestris deposited with eggs of the herbivorous sawfly Diprion pini. This species cuts a slit into pine needles and inserts its eggs into the needle tissue. About a quarter of egg-deposited needles show chlorotic tissue at the oviposition sites, indicating hypersensitive response-like direct defence responses resulting in reduced larval hatching from eggs. Hydrogen peroxide and peroxidase sensitive staining of sections of egg-deposited pine needles revealed the presence of hydrogen peroxide and peroxidase activity in needle tissue close to the eggs. Activity of ROS-producing NADPH-oxidase did not increase after egg deposition. However, the activity of the ROS-detoxifying enzyme catalase decreased after egg deposition and ovipositional wounding of needles. These results show that local ROS accumulation at the oviposition site is not caused by increased NADPH-oxidase activity, but reduced activity of pine needle catalase may contribute to it. However, our data suggest that pine sawflies can counteract the egg deposition-induced hydrogen peroxide accumulation in pine needles by high catalase activity in their oviduct secretion which is released with the eggs into pine tissue.

Resilience and robustness of IPM in protected horticulture in the face of potential invasive pests

Recently, various approaches for the biological control of major pests in protected crops have successfully been adopted. These successes have primarily occurred via the selection, release and/or conservation of generalist native predators that naturally colonize Mediterranean crops that are highly adapted to local environmental conditions. These generalist predators have resulted very effective in controlling key pests; as a result, pesticide use has been reduced considerably. In addition, because these predators are typically highly polyphagous, horticultural crops have become more resilient to newly emerging pests, which are preyed upon by these predators. Possibilities to further strengthen pest management in horticultural crops have recently arisen, such as the use of beneficial microbes (BM) to induce plant resilience. In this review, we used a tomato crop as an example to present two new, highly interrelated plant defense induction strategies: the use of Miridae zoophytophagous predators and the use of Trichoderma spp. microbial biological control agents. Both the mirid predators and Trichoderma symbiotic fungi can activate direct and indirect plant defense responses, which may increase the robustness of pest management against invading organisms. The practical use of these new approaches is discussed.