Heterorhabditis sp. not attracted to synthetic (E)‐β‐caryophyllene,a volatile emitted by roots upon feeding by corn rootworm

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a destructive pest of maize, Zea mays L. in North America and Europe. Larvae and pupae can be controlled with entomopathogenic nematodes (EPN) of the genus Heterorhabditis. When maize is attacked by WCR, the roots of some maize varieties emit (E)‐β‐caryophyllene that attract EPN to the pest larvae. To use synthetic (E)‐β‐caryophyllene in bioassays for the genetic selection of EPN strains with enhanced chemotactic response to a volatile emitted from the damaged root when attacked by larvae of the WCR, different laboratory bioassays were tested. Three sand assays and one agar assay used synthetic (E)‐β‐caryophyllene as an attractant for H. megidis. In none of the assays, attraction of the nematodes to (E)‐β‐caryophyllene was recorded. Possible reasons why (E)‐β‐caryophyllene was not attracting EPN in the bioassays are discussed.     

Demonstration and characterization of (E)-nerolidol synthase from maize: a herbivore-inducible terpene synthase participating in (3E)-4,8-dimethyl-1,3,7-nonatriene biosynthesis

Upon herbivore attack, maize (Zea mays L.) emits a mixture of volatile compounds that attracts herbivore enemies to the plant. One of the major components of this mixture is an unusual acyclic C₁₁ homoterpene, (3E )-4,8-dimethyl-1,3,7-nonatriene (DMNT), which is also emitted by many other species following herbivore damage. Biosynthesis of DMNT has been previously shown to proceed via the sesquiterpene alcohol, (E )-nerolidol. Here we demonstrate an enzyme activity that converts farnesyl diphosphate, the universal precursor of sesquiterpenes, to (3S)-(E )-nerolidol in cell-free extracts of maize leaves that had been fed upon by Spodoptera littoralis. The properties of this (E )-nerolidol synthase resemble those of other terpene synthases. Evidence for its participation in DMNT biosynthesis includes the direct incorporation of deuterium-labeled (E )-nerolidol into DMNT and the close correlation between increases in (E )-nerolidol synthase activity and DMNT emission after herbivore damage. Since farnesyl diphosphate has many other metabolic fates, (E )-nerolidol synthase may represent the first committed step of DMNT biosynthesis in maize. However, the formation of this unusual acyclic terpenoid appears to be regulated at both the level of (E )-nerolidol synthase and at later steps in the pathway. Received: 20 August 1999 / Accepted: 27 October 1999     

Adjusting the scent ratio: using genetically modified Vitis vinifera plants to manipulate European grapevine moth behaviour

Herbivorous insects use olfactory cues to locate their host plant within a complex olfactory landscape. One such example is the European grapevine moth Lobesia botrana, a key pest of the grape in the Palearctic region, which recently expanded both its geographical and host plant range. Previous studies have showed that a synthetic blend of the three terpenoids, (E)‐β‐caryophyllene, (E)‐β‐farnesene and (E)‐4,8‐dimethyl‐1,3,7‐nonatriene (DMNT), was as attractive for the moth as the complete grape odour profile in laboratory conditions. The same studies also showed that the specific ratio of these compounds in the grape bouquet was crucial because a percentage variation in any of the three volatiles resulted in almost complete inhibition of the blend's attractiveness. Here, we report on the creation of stable grapevine transgenic lines, with modified (E)‐β‐caryophyllene and (E)‐β‐farnesene emission and thus with an altered ratio compared to the original plants. When headspace collections from these plants were tested in wind tunnel behavioural assays, they were less attractive than control extracts. This result was confirmed by testing synthetic blends imitating the ratio found on natural and transformed plants, as well as by testing the plants themselves. With this evidence, we suggest that a strategy based on volatile ratio modification may also interfere with the host‐finding behaviour of L. botrana in the field, creating avenues for new pest control methods.     

Herbivore‐induced plant responses in Brassica oleracea prevail over effects of constitutive resistance and result in enhanced herbivore attack

1. Plant responses to herbivore attack may have community‐wide effects on the composition of the plant‐associated insect community. Thereby, plant responses to an early‐season herbivore may have profound consequences for the amount and type of future attack. 2. Here we studied the effect of early‐season herbivory by caterpillars of Pieris rapae on the composition of the insect herbivore community on domesticated Brassica oleracea plants. We compared the effect of herbivory on two cultivars that differ in the degree of susceptibility to herbivores to analyse whether induced plant responses supersede differences caused by constitutive resistance. 3. Early‐season herbivory affected the herbivore community, having contrasting effects on different herbivore species, while these effects were similar on the two cultivars. Generalist insect herbivores avoided plants that had been induced, whereas these plants were colonised preferentially by specialist herbivores belonging to both leaf‐chewing and sap‐sucking guilds. 4. Our results show that community‐wide effects of early‐season herbivory may prevail over effects of constitutive plant resistance. Induced responses triggered by prior herbivory may lead to an increase in susceptibility to the dominant specialists in the herbivorous insect community. The outcome of the balance between contrasting responses of herbivorous community members to induced plants therefore determines whether induced plant responses result in enhanced plant resistance.     

Variability and stability of tea weevil‐induced volatile emissions from tea plants with different weevil densities,photoperiod and infestation duration

Abstract After herbivore attack, many plants emit herbivore‐induced plant volatiles (HIPVs). HIPVs can attract carnivores and/or repel herbivores, thereby mediating tritrophic plant–herbivore–carnivore interactions. HIPVs act as chemical information between organisms; hence, their variability and stability are vital. In the present study, variations in the volatile emissions, from the tea plant Camellia sinensis (O. Ktze) damaged by the tea weevil Myllocerinus aurolineatus (Voss) (Coleoptera: Curculionidae), with weevil densities, photoperiod and infestation duration, were investigated. The volatiles induced by high‐density weevils were more abundant in composition and amount than those induced by low‐density weevils, whether at noon, night or after weevil removal. The induced volatile emissions were similar on the second and third day after infestation, and the emissions of the major induced compounds displayed diurnal cycles. Linalool, (E,E)‐α‐farnesene, and benzyl nitrile were emitted mainly at noon, whereas 1,3,8‐p‐menthatriene and (E)‐β‐ocimene were maximally emitted at night. Given the different emission dynamics, significant differences were found between noon‐ and night‐induced volatiles. In summary, tea plants damaged by different weevil densities emitted a relatively stable signal at a particular time. This stability could be attributed to the similarities under the two densities of the main induced volatile compounds, their relative ratios and the emission dynamics of the induced volatiles.     

Root volatiles in plant–plant interactions II: Root volatiles alter root chemistry and plant–herbivore interactions of neighbouring plants

Volatile organic compounds (VOCs) emitted by plant roots can influence the germination and growth of neighbouring plants. However, little is known about the effects of root VOCs on plant–herbivore interactions of neighbouring plants. The spotted knapweed (Centaurea stoebe) constitutively releases high amounts of sesquiterpenes into the rhizosphere. Here, we examine the impact of C. stoebe root VOCs on the primary and secondary metabolites of sympatric Taraxacum officinale plants and the resulting plant‐mediated effects on a generalist root herbivore, the white grub Melolontha melolontha. We show that exposure of T. officinale to C.stoebe root VOCs does not affect the accumulation of defensive secondary metabolites but modulates carbohydrate and total protein levels in T. officinale roots. Furthermore, VOC exposure increases M. melolontha growth on T. officinale plants. Exposure of T. officinale to a major C. stoebe root VOC, the sesquiterpene (E)‐β‐caryophyllene, partially mimics the effect of the full root VOC blend on M. melolontha growth. Thus, releasing root VOCs can modify plant–herbivore interactions of neighbouring plants. The release of VOCs to increase the susceptibility of other plants may be a form of plant offense.     

Substrate specificity of α‐humulene synthase from Zingiber zerumbet Smith and determination of kinetic constants by a spectrophotometric assay

Terpene synthases are the key enzymes in terpene biosynthesis that provide a structurally complex and highly diverse product spectrum. A suitable and reliable analytical assay is indispensable to measure terpene synthase activity accurately and precisely. In this study, a malachite green assay (MG) was adapted to rapidly assay terpene synthase activity and was validated in comparison to an already established gas chromatography assay. A linear correlation between both assays was observed. Kinetic properties for the previously described sesquiterpene synthase α‐humulene synthase (HUM) from Zingiber zerumbet Smith were investigated for the bioconversion of the monoterpene precursors geranyl pyrophosphate (2E‐GPP) and neryl pyrophosphate (2Z‐NPP) as well as for the sesquiterpene precursor farnesyl pyrophosphate (2E,6E‐FPP). Also, gas chromatography mass spectrometry (GS‐MS) was carried out to identify the products of the bioconversion of (2E)‐GPP and (2Z)‐NPP.     

Elevated volatile concentrations in high‐nutrient plants: do insect herbivores pay a high price for good food?

1. A tritrophic perspective is fundamental for understanding the drivers of insect–plant interactions. While host plant traits can directly affect insect herbivore performance by either inhibiting or altering the nutritional benefits of consumption, they can also have an indirect effect on herbivores by influencing rates of predation or parasitism. 2. Enhancing soil nutrients available to trees of the genus Eucalyptus consistently modifies plant traits, typically improving the nutritional quality of the foliage for insect herbivores. We hypothesised that resulting increases in volatile essential oils could have an indirect negative effect on eucalypt‐feeding herbivores by providing their natural enemies with stronger host/prey location cues. 3. Eucalyptus tereticornis Smith seedlings were grown under low‐ and high‐nutrient conditions and the consequences for the release of volatile cues from damaged plants were examined. The influence of 1,8‐cineole (the major volatile terpene in many Eucalyptus species) on rates of predation on model caterpillars in the field was then examined. 4. It was found that the emission of cineole increased significantly after damage (artificial or herbivore), but continued only when damage was sustained by herbivore feeding. Importantly, more cineole was emitted from high‐ than low‐nutrient seedlings given an equivalent amount of damage. In the field, predation was significantly greater on model caterpillars baited with cineole than on unbaited models. 5. These findings are consistent with the hypothesis that any performance benefits insect herbivores derive from feeding on high‐nutrient eucalypt foliage could be at least partially offset by an increased risk of predation or parasitism via increased emission of attractive volatiles.     

Four terpene synthases produce major compounds of the gypsy moth feeding-induced volatile blend of Populus trichocarpa

After herbivore damage, many plants increase their emission of volatile compounds, with terpenes usually comprising the major group of induced volatiles. Populus trichocarpa is the first woody species with a fully sequenced genome, enabling rapid molecular approaches towards characterization of volatile terpene biosynthesis in this and other poplar species. We identified and characterized four terpene synthases (PtTPS1-4) from P. trichocarpa which form major terpene compounds of the volatile blend induced by gypsy moth (Lymantria dispar) feeding. The enzymes were heterologously expressed and assayed with potential prenyl diphosphate substrates. PtTPS1 and PtTPS2 accepted only farnesyl diphosphate and produced (−)-germacrene D and (E,E)-α-farnesene as their major products, respectively. In contrast, PtTPS3 and PtTPS4 showed both mono- and sesquiterpene synthase activity. They produce the acyclic terpene alcohols linalool and nerolidol but exhibited opposite stereospecificity. qRT-PCR analysis revealed that the expression of the respective terpene synthase genes was induced after feeding of gypsy moth caterpillars. The TPS enzyme products may play important roles in indirect defense of poplar to herbivores and in mediating intra- and inter-plant signaling.     

Testing semiochemicals from aphid,plant and conspecific: attraction of Harmonia axyridis

Abstract Harmonia axyridis Pallas (Coleoptera: Coccinellidae) is an invasive specie affecting the dynamics and composition of several guilds. Nowadays, no biological control method is available to reduce the populations of this harmful coccinellid. Attractants and semiochemicals seem to be the best alternative but only few studies have tested the impact of semiochemicals on this Asian lady beetle. In this work, through wind‐tunnel experiments, semiochemicals from aphids (Z,E‐nepetalactone, [E]‐β‐farnesene, α‐pinene and β‐pinene), from coccinellids ([‐]‐β‐caryophyllene) and from the nettle Urtica dioica L. were evaluated as potential attractants. The nettle volatile compounds ([Z]‐3‐hexenol and [E]‐2‐hexenal) were extracted using a Clevenger Apparatus^® and identified by headspace gas chromatography–mass spectroscopy. In the wind‐tunnel experiments, the main components of the aphid alarm pheromone as well as a component of the aphid sexual pheromone strongly attracted both sexes of the Asian lady beetle while (‐)‐β‐caryophyllene only attracted few individuals and had no impact on the males. The nettle extract as well as the (Z)‐3‐hexenol oriented both males and females to the odor source. The (E)‐2‐hexenal was shown to have no effect on females even if this green leaf volatile attracted males. Because Z,E‐nepetalactone was identified as the most efficient attractant in the wind‐tunnel experiments, this volatile was also tested in a potato field where H. axyridis has been showed to respond to this semiochemical. This study highlighted that Z,E‐nepetalactone orientated the Asian lady beetle H. axyridis under natural conditions, indicating that this volatile compound could certainly help for an efficient biological control approach against this invasive specie.     

Direct formation of the sesquiterpeonid ether liguloxide by a terpene synthase in Senecio scandens

Key message

SsLOS directly catalyzed formation of the sesquiterpenoid ether liguloxide in the medicinal plant Senecio scandens.

Abstract

Terpene synthases determine the diversity of terpene skeletons and corresponding terpenoid natural products. Oxygenated groups introduced in catalysis of terpene synthases are important for solubility, potential bioactivity and further elaboration of terpenoids. Here we identified one terpene synthase, SsLOS, in the Chinese medicinal plant Senecio scandens. SsLOS acted as the sesquiterpene synthase and utilized (E,E)-farnesyl diphosphate as the substrate to produce a blend of sesquiterpenoids. GC–MS analysis and NMR structure identification demonstrated that SsLOS directly produced the sesquiterpenoid ether, liguloxide, as well as its alcoholic isomer, 6-epi-guaia-2(3)-en-11-ol. Homology modeling and site-directed mutagenesis were combined to explore the catalytic mechanism of SsLOS. A few key residues were identified in the active site and hedycaryol was identified as the neutral intermediate of SsLOS catalysis. The plausible catalytic mechanism was proposed as well. Altogether, SsLOS was identified and characterized as the sesquiterpenoid ether synthase, which is the second terpenoid ether synthase after 1,8-cineol synthase, suggesting some insights for the universal mechanism of terpene synthases using the water molecule in the catalytic cavity.

     

9种唇形科芳香植物挥发性萜类成分的比较分析

采用正己烷萃取法，结合GC-MS（气相色谱-质谱联用）技术，并通过谱库、保留指数和文献检索定性，内标法定量，分析9种唇形科芳香植物叶片的挥发性萜类成分并比较其差异，所得结果不仅为芳香植物的高效利用、合理开发提供参考，还为植物萜类的代谢研究提供依据。结果显示：从9种芳香植物中共检测到77种挥发性萜类物质，藿香中检测到的种类最多，为46种，迷迭香（35种）、百里香（33种）、药用鼠尾草（33种）和美国薄荷（31种）次之，石竹烯和蛇麻烯为9种植物共有成分。迷迭香中检测到的挥发性萜类的含量最高，其次是药用鼠尾草和藿香。单萜类成上分明显高于倍半萜类，因此迷迭香、药用鼠尾草、藿香、百里香和美国薄荷，较适宜作为提取挥发性萜类的材料；香蜂花只适宜柠檬醛的提取，牛至、凤梨鼠尾草和南欧丹参不适宜作为提取挥发性萜类的材料。上述芳香植物在正常环境中生成单萜类化合物的能力高于生成倍半萜类化合物的能力，可能与其含有的萜类合酶有关。     

Genetic variation for volatile terpenoids in roots of carrot, Daucus carota, backcrosses and F2 generations

Volatile terpenoid levels were measured in segregating carrot populations. Genetic analysis suggested dominance and multigenic control for low total volatile terpenoids, and low terpinolene quantities. The inheritance of caryophyllene quantities differed in different populations. Percent (E)-γ-bisabolene exhibited simple genetic control in several carrot populations with dominance for high levels. Compensatory shifts between the major volatile terpenes indicates an inter-relationship between the mono- and sesquiterpenoid biosynthetic pools.     

Function of defensive volatiles in pedunculate oak (Quercus robur) is tricked by the moth Tortrix viridana

The indirect defences of plants are comprised of herbivore‐induced plant volatiles (HIPVs) that among other things attract the natural enemies of insects. However, the actual extent of the benefits of HIPV emissions in complex co‐evolved plant‐herbivore systems is only poorly understood. The observation that a few Quercus robur L. trees constantly tolerated (T‐oaks) infestation by a major pest of oaks (Tortrix viridana L.), compared with heavily defoliated trees (susceptible: S‐oaks), lead us to a combined biochemical and behavioural study. We used these evidently different phenotypes to analyse whether the resistance of T‐oaks to the herbivore was dependent on the amount and scent of HIPVs and/or differences in non‐volatile polyphenolic leaf constituents (as quercetin‐, kaempferol‐ and flavonol glycosides). In addition to non‐volatile metabolic differences, typically defensive HIPV emissions differed between S‐oaks and T‐oaks. Female moths were attracted by the blend of HIPVs from S‐oaks, showing significantly higher amounts of (E)‐4,8‐dimethyl‐1,3,7‐nonatriene (DMNT) and (E)‐β‐ocimene and avoid T‐oaks with relative high fraction of the sesquiterpenes α‐farnesene and germacrene D. Hence, the strategy of T‐oaks exhibiting directly herbivore‐repellent HIPV emissions instead of high emissions of predator‐attracting HIPVs of the S‐oaks appears to be the better mechanism for avoiding defoliation.     

Influence of drought on plant performance through changes in belowground tritrophic interactions

Climate change is predicted to increase the risk of drought in many temperate agroecosystems. While the impact of drought on aboveground plant‐herbivore‐natural enemy interactions has been studied, little is known about its effects on belowground tritrophic interactions and root defense chemistry. We investigated the effects of low soil moisture on the interaction between maize, the western corn rootworm (WCR, Diabrotica virgifera), and soil‐borne natural enemies of WCR. In a manipulative field experiment, reduced soil moisture and WCR attack reduced plant performance and increased benzoxazinoid levels. The negative effects of WCR on cob dry weight and silk emergence were strongest at low moisture levels. Inoculation with entomopathogenic nematodes (EPNs, Heterorhabditis bacteriophora) was ineffective in controlling WCR, and the EPNs died rapidly in the warm and dry soil. However, ants of the species Solenopsis molesta invaded the experiment, were more abundant in WCR‐infested pots and predated WCR independently of soil moisture. Ant presence increased root and shoot biomass and was associated with attenuated moisture‐dependent effects of WCR on maize cob weight. Our study suggests that apart from directly reducing plant performance, drought can also increase the negative effects of root herbivores such as WCR. It furthermore identifies S. molesta as a natural enemy of WCR that can protect maize plants from the negative impact of herbivory under drought stress. Robust herbivore natural enemies may play an important role in buffering the impact of climate change on plant‐herbivore interactions.