Engelmann Spruce Chemotypes in Colorado and their Effects on Symbiotic Fungi Associated with the North American Spruce Beetle

Conifer secondary metabolites play a key role in mechanisms of resistance to biotic disturbance, especially by bark beetles and beetle-associated microorganisms. Here, we describe variation in constitutive monoterpenes isolated from Engelmann spruce, Picea engelmannii, phloem across fourteen high-elevation populations in the Rocky Mountains of Colorado, and test interactions between phloem monoterpenes and an endophloedic symbiotic fungus, Leptographium abietinum, associated with the North American spruce beetle, Dendroctonus rufipennis. We consistently identified ten monoterpenes in Engelmann spruce phloem, and the trees in our samples could be classified into two geographically interspersed chemical phenotypes, or ‘chemotypes’: one in which α- and β-pinene were the most abundant monoterpenes, and one in which 3-carene was the most abundant monoterpene. Media amended with low concentrations of α-pinene, β-pinene, 3-carene, myrcene, and terpinolene stimulated growth of L. abietinum. Increasing monoterpene concentrations uniformly retarded fungal growth. Linalool completely suppressed fungal growth at all concentrations, while terpinolene completely suppressed growth at low and intermediate concentrations, indicating relatively high toxicity of these compounds. Tests with monoterpene blends representing the ‘average’ monoterpene composition of each chemotype indicated that representative chemotypes are equivalent in fungistatic activity, with chemotype blends being inhibitory even at low concentrations. Total constitutive monoterpene abundances in Engelmann spruce phloem ranged from 42 to 1796 μg/g. Induction of Engelmann spruce phloem monoterpenes in response to L. abietinum or other biotic agents has yet to be quantified, but is important for further understanding Engelmann spruce resistance to the D. rufipennis-L. abietinum complex.     

Both Gas Chromatography and an Electronic Nose Reflect Chemical Polymorphism of Juniper Shrubs Browsed or Avoided by Sheep

Chemical polymorphism may contribute to variation in browsing damage by mammalian herbivores. Earlier, we demonstrated that essential oil concentration in juniper, Juniperus communis, was negatively associated with herbivore browsing. The aim of the present study was to characterize the volatile chemical composition of browsed and non-browsed J. communis. By using either gas chromatography with flame ionization detection (GC-FID) or an electronic nose device, we could separate sheep-browsed or non-browsed juniper shrubs by their essential oil pattern and complex odor matrix. The main components of the essential oil from J. communis were monoterpenes. We distinguished three chemotypes, dominated either by α-pinene, sabinene, or δ-3-carene. Shrubs belonging to the α-pinene- or sabinene-dominated groups were browsed, whereas all individuals with the δ-3-carene chemotype were unused by the local herbivores. The electronic nose also separated the browsed and non-browsed shrubs indicating that their odor matrix could guide sheep browsing. Responses of sheep could integrate the post-ingestive effects of plant secondary metabolites with sensory experience that stems from odor–phytotoxin interactions. Chemotype diversity could increase the survival rate in the present population of J. communis as certain shrubs could benefit from relatively better chemical protection against the herbivores.     

Weak responses of pine sawfly larvae to high needle flavonoid concentrations in scots pine

Responses of sawfly larvae (Hymenoptera, Diprionidae) to the flavonoid taxifolin glucoside in their host plant were studied in a laboratory experiment. Larvae ofNeodiprion sertifer andDiprion pini were raised from egg hatch to cocoon spinning on two Scots pine (Pinus sylvestris) chemotypes, one without needle taxifolin glucoside (–) and the other containing 2–4% taxifolin glucoside (+). The (+) chemotype had somewhat lower concentrations of needle terpenoids (resin acids) than the (–) chemotype. Current-year needles had higher taxifolin glucoside concentrations than mature needles. There were no differences in survival or body size betweenN. sertifer larvae that fed on the (+) chemotype and those that fed on the (–) chemotype. FemaleD. pini larvae raised on (+) needles developed 6% more slowly than larvae fed (–) needles. The results from this study are contrary to earlier findings showing that flavonoid glucosides have strong negative effects on insect performance. Possible explanations for the different outcomes are discussed.     

Changes in Monoterpene Emission Rates of <Emphasis Type="Italic">Quercus ilex</Emphasis> Infested by Aphids Tended by Native or Invasive <Emphasis Type="Italic">Lasius</Emphasis> Ant Species

The emission of volatile organic compounds (VOCs) depends on temperature and light. Other factors such as insect herbivory also may modify VOC emission. In particular, aphid feeding promotes the release of new compounds and changes the composition of plant volatile blends. Given that some aphids are tended by ants, we investigated whether ants change the emission of VOCs indirectly through attendance on aphids. The effect of Lachnus roboris aphids and two different tending ant species on terpene emission rates of 4-year-old holm oak (Quercus ilex) saplings was investigated during a field experiment. There were five treatments: saplings alone (T1), saplings infested with L. roboris aphids (T2), saplings infested with aphids tended by the local ant Lasius grandis (T3), those tended by small colonies of the invasive ant Lasius neglectus (T4), and those tended by large colonies of the same invasive ant species (T5). The infestation by L. roboris elicited the emission of Δ³-carene and increased the emission of myrcene and γ-terpinene. Terpene emissions were modified depending on the tending ant species. Attendance by the local ant L. grandis increased α and β-pinene and sabinene. Attendance by the invasive ant L. neglectus only decreased significantly the emission of myrcene, one of the major compounds of the Q. ilex blend. Aphid abundance decreased with time for all treatments, but there was no difference in aphid abundance among treatments. Total terpene emission rates were not correlated with aphid abundance. These results highlight that aphids and tending ants may change terpene emission rates, depending on the ant species.     

Qualitative and quantitative variation in monoterpene co-occurrence and composition in the essential oil of Thymus vulgaris chemotypes

Thymus vulgaris has a chemical polymorphism with six different chemotypes that show marked spatial segregation in nature. Although some populations have a single chemotype in majority, many have two or three chemotypes. In this study we analyze the quantitative variation among T. vulgaris populations in the percentage of oil composed of the dominant monoterpene(s) for each chemotype. In general, phenolic chemotypes (thymol and carvacrol), which occur at the end of the biosynthetic chain, have a significantly lower proportion of their oil composed of their dominant monoterpene than nonphenolic chemotypes (geraniol, -terpineol, and linalool). This is due to the presence of high amounts of precursors (-terpinene and paracymene) in the oil of phenolic chemotypes. The essential oil of the nonphenolic thuyanol chemotype has four characteristic monoterpenes that together make up a lower proportion of the oil than the single dominant monoterpene of the other nonphenolic chemotypes. For all chemotypes, the percentage composition of the dominant monoterpene decreased significantly at sites where the chemotype is not the majority type. This decrease is correlated with a significant increase in either the proportion of the two precursors for the thymol chemotype or the monoterpenes characteristic of the other chemotypes at the site. The latter result suggests that a plant with dominant genes is responsible for the production of different monoterpenes can produce several molecules.     

Comparing the Variation of Needle and Wood Terpenoids in Scots Pine Provenances

We determined variation in both the concentration and composition of terpenoids in needles and wood within nine Scots pine (Pinus sylvestris) provenances. Seedlings of different provenances representing a 1200-km N–S transect from Estonia to northern Finland were cultivated in Suonenjoki nursery field, central Finland, for seven years. Growth of seedlings and the number of vertical resin ducts in wood were also determined. -Pinene and 3-carene were the major monoterpenes both in the needles and wood. The total monoterpene concentration was about five times higher in the needles than in the wood. A strong positive correlation was found between proportional quantities of several terpenes of the needles and wood, particularly for 3-carene, sabinene, and terpinolene. The needles contained both labdane-type and tricyclic resin acids, whereas the wood contained only tricyclic ones. The wood had a four times higher tricyclic resin acid concentration than the needles. The highest total monoterpene concentration in the needles and in the wood occurred in the most northern Muonio provenance and in the most southern Saaremaa provenance plants, respectively. The amount of high 3-carene genotype trees decreased among the northern provenances. The wood of the most northern Muonio provenance showed the lowest total resin acid concentration, but provenance did not affect total tricyclic resin acids in the needles. Korpilahti provenance trees from central Finland had the best growth in height. In addition, Korpilahti and Ruokolahti provenance trees showed largest radial growth of stem and smallest number of vertical resin ducts. The results suggest that especially the proportional quantity of 3-carene in the needles could be used in estimating the amount of this compound in the wood and vice versa.     

Gas‐ versus liquid‐phase α-pinene transformation over faujasite‐like zeolites and an amorphous silicoaluminate

The transformation of α-pinene was studied in both gas and liquid phase over a series of faujasite‐like zeolites having different framework composition. A comparison with an amorphous silicoaluminate having 13 wt% of Al₂O₃ (SA) was made. Camphene + limonene were the major products observed in liquid phase at 393 K at conversion levels lower than 90%. At 473 K, where the reaction in gas phase took place, limonene suffered an ulterior transformation mainly into terpinenes and terpinolenes and a faster deactivation of the zeolite catalysts as compared to the one in liquid phase was observed. This behaviour contrasts with the high stability shown by SA in gas phase. Over the wider pore system of SA undesired heavy products were formed in liquid phase at the expenses of the terpene isomers. However, in gas phase this ulterior transformation of monoterpenes was minimised. A maximum activity for the zeolite catalysts was observed when the number of Al^IV per unit cell reached about 17, a value where the population of Si(0Al) sites has been shown to reach the maximum. This revised version was published online in July 2006 with corrections to the Cover Date.     

Seasonal Variation of Chemical Composition and Antioxidant Activity of Essential Oil from <Emphasis Type="Italic">Pistacia atlantica</Emphasis> Desf. Leaves

The composition and antioxidant activities of Pistacia atlantica Desf. essential oil were investigated. Qualitative and quantitative differences in compositions and in antioxidant activities of male and female leaf essential oils were observed during the season. The essential oils obtained by hydrodistillation were analysed by GC and GC–MS. The oils were rich in monoterpenes hydrocarbons and oxygenated sesquiterpenes. The main components of male essential oil were α-pinene/α-thujene, spathulenol and bicyclogermacrene. The major component of female essential oil was δ-3-carene. The seasonal variation showed that most of the main components of the oils reached theirs highest values in September. The antioxidant activity of the oil was investigated in vitro using two assays: DPPH^· (2,2-di-phenyl-1-picrylhydrazyl) free radical-scavenging and FRAP (Ferric Reducing Antioxidant Power). The highest antioxidant capacity to scavenge free DPPH radicals was reached in the month of June for male oils and during the months of September–October for the female oils. The high reducing power for male oil was observed during the month of June and for the female oil it was in August. The female oil was more active than the male oil. The antioxidant capacity of the female oil was almost ten times higher than Ascorbic acid in the FRAP assay.     

Sources of Variation in Concentration and Composition of Foliar Monoterpenes in Tamarack (Larix laricina) Seedlings: Roles of Nutrient Availability, Time of Season, and Plant Architecture

The effects of foliage class, time of season, and nutrient availability on monoterpene composition and concentration were evaluated. In the first experiment, we compared foliar monoterpenes of long shoots versus short shoots, which differ in age and origin, and differences due to nutrient availability and time of season. Both the concentrations and compositions of these monoterpenes were higher in the foliage of the long shoots. The only exceptions were the concentration of -3-carene and the composition of -pinene. Within a foliage class, the concentrations of seven monoterpenes and the percentages of five monoterpenes changed over the season. Of these, almost all declined, with the exceptions of -pinene and an unknown. Nutrient availability significantly affected the concentration or composition of some monoterpenes, but only to a slight extent. -Pinene and the unknown increased with nutrient availability, whereas -pinene and myrcene decreased. A more comprehensive analysis of four foliage classes on a separate cohort of trees showed that all monoterpenes, except sabinene and terpinolene, differed between foliage classes, but there were few general patterns. Overall, within-tree variation in tamarack monoterpenes related to foliage class and age appears more important than between-tree variation due to nutrient availability. These results suppport the view that products of mevalonic acid biosynthesis, such as terpenes, do not follow predictions of the carbon–nutrient balance and growth differentiation hypotheses. Our overall results show that individual hosts need to be considered as phytochemical mosaics when interpreting herbivore–plant interactions.     

Conifer seed oils: Distribution of Δ5 acids between α and β chains by13C nuclear magnetic resonance spectroscopy

Nine seed oils from three different conifer families, already examined by gas chromatography and known to contain diene, triene, and tetraene C₁₈ and C₂₀Δ5 acids, have been reexamined by high-resolution¹³C nuclear magnetic resonance spectroscopy. The Δ5 acids are apparent only in the α-chains. This location is independent of chainlength, double-bond number, and the species considered and is probably a general factor of conifer seed oils. The spectra confirm the presence of oleic, linoleic, α-linolenic, and of Δ5 acids and give quantitative information about (total) n-6, n-3, and Δ5 acids that is in accord with that obtained by gas chromatography.