Terpene response of Picea abies and Abies alba to infection with Heterobasidion s.l.

The monoterpene composition of Picea abies and Abies alba resin was analysed in relation to growth by Heterobasidion spp. Fifteen‐year‐old P. abies and A. alba trees were inoculated on branches with three species of Heterobasidion annosum s.l. After 4 months of incubation, each host was colonized to a significantly greater degree by the pathogen specific to that host (H. parviporum on P. abies, H. abietinum on A. alba) than by the other fungi. Analysis of the enantiomeric monoterpene profiles in the spruce and fir showed that the response in terms of the relative proportions of the monoterpene compounds in the resin differed between tree species. Following challenge with Heterobasidion spp., A. alba trees did not show changes in monoterpene composition in addition to those in the wounding response (increase in (−)‐α‐pinene and (−)‐camphene, and decrease in β‐phellandrene). In P.abies, (−)‐α‐pinene, (+)‐α‐pinene and δ‐3‐carene increased following Heterobasidion attack but not after wounding alone.     

Biochemical responses associated with induced resistance to Colletotrichum acutatum in Pinus radiata seedlings treated with methyl jasmonate and Trichoderma spp.

The effect of Trichoderma (T. atrobrunneumFCC320 and T. atrovirideLU633) and/or methyl jasmonate (MJ) on resistance to terminal crook (Colletotrichum acutatum) and on seedling biochemistry was investigated in radiata pine (Pinus radiata) seedlings. Seedlings were germinated and grown in Trichoderma‐amended or non‐amended media for 3 months and then sprayed with 2.25 mM MJ 1 week before inoculation with C. acutatum. The incidence and severity of terminal crook in the seedlings treated with MJ and Trichoderma+MJ were lower than in Trichoderma‐treated and Trichoderma‐untreated seedlings. The MJ‐induced resistance response was concomitant with an increase in the concentrations of the monoterpenes α‐pinene, β‐pinene, β‐phellandrene, camphene and myrcene in needles, and also α‐pinene, β‐pinene and camphene in stems. The concentrations of α‐pinene, β‐pinene and camphene were elevated from at least 1 week until 4 weeks after MJ application, compared with those in non‐MJ counterparts. Trichoderma alone did not affect monoterpenes, but the concentrations of α‐pinene, β‐pinene and camphene were greater in needles of Trichoderma+MJ than in MJ‐treated seedlings after 28 days. Total phenolic concentration in needles and peroxidase activity in stems were twofold greater in MJ‐treated seedlings than in non‐MJ seedlings over the same period. None of the treatments affected the activity of peroxidase in needles. It is proposed that the accumulation of monoterpene and phenolics and the induction of peroxidases contribute, in part, to MJ‐induced resistance to terminal crook in radiata pine seedlings.     

Toxicity of monoterpenes from Pinus radiata cortical oleoresin to Diplodia pinea spores1

The effect on spore germmation and germ tube growth of Diplodia pinea produced by (+)- α-pinene, (?)-β-pinene, (+)-Δ³-carene, (?)-limonene, β-phellandrene, and terpinolene was determined over a range of vapour concentrations. Spore germination was most inhibited by A³-carene; germ tube growth was inhibited by all these monoterpenes except (?)-limonenc and ß-phellandrene. In contrast to the low toxicity of (?)-limonene, (+)-limonene (not known to be present in Pinus radiata cortical oleoresin) was extremely toxic to both spore germination and germ tube growth. The results arc discussed in rotation to an earlier observation that trees free of dieback in the field had high levels of certain monoterpenes.     

The effect of nitrogen fertilization on fungistatic phenolic compounds in roots of beech (Fagus sylvatica) and Norway spruce (Picea abies)

The effect of nitrogen fertilization on fungistatic phenolic compounds in fine roots of beech and Norway spruce growing in afforestation plots was analysed. The plots were situated at two sites in Switzerland on acidic soil with low base saturation. For 9 years, the trees have been treated with dry ammonium nitrate to give 0, 10, 20, 40, 80, 160 kg N ha^?1 year^?1, respectively. The phenolic compounds responded differently to fertilization. Fine roots of beech showed a significant decrease of (?)‐epicatechin and piceatannol with increasing nitrogen fertilization. The concentration of protocatechuic acid was increased with fertilization. Roots of fertilized Norway spruce showed significantly decreased concentrations of 4‐hydroxyacetophenone and piceatannol. The mycelial growth of three isolates each of Heterobasidion annosum s.l. and Cylindrocarpon destructans was tested on agar media containing various phenolic compounds in concentrations found in fine roots of Norway spruce (Picea abies) and beech (Fagus sylvatica). All three H. annosum isolates were inhibited by p‐coumaric acid and (?)‐epicatechin. Two isolates were inhibited by another four phenolic compounds (p‐hydroxybenzoic acid, 4‐hydroxyacetophenone, piceatannol and protocatechuic acid), one by (+)‐catechin. Two of three C. destructans isolates were inhibited by all phenolic compounds except for (+)‐catechin which affected only one isolate, one isolate did not respond at all.     

A method for trapping hylobius abietis (L.) with a standardized bait and its potential for forecasting seedling damage

A method for trapping walking Hylobius abietis (L.) (Coleoptera: Curculionidae) was developed and evaluated. Synergism between α‐pinene and ethanol in attracting H. abietis was confirmed. The effects of varying release rates of these substances and of adding other host terpenes were studied. A simple bait was developed containing α‐pinene and ethanol released from separate vials. The correlation between trap catch and seedling damage was estimated during May, June, and August on 1‐, 2‐, and 3‐year‐old clear‐cuttings. In May and June, population levels were relatively high on all clear‐cuttings and no significant correlations were found. The August experiment showed a strong positive correlation between catch and damage. The percentage damaged seedlings per captured weevil was considerably higher in August than in May or June. Possibilities for developing a system for forecasting seedling damage using this trapping method are discussed.     

Low-density Ceratocystis polonica inoculation of Norway spruce (Picea abies) triggers accumulation of monoterpenes with antifungal properties

Among the most devastating pests of Norway spruce (Picea abies) are the European spruce bark beetle (Ips typographus) and the associated pathogenic blue-stain fungus Ceratocystis polonica. Following attack and colonization, the beetle and the fungus must cope with induced host chemical defenses, such as monoterpenes that are generally thought to be toxic to both symbionts. The goal of this study was to better understand the response of Norway spruce following C. polonica inoculation at low density that does not overwhelm the tree and to identify monoterpenes mobilized toward the fungus. We inoculated healthy mature trees and monitored monoterpene profiles 2, 3, and 5 months post-inoculation. We also exposed three different C. polonica strains to the most abundant or significantly up-regulated monoterpenes to determine differences in monoterpene toxicity and resistance among strains. Total monoterpene levels, including limonene, were increased at 2 and 3 months after inoculation and had dropped after 5 months. In in vitro assays, all monoterpenes were inhibitory to C. polonica. Limonene and β-pinene were the most potent inhibitors of fungal growth. The extent of inhibition varied between the three strains tested. These results showed a defense response of Norway spruce to C. polonica, in which limonene may play a critical role in inhibiting the spread of the fungus. We also showed that differences between strains of C. polonica must be taken into account when assessing the role of the fungus in this bark beetle–symbiont system.     

Individual variation in low boiling point monoterpenes emitted from hinoki (Chamaecyparis obtusa) needles

Variations in the compositions of low-boilingpoint (LBP) monoterpenes in needle samples of 50 hinoki (Chamaecyparis obtusa) trees were investigated using the headspace technique. Considerable compositional variations were revealed, especially in sabinene composition. The sabinene composition varied from 4.9% to 78.0% of the total LBP monoterpenes.-Pinene, myrcene, and limonene also showed considerable variations (9.0%–32.7%, 5.5%–22.6%, 3.6%–29.0% respectively). Analysis of the monoterpene composition allowed definition of four chemotypes based on the contingency table test. No correlation was observed between tree size and LBP monoterpene composition, indicating that the compositional variation in LBP monoterpene exists genetically in this population of hinoki.Part of this paper was presented at the 11th annual meeting of the Chugoku Shikoku branch of the Japan Wood Research Society, Matsue, September 1999     

Cortical monoterpene variation in natural populations of Pinus sylvestris in Sweden

Cortical oleoresins were obtained from 10 natural populations of Pinus sylvestris L. from different geographical localities. Gas chromatography was used to determine quantities of individual monoterpenes. Considerable variation was found in monoterpene composition among different populations. High proportions of limonene occurred in greater percentages in trees of northern origins, while high sabinene was frequently found in southern trees. Mean proportions of limonene and sabinene in the populations were clinal with latitude. For some monoterpenes, variation coefficients as a measure of variability within populations appeared to be clinal with latitude. 85.4% of the variance in monoterpene composition was found within populations and 14.6 between populations. Discriminant analysis differentiates populations from different geographical localities into different groups. The differences in monoterpene patterns between adjacent populations were statistically nonsignificant. It is recommended that monoterpene composition be used for more general study of genetic variation, geographical distinction between different populations and seed certification purposes.     

Increased atmospheric carbon dioxide concentration: effects on eucalypt rust (Puccinia psidii), C:N ratio and essential oils in eucalypt clonal plantlets

Evaluation of impacts of high CO₂ atmospheric concentration is strategically important for the development of adaptation measures and sustainable crop management. The objective of this study was to evaluate the effects of increased atmospheric CO₂ concentration on eucalypt rust (Puccinia psidii), C:N ratio, yield and chemical composition of essential oils and growth of eucalypt clonal plantlets. Two clones with different levels of rust resistance were studied: a Eucalyptus urophylla × E. camaldulensis hybrid (VM 01) and an E. urophylla (clone MN 463). The experiments were performed in open‐top chambers (OTCs) with CO₂ mean concentrations (μmol mol^?1) of 399 (unenclosed control), 412 (OTCs with ambient CO₂ concentration) and 508 (OTCs with high CO₂ concentration) and in closed chambers (CCs) with CO₂ mean concentrations of 390, 405, 520 and 700. Increased atmospheric CO₂ concentrations resulted in a decrease in rust pustules per leaf, uredinia per leaf area, spores per uredinia and area under the disease progress curve in VM 01 (hybrid) clonal plantlets. The disease did not occur in MN 463 clonal plantlets, which demonstrated that high CO₂ concentrations did not change the level of rust resistance. Plant growth of the two clones was stimulated by up to 23% in height and 26% in stem diameter in OTCs and by 18% for both clones in CCs. An increased C:N ratio in leaves, stems and roots was observed only for the VM 01 clonal plantlets. Essential oils produced by VM 01 (2.8 g 100 g^?1) and MN 463 (1.4 g 100 g^?1), as well as the major essential oil compounds (80% 1.8‐cineole for VM 01; 50% 1.8‐cineole and 32% α‐pinene for MN 463), were not altered. In this study, increased concentrations of atmospheric CO₂ favourably impacted eucalypt growth and reduced rust severity, while not influencing the production of essential oils.     

Hyptis suaveolens and Hyptis spicigera (Lamiaceae) essential oils: qualitative analysis, contact toxicity and repellent activity against Sitophilus granarius (L.) (Coleoptera: Dryophthoridae)

Essential oils from Hyptis suaveolens (L.) Poiteau (Lamiaceae) and Hyptis spicigera Lamarck (Lamiaceae) were first analysed by gas chromatography and by gas chromatography/electron impact mass spectroscopy and then evaluated for toxicity and repellent activity against Sitophilus granarius (L.) (Coleoptera: Dryophthoridae), one of the most serious worldwide stored grain pests. Fifty-six compounds have been identified in the essential oil of H. suaveolens: monoterpene hydrocarbons were the most represented volatiles (64.1%), followed by sesquiterpene hydrocarbons (24.0%), oxygenated monoterpenes (8.1%) and oxygenated sesquiterpenes (2.4%). Diterpenes and non-terpene derivatives were scarcely represented. Sixty compounds have been identified in the essential oil of H. spicigera: monoterpene hydrocarbons were the most represented class of volatiles (70.4%), followed by sesquiterpene hydrocarbons (22.6%). Results from topical applications on insects showed that both essential oils had an effective insecticidal activity. The complete kill of S. granarius was observed 24 h after treatment at a minimum effective dose of 0.4 and 0.6 μl per insect with H. suaveolens and H. spicigera oil, respectively. Results from repellency tests showed that the two essential oils had a repellent activity on S. granarius adults: at the lowest dose (2 × 10⁻⁴ μl oil per cm²), H. spicigera essential oil exhibited a higher repellent effect in comparison to H. suaveolens. No significant differences were observed for the repellent effect between the two essential oils at the highest dose (2 × 10⁻² μl oil per cm²).     

Genetic parameters and expected gains from selection for monoterpene yields in Petford Eucalyptus camaldulensis

Genetic parameters were estimated for yield of 1,8-cineole and total monoterpenes as a fraction of leaf weight in nineteen open-pollinated families of Petford origin at 3.75 years in two progeny/provenance trials of E. camaldulensis in Zimbabwe. Both traits appear to be highly heritable and, as expected, were strongly genetically correlated, with narrow-sense individual heritabilities near 0.50. Expected gain in the first generation following individual selection in the trials of one tree in 10 for either trait is about 25–32%.Genetic correlations between growth traits and 1,8-cineole yield were small. This indicates that both traits might be improved concurrently thus enhancing the economics of growing Petford E. camaldulensis for wood and medicinal oil. However, the presence of moderate and unfavourable genetic correlations between growth traits and total yield of monoterpenes warrants further study. It was not possible to gauge the significance of apparent family × site interaction for 1,8-cineole yield in this study. However, a test of rank correlation showed an association (Ip<0.01) between family rankings on each site and the highest-yielding trees on both sites came from the same families. This raises the possibility of being able to select individuals for cloning that yield well over a range of site conditions.     

Individual variations in monoterpenes released from Cryptomeria japonica and Pinus thunbergii needles

Variations in the compositions of low boiling point (LBP) monoterpenes in needle samples from 99 sugi (Cryptomeria japonica) and 100 kuromatsu (Pinus thunbergii) trees were investigated using a headspace technique. Considerable variations in the proportions of monoterpenes were revealed in both species. In sugi, the proportions of sabinene and α-pinene in the total LBP monoterpenes, ranging from 8.8% to 73.3% and from 14.5% to 73.3%, respectively, showed enormous variations among nine monoterpenes. The proportions of 3-carene and limonene, ranging from 0.1% to 29.5% and from 0.2% to 20.4%, respectively, also showed very specific variations. In kuromatsu, the proportions of β-pinene and α-pinene in the total LBP monoterpenes, ranging from 26.5% to 66.3% and from 18.7% to 46.9%, respectively, showed considerable variations among ten monoterpenes. The proportions of myrcene and 1,8-cineole, ranging from 0.9% to 18.5% and from 0.8% to 12.3%, respectively, also showed specific variations. Part of this article was presented at the 50th Annual Meeting of the Japan Wood Research Society, Kyoto, April, 2000, and the 13th Annual Meeting of the Chugoku Shikoku Branch of the Japan Wood Research Society, Okayama, September 2001     

Chemical analysis of essential oils of Eupatorium adenophorum and their antimicrobial,antioxidant and phytotoxic properties

Essential oils from inflorescences and roots of Eupatorium adenophorum Spreng (Asteraceae) have been investigated for their antimicrobial, phytotoxic and antioxidant activities. Based on GC–MS, the oil from inflorescences is dominated by sesquiterpenes (55.9 %) with γ-cadinene (18.4 %), γ-muurolene (11.7 %), 3-acetoxyamorpha-4,7(11)-diene-8-one (7.4 %) and bornyl acetate (6.3 %) as the major constituents. The oil obtained from the roots contained both sesquiterpenes (34.3 %) and monoterpenes (32.5 %) in almost equal proportions with E,E-cosmene (19.9 %), γ-muurolene (10.1 %), isothymol (7.5 %), β-cadinene (7.0 %) and α-phellandren-8-ol (5.9 %) as the major constituents. Both oils exhibited significant antifungal activity against five phytopathogenic fungi. The inflorescence oil showed higher antibacterial activity against Klebsiella pneumoniae, while the root oil was more effective against Staphylococcus aureus. The oils strongly inhibited or delayed germination and seedling growth of the weed Phalaris minor in a dose-dependent manner. As evidenced by a DPPH assay, the essential oils also exhibited significant free radical scavenging activity.     

Chemical structures of p-menthane monoterpenes with special reference to their effect on seed germination and termite mortality

A series of p-menthane monoterpenes was investigated to confirm any correlation between their bioactivity (effect on seed germination and termite mortality) and chemical structure. The germination percentages of Brassica rapa seeds at a concentration of 0.1 mg/Petri dish of (+)-pulegone, isopulegol, piperitone, (?)-dihydrocarveol, terpinen-4-ol and (?)-menthol were found to be 21.6, 27.3, 27.3, 29.1, 42.9 and 43.4, respectively. The lethal concentration 50 values of carvacrol, (+)-pulegone, thymol, (?)-menthol and (?)-terpinen-4-ol for termites (Reticulitermes speratus) were 0.34, 0.50, 0.65, 0.92 and 1.26 (mg/Petri dish), respectively. Of all the compounds tested, phenols produced the highest levels of termite mortality, with ketones and alcohols also showing bioactivity. An assessment of the bioactivity revealed that the presence of a phenol group was effective for termite mortality, with a carbonyl group also showing strong bioactivity. The presence of an alcohol or isopropyl group in a ring also contributed to the bioactivity, whereas the presence of an isopropenyl group at the same position, however, exhibited an inhibitory effect on seed germination. In conclusion, the bioactivity of the p-menthane monoterpenoids was dependent upon the presence and position of certain functional groups and the degree of saturation in the functional group of the side chain.     

Tree-to-tree and clone-to-clone variations of monoterpenes emitted from needles of hinoki (Chamaecyparis obtusa)

Variations in the composition of low boiling point (LBP) monoterpenes emitted from needle samples of 150 hinoki (Chamaecyparis obtuse) trees (30 strains, each with five clones) native to Shimane Prefecture, Japan, were investigated using a headspace technique. The assays revealed considerable proportional variations especially in the amount of sabinene, which ranged from 24% to 78% of the total LBP monoterpenes. The proportions of α-pinene, myrcene, and limonene negatively correlated with that of sabinene overall. In particular, the proportion of limonene showed clear negative correlation with that of sabinene (r = −0.98). Differences in the proportion of sabinene among five clones in each strain were less than 15% in 22 out of 30 strains, indicating that monoterpene composition is constitutively steady in most strains. In a few strains, however, considerable variation in the composition was observed among clones.     

In vitro inhibition of Heterobasidion annosum by Phaeotheca dimorphospora

The antagonistic fungus Phaeotheca dimorphospora has been tested in vitro on agar plates and wood discs to evaluate its potential use against P (Eastern Canada) and S (Finland) intersterility groups strains of Heterobasidion annosum using two methods. Production of antifungal metabolites by P. dimorphospora was demonstrated on agar plates as well as on Pinus resinosa and Picea abies discs using the bi‐layer technique. Diffusible metabolites inhibited the growth of the pathogen on both substrates. Direct confrontation on wood discs showed that a 7‐day period of advanced growth by P. dimorphospora completely prevented the colonization of the tissue by H. annosum. Control was partial when the antagonist had only one day of advanced growth. Growth inhibition was superior against S group strains with less than 1% of the samples colonized by H. annosum compared with 22% for P group strains. Moreover, P. dimorphospora was isolated in more than 83% of the samples in the absence of the pathogen in both wood species. In vitro tests demonstrated that P. dimorphospora is able to colonize red pine as well as Norway spruce tissue and to use wood components for the production of antifungal metabolites.     

Chemical composition,phytotoxic and antifungal activities of <Emphasis Type="Italic">Pinus pinea</Emphasis> essential oil

The chemical composition of essential oils isolated by hydrodistillation from the aerial parts of Tunisian Pinus pinea Linn. was analyzed by GC and GC/MS. Sixty-six compounds were identified, representing 98.5% of total oil, which was found to be rich in monoterpene hydrocarbons (73.1%) particularly limonene (54.1%), α-pinene (7.7%), and β-pinene (3.4%). The yield and the physico-chemical properties were determined. Results of the antifungal activity study by in vitro contact assay showed that P. pinea oil significantly inhibited the growth of ten plant pathogenic fungi. Moreover, herbicidal properties of the oil, tested on Sinapis arvensis L., Lolium rigidum Gaud., and Raphanus raphanistrum L., indicated that the oil completely inhibited the seed germination at a high concentration, while at low doses the oil acted by decreasing and delaying the germination and inhibiting the seedling growth of all tested weeds unlike the commercial herbicide. Our results showed that P. pinea essential oil could be valorized as bioproduct for biocontrol of weeds and fungal plant diseases.     

Potential of zimtaldehyde, 4-allyl-anisol, linalool, terpineol and other phytochemicals for the control of the confused flour beetle (Tribolium confusum J. d. V.) (Col., Tenebrionidae)

Toxicity and repellency effects of several pure compounds (of plant origin) against the confused flour beetle (Tribolium confusum) were investigated in a filter paper test. The compounds were α-pinen, β-pinen, 4-allyl-anisol, camphor, 1,8-cineol, eugenol, linalool, menthol, piperin, terpineol, thymol and zimtaldehyde. Five compounds (4-allyl-anisol, linalood, terpineol, thymol and zimtaldehyde) caused enough mortality of this insect to allow the determination of LD₅₀ and LD₉₅ values. Zimtaldehyde and 4-allyl-anisol showed the strongest toxicity effects with LD₅₀ values of 0.04–0.05 μl/cm² within an exposure period of 24 h. When the isolated fumigant effects were tested by separating the insects from the filter paper, the toxicity of zimtaldehyde (LD₅₀=0.29 μl/cm²) was much lower than that of 4-allyl-anisol (LD₅₀=0.10μl/cm²).     

Induction of tumours by various strains of Agrobacterium tumefaciens on Abies nordmanniana and Picea abies

Abies nordmanniana and Picea abies seedlings were inoculated with a wild‐type (C 58) or an attenuated strain (rooter or shooter mutants) of Agrobacterium tumefaciens. Large tumours were formed on A. nordmanniana in response to the wild‐type and rooter strains, 60–65 % of the seedlings being susceptible. Smaller tumours were formed on Picea abies in response to the wild‐type strain, at least 12% of the seedlings being susceptible. Tumours from both species induced by the wild‐type strain grew in vitro on medium without added phytohormones.