Use of solid phase microextraction (SPME) for profiling fungal volatile metabolites

AIMS: The influence of isolation methods: solid phase microextraction (SPME) with different fibres and simultaneous distillation extraction (SDE) on the profile of isolated fungal volatile metabolites was investigated. METHODS AND RESULTS: Four SPME fibre types: Polydimethylsiloxane, Polyacrylate, Carboxen/PDMS and Carboxen/Divinylbenzene/PDMS were evaluated in terms of their efficiency in extracting volatile metabolites emitted by Penicillium roqueforti grown on wheat kernel medium. All fibres showed varied efficiency and selectivity in extracting volatile compounds. Sesquiterpene hydrocarbons were the predominant fraction of volatile compounds isolated by all fibres, and ranged from 55.4 to 93.7% of all volatiles depending on the type of fibre used. Alcohols and ketones ranged from 2.7 to 20.5%, esters from 1.2 to 12.8%, and monoterpene hydrocarbons from 1.2 to 5.4%. Profile of volatile compounds obtained by SDE differed from SPME and the oxygenated sesquiterpenes formed the predominant fraction of volatiles isolated using SDE. SIGNIFICANCE AND IMPACT OF THE STUDY: The data in this study show that analysed profile of volatile compounds emitted by fungi is highly dependent on the extraction method.     

Feeding of large pine weevil on Scots pine stem triggers localised bark and systemic shoot emission of volatile organic compounds

Feeding by the large pine weevil (Hylobius abietis L.) causes severe damage to the bark of Scots pine (Pinus sylvestris L.) seedlings. We measured photosynthesis, the emission of volatile organic compounds from intact and weevil-damaged bark and systemic emissions from undamaged foliage. Feeding damage did not affect photosynthesis. Monoterpenes dominated the emissions from the feeding site, although some sesquiterpenes were also emitted. Weevil feeding increased bark emission of monoterpenes by nearly 4-fold and sesquiterpenes by 7-fold. The influence of weevil damage on systemic monoterpene emissions from shoots was more profound. Several compounds were substantially induced, including linalool, β-phellandrene, limonene and 1,8-cineole. Sesquiterpenes contributed only 1.2% of the total foliage emission, but comprised eight different compounds including (E,E)-α-farnesene, β-bourbonene and (E)-β-farnesene. The total emission of monoterpenes and sesquiterpenes from shoots was respectively 2.8-fold and 2.9-fold higher in the pine weevil damaged plants than the undamaged plants. As many of the induced compounds are highly reactive in the atmosphere and form organic aerosol particles, our results suggest that conifers damaged by insects could become a more important source of secondary organic aerosols than healthy trees.     

Sesquiterpenes of six Porella species (hepaticae)

The distribution in six Porella species of drimane-, aromadendrane- and pinguisane-type sesquiterpenes and norsesquiterpenes is described. The sharp pungent substance of P. gracillissima, P. fauriei and P. macroloba is (+)-tadeonal.     

Within-plant distribution and emission of sesquiterpenes from Copaifera officinalis

Copaifera officinalis, the diesel tree, is known for massive production of oleoresin, mainly composed of sesquiterpene hydrocarbons. In this study, composition of these sesquiterpenes and their concentrations in leaves, stems and roots of C. officinalis at two developmental stages, including the three-week old (TW) seedlings and two-year old (TY) trees, were determined. The leaves of TW seedlings and TY trees contained similar number of sesquiterpenes, which also had comparable concentrations. The stems of TW seedlings had higher concentrations of sesquiterpenes than those of TY trees. In contrast, the number of sesquiterpene species and their concentrations in the roots of TW seedlings were much lower than those in the roots of TY trees. Cluster analysis of sesquiterpenes estimated that there are at least four terpene synthase genes involved in the production of sesquiterpenes in C. officinalis. Because sesquiterpenes are highly volatile, emissions of sesquiterpenes from healthy and wounded TW seedlings were examined using headspace analysis. Whereas very low emission of sesquiterpenes was detected from undamaged plants, the physically injured seedlings emitted a large number of sesquiterpenes, the quality and the relative quantity of which were similar to those in leaves determined using organic extraction. The implications of our findings to the biosynthetic pathways leading to the production of sesquiterpenes as well as their biological roles in C. officinalis are discussed.     

Diversity and distribution of leaf mining insects on birches (<Emphasis Type="Italic">Betula</Emphasis> spp.) in Siberia

The diversity and distribution of leaf mining insects developing on birches (Betula spp.) in Siberia were reviewed based on published records and our observations. Analysis of the literature revealed 52 species of leaf miners recorded as feeding on different Betula species in Siberia. Among them, three species were listed under different names and six species were erroneously recorded as birch consumers. Thus, the revised list of birch leaf miners contains 44 species. Five moth and four sawfly species are mentioned in the literature as pests of Betula. Four sawflies are known to be invasive in North America. Our collections comprised 25 species, including the micro-moth Stigmella continuella (Lepidoptera, Nepticulidae), a new species for Siberia found in Novosibirsk. Immature stages of 15 species were identified using DNA barcoding. Twenty species were recorded from several regions of Siberia for the first time. The dominant group is Lepidoptera (31 species), followed by Coleoptera (7), Hymenoptera (5), and Diptera (1). Two-thirds of all the known leaf miners develop exclusively on birches; the remaining species also colonize alders (Alnus, Betulaceae), some Rosaceae, Salicaceae, and Ulmaceae. In our observations, the majority of insects (96%) developed on B. pendula. About half of them were also observed on the East Asian birches B. dahurica, B. divaricata, B. costata, B. ermanii, and B. gmelinii; five species were found on the North American birches B. occidentalis and B. papyrifera. All the leaf mining species listed in our paper for Siberia also occur in Europe. The similarity between the miner faunas of these regions is discussed and it is warned about possible errors in diagnostics of the Siberian species using the keys and catalogues for the European fauna. The importance of DNA barcoding in the study of the local insect faunas of poorly explored regions is also emphasized.     

Structural and functional peculiarities of plants from the genus <Emphasis Type="Italic">Betula</Emphasis> L. at early stages of ontogenesis

The morphological, biochemical, and physiological characteristics of silver birch (Betula pendula Roth), curly birch (B. pendula Roth var. carelica), and downy birch (B. pubescens Ehrh.) at early stages of ontogenesis in natural conditions were investigated. Some intra- and interspecific peculiarities of the morphophysiological properties were determined. Priority development of the underground mass for the seedlings of curly birch and that of the aboveground mass for the seedlings of silver birch and downy birch were found. The leaf of curly birch is developed more actively as compared with those in the other two species under equal potential possibility of the growth of their leaves by elongation. A high similarity of the mineral composition (C, N, P, K) of different forms and species of birches was demonstrated. For silver birch the maximum values of the stomatal conductance, rate of photosynthesis, and transpiration under similar atmospheric and soil conditions were obtained. In downy birch we found a higher efficiency of water use. The highest values of the maximum rate of ribulose 1,5-bisphosphate carboxylase/oxygenase carboxylation were found in curly birch. This may serve as an indicator of its shade tolerance, as compared with silver birch and downy birch.     

Do Insectivorous Birds use Volatile Organic Compounds from Plants as Olfactory Foraging Cues? Three Experimental Tests

Some insectivorous birds orient towards insect‐defoliated trees even when they do not see the foliar damage or the herbivores. There are, however, only a few studies that have examined the mechanisms behind this foraging behaviour. Previous studies suggest that birds can use olfactory foraging cues (e.g. volatile organic compounds (VOCs) emitted by defoliated plants), indirect visual cues or a combination of the two sensory cues. VOCs from insect‐defoliated plants are known to attract natural enemies of herbivores, and researchers have hypothesized that VOCs could also act as olfactory foraging cues for birds. We conducted three experiments across a range of spatial scales to test this hypothesis. In each experiment, birds were presented with olfactory cues and their behavioural responses or foraging outcomes were observed. In the first experiment, two different VOC blends, designed to simulate the volatile emissions of mountain birch (Betula pubescens ssp. czerepanovii) after defoliation by autumnal moth (Epirrita autumnata) larvae, were used in behavioural experiments in aviaries with pied flycatchers (Ficedula hypoleuca). The second experiment was a field‐based trial of bird foraging efficiency; the same VOC blends were applied to mountain birches, silver birches (B. pendula) and European white birches (B. pubescens) with plasticine larvae attached to the trees to serve as artificial prey for birds and provide a means to monitor predation rate. In the third experiment, the attractiveness of silver birch saplings defoliated by autumnal moth larvae versus intact controls was tested with great tits (Parus major) and blue tits (Cyanistes caeruleus) in an aviary. Birds did not orient towards either artificial or real trees with VOC supplements or towards herbivore‐damaged saplings when these saplings and undamaged alternatives were hidden from view. These findings do not support the hypothesis that olfactory foraging cues are necessary in the attraction of birds to herbivore‐damaged trees.     

Evolutionary patterns of volatile terpene emissions across 202 tropical tree species

Plant responses to natural enemies include formation of secondary metabolites acting as direct or indirect defenses. Volatile terpenes represent one of the most diverse groups of secondary metabolites. We aimed to explore evolutionary patterns of volatile terpene emission. We measured the composition of damage‐induced volatile terpenes from 202 Amazonian tree species, spanning the angiosperm phylogeny. Volatile terpenes were extracted with solid‐phase micro extraction and desorbed in a gas chromatography–mass spectrometry for compound identification. The chemical diversity of the terpene blend showed a strong phylogenetic signal as closely related species emitted a similar number of compounds. Closely related species also tended to have compositionally similar blends, although this relationship was weak. Meanwhile, the ability to emit a given compound showed no significant phylogenetic signal for 200 of 286 compounds, indicating a high rate of diversification in terpene synthesis and/or great variability in their expression. Three lineages (Magnoliales, Laurales, and Sapindales) showed exceptionally high rates of terpene diversification. Of the 70 compounds found in >10% of their species, 69 displayed significant correlated evolution with at least one other compound. These results provide insights into the complex evolutionary history of volatile terpenes in angiosperms, while highlighting the need for further research into this important class of compounds.     

Opportunistic emissions of volatile isoprenoids

Isoprene, monoterpenes and sesquiterpenes are synthesized and emitted by some plant species, but not all plant species have this ability. These volatile, nonessential isoprenoid compounds share the same biochemical precursors as larger essential isoprenoids such as gibberellic acids and carotenoids. They have many protective and ecological functions for the plant species that produce them, but plant species that do not produce these compounds also grow and reproduce successfully. Here, we develop an 'opportunist hypothesis' suggesting that (i) volatile isoprenoid production takes advantage of dimethylallyl diphosphate (DMAPP) and its isomer isopentenyl diphosphate (IPP), which are synthesized primarily to produce essential isoprenoids, and (ii) conditions affecting synthesis of the higher isoprenoids will affect the production and emission of volatile isoprenoids.     

Foraging by moose on two species of birch when these occur in different proportions

The two tree-like birch species Betula pendula and B. pubescens are of medium preference to moose during winter in northern Sweden. Because these birches are abundant in many biotopes, they form a major part of the moose diet. The two birches are very similar in appearance and often occur in mixed stands. Twenty-one birch stands where B. pendula formed from 2 to 97% of the birches were investigated to determine how the relative composition of the stands affected browsing of the two species by moose. In stands composed of 60% or less of B. pendula , there was a preference for this species when regarding 1.5–3.0 m high birches, which carry the largest amount of available browse per tree. At higher proportions B. pendula was still preferred, but use was closer to availability. However, in all stands there were significantly more moose bites per tree of B. pendula than of B. pubescens . In four birch stands browsing on 1.0–1.5 m high birches was also investigated. For this height class there was still a preference for B. pendula , but the difference between the two species was less pronounced than for the 1.5–3.0 m height class.     

The significance of bottom-up effects for host plant specialization in Chrysomela leaf beetles

The leaf beetle species Chrysomela lapponica, which belongs to the so‐called C. interrupta group, forms distinct allopatric populations either on willows (Salicaceae) or birches (Betulaceae). It was recently suggested that, on several occasions, host plant shifts from Salicaceae to Betulaceae occurred independently within the C. interrupta group. Our study aims to elucidate bottom‐up effects of the host plants that might have shaped the evolution of host plant specialization in the populations of C. lapponica, and thus, to shed some light on the driving forces of host shifts within the C. interrupta group, too. We compared the oviposition behaviour and performance of two C. lapponica populations, one of which has adapted to birches and the other to willows. The studies were conducted under laboratory conditions, eliminating the impact of natural enemies. Experiments involving the transfer of individuals of the birch‐specialized population to willows and vice versa with individuals of the willow‐specialized population to birches aimed to examine the plasticity in host plant use. Females of each population almost exclusively chose their natural host plant for oviposition, when offering birch and willow in dual choice experiments. When specimens of the two C. lapponica populations were reared on their natural host plants, the birch specialists suffered higher mortality, needed a longer period of development and produced less larval defensive secretion than the willow specialists. When the birch specialists were fed with willow, these performance parameters decreased even more. Other parameters, such as body weight and fecundity, did not differ between birch and willow specialists when they were fed with their natural host plant. While individuals of the birch‐specialized population could be reared on willow, all neonate larvae from the willow‐specialized population died after being transferred to birch. The significance of these bottom‐up effects for the evolution of host plant specialization in C. lapponica is discussed.     

Biochemical analysis of ‘kerosene tree’ Hymenaea courbaril L. under heat stress

Hymenaea courbaril or jatoba is a tropical tree known for its medically important secondary metabolites production. Considering climate change, the goal of this study was to investigate differential expression of proteins and lipids produced by this tree under heat stress conditions. Total lipid was extracted from heat stressed plant leaves and various sesquiterpenes produced by the tree under heat stress were identified. Gas chromatographic and mass spectrometric analysis were used to study lipid and volatile compounds produced by the plant. Several volatiles, isoprene, 2-methyl butanenitrile, β ocimene and a numbers of sesquiterpenes differentially produced by the plant under heat stress were identified. We propose these compounds were produced by the tree to cope up with heat stress. A protein gel electrophoresis (2-D DIGE) was performed to study differential expression of proteins in heat stressed plants. Several proteins were found to be expressed many folds different in heat stressed plants compared to the control. These proteins included heat shock proteins, histone proteins, oxygen evolving complex, and photosynthetic proteins, which, we believe, played key roles in imparting thermotolerance in Hymenaea tree. To the best of our knowledge, this is the first report of extensive molecular physiological study of Hymenaea trees under heat stress. This work will open avenues of further research on effects of heat stress in Hymenaea and the findings can be applied to understand how global warming can affect physiology of other plants.     

Differential volatile emissions and salicylic acid levels from tobacco plants in response to different strains of<Emphasis Type="Italic"> Pseudomonas syringae</Emphasis>

Pathogen-induced plant responses include changes in both volatile and non-volatile secondary metabolites. To characterize the role of bacterial pathogenesis in plant volatile emissions, tobacco plants, Nicotiana tabacum L. K326, were inoculated with virulent, avirulent, and mutant strains of Pseudomonas syringae. Volatile compounds released by pathogen-inoculated tobacco plants were collected, identified, and quantified. Tobacco plants infected with the avirulent strains P. syringae pv. maculicola ES4326 (Psm ES4326) or pv. tomato DC3000 (Pst DC3000), emitted quantitatively different, but qualitatively similar volatile blends of (E)-beta-ocimene, linalool, methyl salicylate (MeSA), indole, caryophyllene, beta-elemene, alpha-farnesene, and two unidentified sesquiterpenes. Plants treated with the hrcC mutant of Pst DC3000 (hrcC, deficient in the type-III secretion system) released low levels of many of the same volatile compounds as in Psm ES4326- or Pst DC3000-infected plants, with the exception of MeSA, which occurred only in trace amounts. Interaction of the virulent pathogen P. syringae pv. tabaci (Pstb), with tobacco plants resulted in a different volatile blend, consisting of MeSA and two unidentified sesquiterpenes. Overall, maximum volatile emissions occurred within 36 h post-inoculation in all the treatments except for the Pstb infection that produced peak volatile emissions about 60 h post-inoculation. (E)-beta-Ocimene was released in a diurnal pattern with the greatest emissions during the day and reduced emissions at night. Both avirulent strains, Psm ES4326 and Pst DC3000, induced accumulation of free salicylic acid (SA) within 6 h after inoculation and conjugated SA within 60 h and 36 h respectively. In contrast, SA inductions by the virulent strain Pstb occurred much later and conjugated SA increased slowly for a longer period of time, while the hrcC mutant strain did not trigger free and conjugated SA accumulations in amounts significantly different from control plants. Jasmonic acid, known to induce plant volatile emissions, was not produced in significantly higher levels in inoculated plants compared to the control plants in any treatments, indicating that induced volatile emissions from tobacco plants in response to P. syringae are not linked to changes in jasmonic acid.     

In situ modification of herbivore-induced plant odors: a novel approach to study the attractiveness of volatile organic compounds to parasitic wasps

Many parasitic wasps (parasitoids) exploit volatile organic compounds (VOCs) emitted by herbivore-infested plants in order to locate their hosts, but it remains largely unknown which specific compounds within the volatile blends elicit the attractiveness to parasitoids. One way of studying the importance of specific VOCs is to test the attractiveness of odor blends from which certain compounds have been emitted. We used this approach by testing the attraction of naive and experienced females of the two parasitoids Cotesia marginiventris and Microplitis rufiventris to partially altered volatile blends of maize seedlings (Zea mays var. Delprim) infested with Spodoptera littoralis larvae. Adsorbing filter tubes containing carbotrap-C or silica were installed in a four-arm olfactometer between the odor source vessels and the arms of the olfactometer. The blends breaking through were tested for chemical composition and attractiveness to the wasps. Carbotrap-C adsorbed most of the sesquiterpenes, but the breakthrough blend remained attractive to naive C. marginiventris females. Silica adsorbed only some of the more polar VOCs, but this essentially eliminated all attractiveness to naive C. marginiventris, implying that among the adsorbed compounds there are some that play key roles in the attraction. Unlike C. marginiventris, M. rufiventris was still attracted to the latter blend, showing that parasitoids with a comparable biology may employ different strategies in their use of plant-provided cues to locate hosts. Results from similar experiments with modified odor blends of caterpillar-infested cowpea (Vigna unguiculata) indicate that key VOCs in different plant species vary greatly in quality and/or quantity. Finally, experienced wasps were more strongly attracted to a specific blend after they perceived the blend while ovipositing in a host. Considering the high number of distinct adsorbing materials available today, this in situ modification of complex volatile blends provides a new and promising approach pinpointing on key attractants within these blends. Advantages and disadvantages compared to other approaches are discussed.     

Structural diversity and geographical distribution of halogenated secondary metabolites in red algae,Laurencia nangii Masuda (Rhodomelaceae,Ceramiales), in the coastal waters of North Borneo Island

The red algae genus Laurencia (Rhodomelaceae, Ceramiales) is known as a prolific producer of halogenated secondary metabolites with a high level of species diversity and geographical distribution. In North Borneo Island, Malaysia, there are four main Laurencia species: Laurencia snackeyi, Laurencia majuscula, Laurencia similis and L. nangii. Although the chemistry of Laurencia is well studied, the diversity of compounds in L. nangii has not been thoroughly investigated. Therefore, we studied the chemical constituents of seven populations of L. nangii from Tunku Abdul Rahman Marine Park (two populations), Dinawan Island (one population), Tun Mustapha Marine Park (two populations) and Tun Sakaran Marine Park (two populations). Halogenated compounds were isolated and the structures determined via spectroscopic methods. A total of 20 metabolites belonging to the classes of sesquiterpenes, acetylenes, bromoallenes, diterpenes and triterpenes were identified. Populations from Tunku Abdul Rahman Marine Park and Dinawan Island contained non-chamigrane-type sesquiterpenes, acetylenes and diterpenes. Populations from Tun Mustapha Marine Park contained chamigrane-type sesquiterpenes, acetylenes and diterpenes. However, the chemical compositions of populations from Tun Sakaran Marine Park were found to differ significantly, containing chamigrane-type and non-chamigrane-type sesquiterpenes, bromoallenes and triterpenes. This investigation has revealed the presence of interesting chemotaxonomical markers in populations of L. nangii and the existence of chemical races in this species.     

Emitted and endogenous floral scent compounds of Prunus mume and hybrids

Prunus mume is the only species of Prunus known to produce a strong floral fragrance. Most interspecific hybrids between P. mume and other species of Prunus lack the fragrance. The analysis of variations in emitted and endogenous compounds among genetically close cultivars is a powerful approach for revealing the mechanisms underlying floral scent emission. Compounds emitted by flowers from five cultivars were collected using the static headspace method, and endogenous compounds in the flowers were extracted with ethyl acetate. Samples were analysed quantitatively and qualitatively using gas chromatography-mass spectrometry. The result showed that benzenoids were the dominant compounds, of which benzyl acetate was the principal component contributing to the floral scent of P. mume. A clustering analysis of the floral volatiles from the different cultivars suggested that the scent traits of hybrids are related to the taxonomic relationship between their parents. The correlations between the amount of the endogenous and emitted compounds revealed that benzyl acetate had a stronger tendency to be volatile than the other compounds and the volatilisation rate of volatile compounds varied greatly among different cultivars. The importance of the biosynthetic pathway and the function of benzaldehyde are discussed.     

Ecological role of reindeer summer browsing in the mountain birch (<Emphasis Type="Italic">Betula pubescens</Emphasis> ssp. <Emphasis Type="Italic">czerepanovii</Emphasis>) forests: effects on plant defense,litter decomposition,and soil nutrient cycling

Mammalian herbivores commonly alter the concentrations of secondary compounds in plants and, by this mechanism, have indirect effects on litter decomposition and soil carbon and nutrient cycling. In northernmost Fennoscandia, the subarctic mountain birch (Betula pubescens ssp. czerepanovii) forests are important pasture for the semidomestic reindeer (Rangifer tarandus). In the summer ranges, mountain birches are intensively browsed, whereas in the winter ranges, reindeer feed on ground lichens, and the mountain birches remain intact. We analyzed the effect of summer browsing on the concentrations of secondary substances, litter decomposition, and soil nutrient pools in areas that had been separated as summer or winter ranges for at least 20 years, and we predicted that summer browsing may reduce levels of secondary compounds in the mountain birch and, by this mechanism, have an indirect effect on the decomposition of mountain birch leaf litter and soil nutrient cycling. The effect of browsing on the concentration of secondary substances in the mountain birch leaves varied between different years and management districts, but in some cases, the concentration of condensed tannins was lower in the summer than in the winter ranges. In a reciprocal litter decomposition trial, both litter origin and emplacement significantly affected the litter decomposition rate. Decomposition rates were faster for the litter originating from and placed into the summer range. Soil inorganic nitrogen (N) concentrations were higher in the summer than in the winter ranges, which indicates that reindeer summer browsing may enhance the soil nutrient cycling. There was a tight inverse relationship between soil N and foliar tannin concentrations in the winter range but not in the summer range. This suggests that in these strongly nutrient-limited ecosystems, soil N availability regulates the patterns of resource allocation to condensed tannins in the absence but not in the presence of browsing.     

Abscisic acid promotes root system development in birch tissue culture: a comparison to aspen culture and conventional rooting‐related growth regulators

The research aim was to assess the effects of the plant hormone abscisic acid (ABA) and the growth regulator paclobutrazol (PBZ) on root system development during the in vitro culture of different birch and aspen genotypes. The studied genotypes involved two aspen (Populus tremula and Populus tremuloides × P. tremula) and two silver birch (Betula pendula) trees, with one of the birches characterized by its inability to root in vitro. For experiments, apical shoot segments were cultured on nutrient medium enriched with either ABA or PBZ. Additionally, the analysis of the endogenous hormones in shoots developed on hormone‐free medium was conducted by high‐performance liquid chromatography. The endogenous concentration of auxin indole‐3‐acetic acid was much higher in the aspens than that in the birches, while the highest concentration of ABA was found in the root‐forming birch. The culturing of this birch genotype on medium enriched with ABA resulted in an increased root length and a higher number of lateral roots without any negative effect on either shoot growth or adventitious root (AR) formation, although these two processes were largely inhibited by ABA in the aspens. Meanwhile, PBZ promoted AR formation in both aspen and birch cultures but impaired secondary root formation and shoot growth in birches. These results suggest the use of ABA for the in vitro rooting of birches and PBZ for the rooting of aspens.