Phenolic compounds of willow bark as deterrents against feeding by mountain hare

Summary Overwintering mountain hares (Lepus timidus) fed selectively on the shoots of a number of northern willow (Salix spp.) species. The hares preferred certain species over others and mature shoots over juvenile ones. There was a negative correlation between the phenolic glycoside concentration in the twigs and the hares' feeding. This correlation was substantiated by feeding experiments in which oat grains treated with purified phenoglycoside and catechin fractions of willow bark were offered along with untreated control oat grains to free-ranging mountain hares. Both fractions in concentrations normally found in willow twigs inhibited hare feeding. The results suggest that these phenolic compounds play a crucial role in the building up of resistance patterns among the willows. The decline in resistance in mature shoots of tall willows indicates that the juvenile resistance can be, perhaps secondarily, an adaptation against mammals browsing from ground level. Accordingly, low willow species retain a high level of resistance also in maturity.     

Simultaneous determination of five characteristic stilbene glycosides in root bark of Morus albus L. (Cortex Mori) using high-performance liquid chromatography

Introduction – Cortex Mori, one of the well‐known traditional Chinese herbal medicines, is derived from the root bark of Morus alba L. according to the China Pharmacopeia. Stilbene glycosides are the main components isolated from aqueous extracts of Morus alba and their content varies depending on where Cortex Mori was collected. We have established a qualitative and quantitative method based on the bioactive stilbene glycosides for control of the quality of Cortex Mori from different sources. Objective – To develop a high‐performance liquid chromatography coupled with ultraviolet absorption detection for simultaneous quantitative determination of five major characteristic stilbene glycosides in 34 samples of the root bark of Morus alba L. (Cortex Mori) from different sources. Methodology – The analysis was performed on an ODS column using methanol‐water‐acetic acid (18: 82: 0.1, v/v/v) as the mobile phase and the peaks were monitored at 320 nm. Results – All calibration curves showed good linearity (r ≥ 0.9991) within test ranges. This method showed good repeatability for the quantification of these five components in Cortex Mori with intra‐ and inter‐day standard deviations less than 2.19% and 1.45%, respectively. Conclusion – The validated method was successfully applied to quantify the five investigated components, including a pair of cis‐trans‐isomers 1 and 2 and a pair of isomers 4 and 5 in 34 samples of Cortex Mori from different sources. Copyright © 2010 John Wiley & Sons, Ltd.     

Phenolic compounds of Siberian and Dahurian larch phloem

The paper presents results of the investigation of phenolic compounds of the bark of the Siberian larch (Larix sibirica Ledeb.) and the Dahurian larch (Larix gmelini (Rupr.) Rupr). The flavonoids, stilbenes, phenolic acids have been identified on the basis of chemical, chromatographic and spectral data. Quercetin-3-O-??-L-rhamnopyranoside has been found for the first time in the bark of the Larix genus. Phenolic compounds with pyrocatechol type of substitution of aromatic ring have been found to dominate in the phloem of the larch species considered.     

Aqueous and ethanolic extracts of Galinsoga parviflora and Galinsoga ciliata. Investigations of caffeic acid derivatives and flavonoids by HPTLC and HPLC-DAD-MS methods

Galinsoga ciliata Raf. Blake like Galinsoga parviflora Cav., comes from the Andes region. The chemical composition, activity and use are similar for both species. Galinsoga species are used in folk medicine as anti-inflammatory agents and accelerators for wound healing. Extracts are applied topically onto the skin to treat dermatological diseases, eczemas, lichens and hard-healing-wounds, and also to treat snakebites. Orally they used to cure flu and colds.In the studies using HPTLC method, different stationary phases, including unmodified silica gel, silica gels modified with CN, NH₂, DIOL and RP18 groups were tried. The best separation of the tested compounds was achieved on silica gel plates, when as mobile phases mixtures – ethyl acetate–acetic acid–formic acid–water (100:11:11:26, v/v/v/v), ethyl acetate–methanol–formic acid–water (50:3:4:6, v/v/v/v) and ethyl acetate–methyl ethyl ketone–formic acid–water (30:9:3:3, v/v/v/v) – were used. Using reference substances, in the examined extracts the presence of flavonoids: patulitrin, quercimeritrin, quercitagetrin, and phenolic acids – caffeic and chlorogenic acids was found.HPLC analyses of extracts were carried out on a reversed-phase Zorbax SB column (150 mm × 2.1 mm, 1.9 μm). The mobile phase (A) was water/acetonitrile/formic acid (95:5:0.1, v/v/v) and the mobile phase (B) was acetonitrile/formic acid (100:0.1, v/v). A linear gradient system was used: 0–30 min, 1–30% B. Application of HPLC-DAD-MS method confirmed the results obtained by HPTLC method. Moreover, in the tested extracts the presence of caffeoylglucaric acids as dominating compounds was detected.     

Phytoextraction of cadmium and zinc by Salix from soil historically amended with sewage sludge

Short rotation coppice (SRC) such as Salix spp. can be grown as an energy crop and offers some potential for economic and practical phytoextraction of marginally contaminated arable soil. This study tested various soil amendments intended to increase soil metal availability to Salix, investigated the distribution of metal between different tree fractions and assessed the viability of phytoextraction using SRC on arable soils. Several Salix genotypes were grown in field trials over 4 years. Cd and Zn concentrations were generally ranked in the order leaves > bark > wood. Metal concentrations in wood increased towards the top of the willow stems, whereas concentrations in leaves showed the opposite trend. None of the amendments significantly increased uptake of Zn by willow. However, in response to a range of soil HCl treatments, mean Cd concentrations in stems and leaves were 112% and 130% of control values. Data from the current experiment, and previous studies, were combined to develop a predictive model of Cd and Zn stem uptake by Salix. The minimum biological concentration factor (BCF) required to achieve a prescribed soil metal target was also calculated based on typical proportions of bioavailable Cd in sludge-amended soils for a 25-year Salix rotation. The best Salix genotypes investigated achieved less than 20% of the uptake rate required to remove one third of the soil Cd content (equivalent to the average isotopically exchangeable Cd fraction in soils at the study site).     

Size class structure and tree dispersion patterns in old-growth cedar-hemlock forests of the northern Rocky Mountains (USA)

Summary The relationship between the food selection of four leaf beetle species (Phratora vitellinae, Plagiodera versicolora, Lochmaea capreae, Galerucella lineola) and the phenolic glycosides of willow (Salix spp.) leaves was tested in laboratory food choice experiments. Four willow species native to the study area (Eastern Finland) and four introduced, cultivated willows were tested.The willow species exhibited profound differences in their phenolic glycoside composition and total concentration. The food selection patterns of the leaf beetles followed closely the phenolic glycoside spectra of the willow species. Both the total amount and the composition of phenolic glycosides affected the feeding by the beetles. Phenolic glycosides apparently have both stimulatory and inhibitory influences on leaf beetle feeding depending on the degree of adaptation of a particular insect. Very rare glycosides or exceptional combination of several glycoside types seem to provide certain willow species with high level of resistance against most herbivorous insects. Analogously the average absolute amount of leaf beetle feeding was lower on the introduced willows than on the native species to which the local herbivores have a good opportunity to become adapted.     

Effects of different leaf traits on growth rates of insect herbivores on willows

We examined relative effects of traits of leaf quality of ten willow species (Salix: Salicaceae) on growth rates of five species of insect herbivores found in interior Alaska (a willow sawfly, Nematus calais; the tiger swallowtail butterfly, Papilio canadensis; and three species of chrysomelid beetles, Gonioctena occidentalis, Calligrapha verrucosa, and Chrysomela falsa). Leaf traits examined were water content, toughness, total nitrogen contnet, pubescence, and presence or absence of phenolic glycosides. Of ten Salix species, four species contain phenolic glycosides in their leaves. We examined relative effects of water content, toughness, and nitrogen content of the Salix leaves on larval growth rates at three different levels, i.e., on a single host species, between different host species, and between herbivore species. The within-host analyses showed that effects of water content, toughness and/or nitrogen content on herbivore growth rates were generally significant in early-season herbivores but not in late-season herbivores. For each herbivore species, differences in growth rates between hosts were not explained by differences in water content, toughness, or nitrogen content. The between-herbivore analysis showed that the interspecific difference in larval growth rates were related to difference in water and nitrogen content of the hosts. Pubescence of Salix leaves had little effects on herbivore growth rates. Presence of phenolic glycosides had a positive effects on growth rates of a specialist, N. calais, but no effect on the other specialist, Ch. falsa. Presence of phenolic glycosides had, in general, negative effects on growth rates of nonspecialists, G. occidentalis, C. verrucosa, and P. canadensis.     

不同能源柳无性系对土壤镉污染的抗性研究

运为了比较不同能源柳无性系对土壤镉抗性的差异,采用盆栽方法,设置土壤镉含量5个梯度(0、10、20、40、80 mg·kg^-1),以乡土旱柳为对照,对能柳1、能柳2、能柳E、能柳C的扦插幼苗对土壤镉污染的生理生态反应进行了系统测定,分析了不同无性系的抗性。结果显示,低浓度处理（≤20 mg·kg^-1）促进苗木生长,其中能柳2在10 mg·kg^-1处理下根和单株生物量增幅最大,可达到对照的132.0%和120.0%;随着镉浓度的增加,生长量下降,在最高浓度处理下,能柳C生长量降低最少,根、枝、叶和单株生物量分别为对照的86.1%、85.3%、82.9%、84.9%,旱柳的枝、叶生物量降低最多,为对照（无镉）的43.8%、45.0%,而能柳1的根、单株生物量降低最多,仅为对照的36.1%、44.4%;随着镉浓度的增加,不同能源柳无性系叶片的SOD、POD、CAT活性和根系活力基本呈现先升后降的趋势,其中,CAT活性变化较为平缓;关于SOD活性和根系活力,能柳1、能柳2在20 mg·kg^-1时已显著下降,旱柳在40 mg·kg^-1时显著下降,而能柳E、能柳C在40 mg·kg^-1时才显著下降。采用隶属函数对5个无性系在镉胁迫条件下的生长和酶指标进行综合分析,不同无性系对土壤镉污染的抗性顺序为能柳C>能柳2>能柳1>旱柳>能柳E。不同无性系对镉均有抗性,都可在镉污染区推广种植,栽培中,应根据土壤污染和绿化目标,做出适当选择。     

Biodiversity of Salix spp. honeydew and nectar honeys determined by RP-HPLC and evaluation of their antioxidant capacity

Rare unifloral willow (Salix spp.) honeys obtained from nectar or honeydew were investigated by direct RP‐HPLC‐DAD method in order to identify and quantify compounds that can be used as possible markers of their origin. Antioxidant and antiradical activities of willow honeys were evaluated using FRAP (=ferric reducing antioxidant assay) and DPPH (=1,1‐diphenyl‐2‐picrylhydrazyl radical) tests, respectively. Also HMF (=5‐(hydroxymethyl)furfural), diastase activity, and CIE L*a*b*C*h* chromatic coordinates were evaluated. Abscisic acids (ABA) are typical of willow nectar honey, with a predominance of (Z,E)‐ABA on (E,E)‐ABA (98.2 and 31.7 mg/kg, resp.). Kinurenic acid and salicylic acid are useful to mark willow honeydew honey. The proposed HPLC‐DAD method proved to be easy and reliable to identify the two different Salix spp. honeys, being not affected from any sample preparation artifact. Total antioxidant activity measured with the FRAP assay ranged from 3.2 to 12.6 mmol Fe²⁺/kg, and the antiradical activity measured with the DPPH assay ranged from 0.6 to 3.0 mmol TEAC (=Trolox equivalent antioxidant capacity)/kg in nectar and honeydew honeys, respectively. Salix spp. nectar and honeydew honeys proved to be two completely different honeys, because, besides color attributes, they show different antioxidant properties and specific compounds.     

Alignment of a <Emphasis Type="Italic">Salix</Emphasis> linkage map to the <Emphasis Type="Italic">Populus</Emphasis> genomic sequence reveals macrosynteny between willow and poplar genomes

Poplars (genus Populus) and willows (genus Salix) are members of the Salicaceae, a family of catkin-bearing trees, shrubs and sub-shrubs. Poplar is considered the model system for biological studies in trees and considerable genetic and genomic resources have become available in recent years. The transfer of information to research studies in willow, for which fewer resources are currently available, would be highly beneficial. However, the extent of conservation between poplar and willow genomes has not yet been extensively studied. To address this, we have constructed a linkage map of willow based on a large mapping population derived from a cross between two Salix viminalis × (S. viminalis × S. schwerinii) hybrid sibs, and aligned this to the publicly available poplar genome sequence. A set of genome-wide, expressed poplar sequences was selected and used to design primer sets that efficiently amplified homeologous regions in willow. Direct sequencing of the willow products confirmed homology with the poplar target in the majority of instances and allowed identification of single nucleotide polymorphisms (SNPs) that were used to map these loci. In total, 202 amplified fragment length polymorphisms (AFLPs), 75 microsatellites and 79 SNPs were used to construct a willow consensus map that spanned 1,856.7 cM with an average interval between markers of 6.3 cM. Poplar sequences homologous to those of the mapped willow microsatellite loci were identified and used in addition to the SNP markers to putatively align all but two minor linkage groups to the poplar genome sequence. A high degree of macrosynteny was revealed.     

Systematic characterisation of secondary metabolites from Ixeris sonchifolia by the combined use of HPLC‐TOFMS and HPLC‐ITMS

Introduction – Ixeris sonchifolia (Bunge) Hance, a folk medicine, has been widely used in China for its anti‐inflammatory and haemostatic effects. However, the miscellaneous component composition of this herbal medicine is not well known. Objective – To develop a fast and comprehensive analytical method for the characterisation of various components from I. Sonchifolia, as a tool for the quality control of the herb and its related preparations. Methodology – Ixeris sonchifolia samples were extracted with 60% aqueous methanol, purified by solid‐phase extraction and then analysed by the combinatorial use of HPLC‐TOFMS and HPLC‐ITMS. Results – A total of six sesquiterpene lactones, six phenolic acids and seven flavonoids were identified or tentatively characterised. Five of them were reported for the first time in I. sonchifolia and, in particular, two amino acid‐sesquiterpene lactone conjugates, 11,13‐dihydro‐13‐prolyl‐ixerin Z and 11,13‐dihydro‐13‐prolyl‐ixerin Z₁, that were first found in this plant source. Conclusion – A global profile of I. sonchifolia constituents was described, which could be useful for the quality control of this herb and its related preparations. The employed combination of HPLC‐TOFMS and HPLC‐ITMS could also be a promising tool for the analysis of other herbal medicines containing sesquiterpene lactones, phenolic acids or flavonoids. Copyright © 2010 John Wiley & Sons, Ltd.     

Beetle assemblages on willow trees: do phenolic glycosides matter?

Herbivorous beetles were sampled in central Slovakia and in the Tatra Mountains of northern Slovakia from seven different Salix species which are partly characterized by smooth leaves containing phenolic glycosides and partly by hairy leaves containing tannins. The collection included about 8000 individuals representing 129 species. Of the 129 species, 77 species are able to use the willows as hosts; the remaining 52 ‘tourist’ species comprise less than 3% of the collected individuals. The data on species richness and abundance did not generally support the ‘feeding specialization’ hypothesis. The proportion of specialized (= monophagous and oligophagous) beetles feeding on willows of both morphological and biochemical groups was in the same range. Host plants of the two groups could support high diversity and high evenness values, even when leaf characteristics and plant chemicals largely influenced species assemblage. The region in which the willow trees grow had a considerable impact on host plant use. Generalist beetle species predominated in central Slovakia. By contrast, in the Tatra Mountains, specialist feeders which are able to use phenolic glycosides to their advantage were predominant. The number of species and the total density of individuals collected from willows containing phenolic glycosides (S. fragilis and S. purpurea) did not usually vary between the two regions. In contrast, fewer species and individuals were found in the Tatra Mountains when they settled on willow species containing tannins (S. caprea and S. cinerea). Also, the phylogenetic status of host plants affected species assemblages. In central Slovakia willow species of the subgenus Vetrix (S. purpurea, S. caprea and S. cinerea) generally showed a higher beetle diversity (H_s) than species of the subgenus Salix (S. fragilis, S. alba and S. triandra), although both subgenera comprise species of both morphological and biochemical groups. Furthermore, when the analysis was restricted to beetles of central Slovakia, which should be most adapted to their host plants (i.e. catkin feeders and phyllophages in the adult and larval stage), the phylogenetic status was found to be more important than any single leaf character measured.     

Potential of Salix as phytoextractor for Cd on moderately contaminated soils

Biomass production and Cd uptake in Salix (willow) is generally high. The aim of this study was to quantify the amounts of Cd in different plant and soil compartments in commercial Salix stands grown on different soil types and to use these data for estimation of the long-term effects of Cd removal by Salix at stem harvest on the soil Cd content by calculating Cd balances. Ten commercial short rotation willow coppice stands, planted with clone 78183 of Salix viminalis (L.) were investigated in order to include three different soil types with respect to pH. Stands with high stem biomass production generally had lower Cd concentrations in the plant and grew on more alkaline soils with low Cd availability when compared to stands with low stem biomass production. The highest output of Cd by Salix is therefore not necessarily achieved by stands with the highest yields or the highest Cd concentrations, as both these parameters have to be considered. The net removal of Cd from the plough layer by the Salix crop under different conditions varied between 2.6 and 16.5 g Cd ha^–1 year^–1. The long-term effects of Salix cultivation on soil Cd content, i.e. after 6–7 cutting cycles (25 years), were calculated and we reached the conclusion that there is a high potential in Salix to remove significant amounts of Cd at stem harvest. Salix can therefore presumably be used to counteract Cd pollution of agricultural soils in Sweden provided that the Cd in furnace ashes is taken care of.     

Susceptibility of willow clones (Salix spp.) to herbivory by Phyllodecta vulgatissima (L.) and Galerucella lineola (Fab.) (Coleoptera, Chrysomelidae)

Levels of damage by mixed natural infestations of the leaf-feeding chrysomelid beetles, Phyllodecta vulgatissima (L.) (the blue willow beetle) and Galerucella lineola (Fab.) (the brown willow beetle), were determined in replicated field plots of 24 Salix clones at Long Ashton (Bristol, UK) during 1993–94. Over the same period, the host plant preferences of both chrysomelids were investigated in a standard multiple-choice laboratory procedure, where beetles were enclosed in Petri dishes with leaf discs cut from young pot-grown trees propagated from shoot cuttings taken from 20 of the 24 willow clones represented in the field study. The laboratory experiments indicated that P. vulgatissima and G. lineola had similar host plant preferences in the range of willows examined (r >0.85). In both field and laboratory, the least preferred Salix clones and hybrids were those of 5. eriocephala, followed by S. purpurea, S. burjatica, S. dasyclados and S. triandra. Clones of S. eriocephala and S. purpurea were frequently rejected altogether in laboratory tests. Most preferred were clones of S. viminalis and several hybrids of S. viminalis, S. aurita, S. caprea and S. cinerea. These results substantiate the reports that P. vulgatissima and G. lineola are deterred from feeding on willows which have relatively high concentrations of phenolic (salicylate) glucosides in the leaves. The least preferred willows, particularly S. eriocephala, S. purpurea and S. burjatica, could be of great potential value in plant breeding for resistance to these willow beetle pests.     

Heavy Metal Uptake by Willow Clones from Sewage Sludge-Treated Soil: The Potential for Phytoremediation

Willow (Salix spp.) has shown potential for use in the phytoremediation of soil contaminated with heavy metals. In particular, it can be grown in short rotation coppice systems to produce biomass that can be used for energy production. Twenty different species or varieties of willow, grown over 2 years (1995 to 1997) on a soil that was highly contaminated with heavy metals due to long-term sewage sludge disposal, showed considerable variation in survival, biomass production and metal uptake. The willows could be divided into two groups after the first harvest. One group had relatively low Ni and Cu in the bark and high Cd and Zn in the wood, with a good survival rate and biomass production. This group partitioned Cu, Cd, and Zn into the wood tissue from the bark, whereas Ni was excluded. The second group had relatively high Ni and Cu in the bark and low Cd and Zn in the wood and performed poorly in terms of survival and biomass production. Of the 20 types of willow used, 11 showed potential for use in phytoremediation, combining good survival and biomass production with high metal uptake. Of the others, 2 failed to survive until the second harvest and the other 7 had very poor survival rates.     

Qualitative analysis and simultaneous quantification of phenolic compounds in the aerial parts of Salvia miltiorrhiza by HPLC-DAD and ESI/MS(n)

Introduction – The increasing demands of roots and rhizomes of Salvia miltiorrhiza almost exhausted the wild Salvia sources in China. However, the content and composition of phenolic acids in the aerial parts of the plant and their potential to be used as a substitute has not been explored. Objective – To evaluate the potential of the aerial parts of Salvia miltiorrhiza as new natural sources of phenolic acids. Methodology – HPLC coupled with diode array detection (DAD) and electrospray ionization multistage mass spectrometry (ESI/MSⁿ) has been used for qualitative and quantitative analysis of phenolic compounds. Results – A total of 38 phenolic compounds were identified or tentatively characterized. A quantitative HPLC‐DAD method allowing the simultaneously quantification of six phenolic acids was optimized and validated for linearity, precision, accuracy, and limits of detection and quantification. Calibration curves showed good linear regression (r² > 0.9991) within test ranges; the recoveries ranged between 95.64 and 101.67% and the RSDs were less than 3.01%. Conclusion – The developed methods have been proved to be effective for the identification and quantification of phenolic acids in S. miltiorrhiza. The results obtained suggest that the aerial parts of the plant could be used as an alternative source of sage phenolics. Copyright © 2010 John Wiley & Sons, Ltd.     

Phylogenetic Relationships of American Willows (Salix L., Salicaceae)

Salix L. is the largest genus in the family Salicaceae (450 species). Several classifications have been published, but taxonomic subdivision has been under continuous revision. Our goal is to establish the phylogenetic structure of the genus using molecular data on all American willows, using three DNA markers. This complete phylogeny of American willows allows us to propose a biogeographic framework for the evolution of the genus. Material was obtained for the 122 native and introduced willow species of America. Sequences were obtained from the ITS (ribosomal nuclear DNA) and two plastid regions, matK and rbcL. Phylogenetic analyses (parsimony, maximum likelihood, Bayesian inference) were performed on the data. Geographic distribution was mapped onto the tree. The species tree provides strong support for a division of the genus into two subgenera, Salix and Vetrix. Subgenus Salix comprises temperate species from the Americas and Asia, and their disjunction may result from Tertiary events. Subgenus Vetrix is composed of boreo-arctic species of the Northern Hemisphere and their radiation may coincide with the Quaternary glaciations. Sixteen species have ambiguous positions; genetic diversity is lower in subg. Vetrix. A molecular phylogeny of all species of American willows has been inferred. It needs to be tested and further resolved using other molecular data. Nonetheless, the genus clearly has two clades that have distinct biogeographic patterns.     

Spillover of tent caterpillar (Malacosoma americanum) herbivory onto willow bioenergy crops in an agricultural landscape

The circumstances and potential for insects to damage perennial bioenergy crops is not well understood in the United States. In this study, we evaluated the spillover and herbivory of eastern tent caterpillars (Malacosoma americanum) from host trees onto short rotation coppice (SRC) willow bioenergy crops (Salix sp.). Host trees were all in the Rosaceae family and included Prunus americana, Prunus virginiana and Malus sp. Willow showed greater leaf herbivory with increasing proximity to a defoliated host tree, suggesting that tent caterpillars spilled‐over to willow after denuding their host. More tent caterpillar herbivory was associated with greater mortality of willow. This study suggests that landscape context and spatial position of host trees is important to the early establishment of a willow bioenergy crop.