Tannins, flavonoids and stilbenes in extracts of African savanna woodland trees Terminalia brownii, Terminalia laxiflora and Anogeissus leiocarpus showing promising antibacterial potential

Terminalia laxiflora, Terminalia brownii and Anogeissus leiocarpus are used as decoctions, macerations, infusions and fumigations in East and West African traditional medicine for treatment of infectious diseases and their symptoms. Using this ethnopharmacological information as a guideline for our research and owing to the fact that these species have not been subjected to in depth antibacterial and phytochemical studies, thirty-nine extracts of various polarities of the stem bark, stem wood and roots were studied for growth inhibitory effects against the human pathogenic bacteria Staphylococcus epidermidis, Staphylococcus aureus, Micrococcus luteus and Pseudomonas aeruginosa. Our results indicate that the studied species contain antibacterial compounds of a wide range of polarities. All polar root extracts of T. laxiflora and various polar extracts of T. brownii roots, including hot water decoctions, gave broad-spectrum antibacterial effects and low MIC values of 39 mu g/ml. The main ellagitannins in an ethyl acetate extract of the root of T. laxiflora were found to be corilagin and its derivative and punicalagin. A methanol extract of the roots of T. brownii contained methyl-(S)-flavogallonate and its derivative as the main identified ellagitannins. Moreover, both Terminalia species were found to contain ellagic acid xylopyranoside and methyl ellagic acid xyloside and pure ellagic acid was present in T. brownii. Pure punicalagin did not give as low MIC as an ethyl acetate extract of the roots of T. laxiflora, containing punicalagin as one of its main compounds, although this ellagitannin totally inhibited the growth of S. aureus at 125 mu g/ml and P. aeruginosa at 500 mu g/ml. Similarly, pure ellagic and gallic acid gave higher MIC values than the methanolic root extract of T. brownii against S. aureus and P. aeruginosa. Moreover, a Sephadex LH-20 fraction of the methanolic extract of the roots of T. brownii, enrichedwithmethyl-(S)-flavogallonate and its isomer, gave higher MIC values than the crude methanolic extract. These results suggest that the polyphenols in the extracts might act synergistically with each other. A methanolic soxhlet extract of the roots of A. leiocarpus, containing ampelopsin, aromadendrin, taxifolin, pinosylvin and 4'-methylpinosylvin gave a low MIC value of 39 mu g/ml against all bacterial strains used in this investigation. Our results demonstrate that the roots, stem bark and stem wood of T. brownii, T. laxiflora and A. leiocarpus are rich sources of (new) antimicrobial compounds and justify the uses of these plants for treatment of infections in African traditional medicine.Peer reviewe

Interannual variation in UV-B and temperature effects on bud phenology and growth in Populus tremula

acceptedVersio

Phenolic compounds and expression of 4CL genes in silver birch clones and Pt4CL1a lines

A small multigene family encodes 4-coumarate:CoA ligases (4CLs) catalyzing the CoA ligation of hydroxycinnamic acids, a branch point step directing metabolites to a flavonoid or monolignol pathway. In the present study, we examined the effect of antisense Populus tremuloides 4CL (Pt4CL1) to the lignin and soluble phenolic compound composition of silver birch (Betula pendula) Pt4CL1a lines in comparison with non-transgenic silver birch clones. The endogenous expression of silver birch 4CL genes was recorded in the stems and leaves and also in leaves that were mechanically injured. In one of the transgenic Pt4CL1a lines, the ratio of syringyl (S) and guaiacyl (G) lignin units was increased. Moreover, the transcript levels of putative silver birch 4CL gene (Bp4CL1) were reduced and contents of cinnamic acid derivatives altered. In the other two Pt4CL1a lines changes were detected in the level of individual phenolic compounds. However, considerable variation was found in the transcript levels of silver birch 4CLs as well as in the concentration of phenolic compounds among the transgenic lines and non-transgenic clones. Wounding induced the expression of Bp4CL1 and Bp4CL2 in leaves in all clones and transgenic lines, whereas the transcript levels of Bp4CL3 and Bp4CL4 remained unchanged. Moreover, minor changes were detected in the concentrations of phenolic compounds caused by wounding. As an overall trend the wounding decreased the flavonoid content in silver birches and increased the content of soluble condensed tannins. The results indicate that by reducing the Bp4CL1 transcript levels lignin composition could be modified. However, the alterations found among the Pt4CL1a lines and the non-transgenic clones were within the natural variation of silver birches, as shown in the present study by the clonal differences in the transcripts levels of 4CL genes, soluble phenolic compounds and condensed tannins

Comparison of quantification methods for the condensed tannin content of extracts of Pinus pinaster bark

Bark from Pinus Pinaster is an interesting source of polyphenolic natural compounds, that can be used successful1y as total or partial replacement of conventional phenolic resins. These compounds, among other applications, are used as adhesives in the wood agglomerate industry. In this kind of application some problems remain to be solved in order to obtain a Pine extract of commercial value. It is necessary to optimise tije extraction procedure and select a suitable method for the quantification of the tannin content of the bark. In order to study these problems, the tannin extraction from the Pine bark was tested with an alkaline solution (NaOH), and with a fractionation procedure based on a sequence of an organic (ethanol) and aqueous extraction. The phenolic content of each extract or fraction was evaluated by the FolinCiocalteu colorimetric assay for total phenols and two procedures using the Stiasny reaction: the gravimetric Stiasny method and the indirect colorimetric procedure that uses the Folin-Ciocalteu reagent to evaluate the total phenols present in the extract solution before and after it condenses with formaldehyde. The yield value when the alkaline extraction is used is substantially higher than the values obtained with organic or aqueous solutions. However, the selectivity of the process is low. In fact, it was found that the alkaline extract Formaldehyde Condensable Phenolic Material (FCPM) content represents 9596 % of the total phenols content of the extract but this fraction is only ~ 40 % of the total mass of extract. So, the alkaline extract is relatively poor in phenolic material, exhibiting a large variety of non-phenolic extractives. On the other end, ethanol provides a very rich phenolic extract, in which 96 % of total phenols are condensable with formaldehyde, but exhibits a relatively low extraction yield. The aqueous extract presents the lowest extraction yield with low content either in phenolic material as in FCPM, but, as most of the phenolics had already been extracted by the previous organic extraction, especially the low molecular weight fractions, this result was predictable

Reduced Population Control of an Insect Pest in Managed Willow Monocultures

BACKGROUND: There is a general belief that insect outbreak risk is higher in plant monocultures than in natural and more diverse habitats, although empirical studies investigating this relationship are lacking. In this study, using density data collected over seven years at 40 study sites, we compare the temporal population variability of the leaf beetle Phratora vulgatissima between willow plantations and natural willow habitats. METHODOLOGY/PRINCIPAL FINDINGS: The study was conducted in 1999-2005. The density of adult P. vulgatissima was estimated in the spring every year by a knock-down sampling technique. We used two measures of population variability, CV and PV, to compare temporal variations in leaf beetle density between plantation and natural habitat. Relationships between density and variability were also analyzed to discern potential underlying processes behind stability in the two systems. The results showed that the leaf beetle P. vulgatissima had a greater temporal population variability and outbreak risk in willow plantations than in natural willow habitats. We hypothesize that the greater population stability observed in the natural habitat was due to two separate processes operating at different levels of beetle density. First, stable low population equilibrium can be achieved by the relatively high density of generalist predators observed in natural stands. Second, stable equilibrium can also be imposed at higher beetle density due to competition, which occurs through depletion of resources (plant foliage) in the natural habitat. In willow plantations, competition is reduced mainly because plants grow close enough for beetle larvae to move to another plant when foliage is consumed. CONCLUSION/SIGNIFICANCE: To our knowledge, this is the first empirical study confirming that insect pest outbreak risk is higher in monocultures. The study suggests that comparative studies of insect population dynamics in different habitats may improve our ability to predict insect pest outbreaks and could facilitate the development of sustainable pest control in managed systems

UHPLC-ESI/TOFMS Determination of Salicylate-like Phenolic Gycosides in Populus tremula Leaves

Associations of salicylate-like phenolic glycosides (PGs) with biological activity have been reported in Salix and Populus trees, but only for a few compounds, and in relation to a limited number of herbivores. By considering the full diversity of PGs, we may improve our ability to recognize genotypes or chemotype groups and enhance our understanding of their ecological function. Here, we present a fast and efficient general method for salicylate determination in leaves of Eurasian aspen that uses ultra-high performance liquid chromatography-electrospray ionization/time-of-flight mass spectrometry (UHPLC-ESI/TOFMS). The time required for the liquid chromatography separations was 13.5 min per sample, compared to around 60 min per sample for most HPLC protocols. In leaf samples from identical P. tremula genotypes with diverse propagation and treatment histories, we identified nine PGs. We found the compound-specific mass chromatograms to be more informative than the UV-visible chromatograms for compound identification and when quantitating samples with large variability in PG content. Signature compounds previously reported for P. tremoloides (tremulacin, tremuloidin, salicin, and salicortin) always were present, and five PGs (2'-O-cinnamoyl-salicortin, 2'-O-acetyl-salicortin, 2'-O-acetyl-salicin, acetyl-tremulacin, and salicyloyl-salicin) were detected for the first time in P. tremula. By using information about the formic acid adduct that appeared for PGs in the LTQ-Orbitrap MS environment, novel compounds like acetyl-tremulacin could be tentatively identified without the use of standards. The novel PGs were consistently either present in genotypes regardless of propagation and damage treatment or were not detectable. In some genotypes, concentrations of 2'-O-acetyl-salicortin and 2'-O-cinnamoyl-salicortin were similar to levels of biologically active PGs in other Salicaceous trees. Our study suggests that we may expect a wide variation in PG content in aspen populations which is of interest both for studies of interactions with herbivores and for mapping population structure

Increased Resistance of Bt Aspens to Phratora vitellinae (Coleoptera) Leads to Increased Plant Growth under Experimental Conditions

One main aim with genetic modification (GM) of trees is to produce plants that are resistant to various types of pests. The effectiveness of GM-introduced toxins against specific pest species on trees has been shown in the laboratory. However, few attempts have been made to determine if the production of these toxins and reduced herbivory will translate into increased tree productivity. We established an experiment with two lines of potted aspens (Populus tremula×Populus tremuloides) which express Bt (Bacillus thuringiensis) toxins and the isogenic wildtype (Wt) in the lab. The goal was to explore how experimentally controlled levels of a targeted leaf beetle Phratora vitellinae (Coleoptera; Chrysomelidae) influenced leaf damage severity, leaf beetle performance and the growth of aspen. Four patterns emerged. Firstly, we found clear evidence that Bt toxins reduce leaf damage. The damage on the Bt lines was significantly lower than for the Wt line in high and low herbivory treatment, respectively. Secondly, Bt toxins had a significant negative effect on leaf beetle survival. Thirdly, the significant decrease in height of the Wt line with increasing herbivory and the relative increase in height of one of the Bt lines compared with the Wt line in the presence of herbivores suggest that this also might translate into increased biomass production of Bt trees. This realized benefit was context-dependent and is likely to be manifested only if herbivore pressure is sufficiently high. However, these herbivore induced patterns did not translate into significant affect on biomass, instead one Bt line overall produced less biomass than the Wt. Fourthly, compiled results suggest that the growth reduction in one Bt line as indicated here is likely due to events in the transformation process and that a hypothesized cost of producing Bt toxins is of subordinate significance

Physiological and cell ultrastructure disturbances in wheat seedlings generated by Chenopodium murale hairy root exudate.

Chenopodium murale L. is an invasive weed species significantly interfering with wheat crop. However, the complete nature of its allelopathic influence on crops is not yet fully understood. In the present study, the focus is made on establishing the relation between plant morphophysiological changes and oxidative stress, induced by allelopathic extract. Phytotoxic medium of C. murale hairy root clone R5 reduced the germination rate (24% less than control value) of wheat cv. Nataša seeds, as well as seedling growth, diminishing shoot and root length significantly, decreased total chlorophyll content, and induced abnormal root gravitropism. The R5 treatment caused cellular structural abnormalities, reflecting on the root and leaf cell shape and organization. These abnormalities mostly included the increased number of mitochondria and reorganization of the vacuolar compartment, changes in nucleus shape, and chloroplast organization and distribution. The most significant structural changes were observed in cell wall in the form of amoeboid protrusions and folds leading to its irregular shape. These structural alterations were accompanied by an oxidative stress in tissues of treated wheat seedlings, reflected as increased level of H2O2 and other ROS molecules, an increase of radical scavenging capacity and total phenolic content. Accordingly, the retardation of wheat seedling growth by C. murale allelochemicals may represent a consequence of complex activity involving both cell structure alteration and physiological processes.This is a post-peer-review, pre-copyedit version of an article published in Protoplasma. The final authenticated version is available online at: [http://dx.doi.org/10.1007/s00709-018-1250-0