Predicting patterns of invasion by black cherry (Prunus serotina Ehrh.) in Flanders (Belgium) and its impact on the forest understorey community

The design of cost-efficient control strategies for invasive species that are too widespread and abundant for complete eradication, at least in the short term, will benefit from a rigorous analysis of invasion patterns and associated effects on native biodiversity. In this paper, the case of the invasive North American tree Prunus serotina in Flanders (Belgium) is presented. Our main objectives were to determine the susceptibility of forest stands to invasion by P. serotina and the subsequent effects of invasion on the understorey community. We used the large database of the first Flemish Forest Inventory. Multiple logistic regressions indicated that P. serotina occurred more frequently in privately owned, younger forest on coarse-textured, dry soils (podzols), and the combination of these factors allowed us to correctly predict presence/absence of P. serotina in 70% of the validation plots. However, locational variables proved to be important as well, indicating that the invasion process is still ongoing. Prediction of P. serotina densities by means of multiple linear regressions was less successful. Effects on the understorey richness were analysed by comparing the number of species and the mean Ellenberg values between pairs of plots, only differing by the presence of P. serotina in the shrub layer. A reduction of the understorey richness following invasion was only pronounced on the more moist soils, while compositional changes mainly occurred on drier soils. It is concluded that priority for control should be given to landscapes with a low fraction of invaded stands and to forest stands located on more moist soils. However, using its potential to threaten native biodiversity as an argument for control should be done with care as further research is needed whether the observed negative effects are due to a species (i.e. native vs. non-native) or a density effect (high vs. low).     

Characterization and purification of a bacteriocin from <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> subsp. <Emphasis Type="Italic">paracasei</Emphasis> BMK2005, an intestinal isolate active against multidrug-resistant pathogens

A strain of Lactobacillus paracasei subsp. paracasei BMK2005 isolated from healthy infant faeces has shown a remarkable antibacterial activity against 32 bacterial pathogenic strains of human clinical isolates. Among them, 13 strains belonging to species of Escherichia coli, Citrobacter freundii, Citrobacter diversus, Klebsiella oxytoca, Enterobacter cloacae and Pseudomonas aeruginosa were resistant to Cefotaxime (CTX) and Ceftazidime (CAZ), and 4 strains of Staphylococcus aureus were resistant to Methicillin (MRSA). This antibacterial activity was attributed to a bacteriocin designated as Paracaseicin A. It was heat-stable up to 120°C for 5 min and active within the pH range of 2–5. Its activity was lost when treated with proteases, which reveals its proteinaceous nature. This bacteriocin was successfully purified only by two steps of reversed phase chromatography. Its molecular mass, determined by mass spectrometry analysis, was 2,462.5 Da. To our knowledge, the present study is the first report on characterization and purification of a bacteriocin, produced by a L. paracasei subsp. paracasei strain exhibiting an antibacterial activity against various multidrug-resistant species of Gram-positive and Gram-negative bacteria, which reveals its potential for use in prevention or treatment of infections caused by multidrug-resistant species especially in cases of antibiotics-associated diarrhea (AAD).     

Diffusion Limitations in Root Uptake of Cadmium and Zinc, But Not Nickel, and Resulting Bias in the Michaelis Constant

It has long been recognized that diffusive boundary layers affect the determination of active transport parameters, but this has been largely overlooked in plant physiological research. We studied the short-term uptake of cadmium (Cd), zinc (Zn), and nickel (Ni) by spinach (Spinacia oleracea) and tomato (Lycopersicon esculentum) in solutions with or without metal complexes. At same free ion concentration, the presence of complexes, which enhance the diffusion flux, increased the uptake of Cd and Zn, whereas Ni uptake was unaffected. Competition effects of protons on Cd and Zn uptake were observed only at a very large degree of buffering, while competition of magnesium ions on Ni uptake was observed even in unbuffered solutions. These results strongly suggest that uptake of Cd and Zn is limited by diffusion of the free ion to the roots, except at very high degree of solution buffering, whereas Ni uptake is generally internalization limited. All results could be well described by a model that combined a diffusion equation with a competitive Michaelis-Menten equation. Direct uptake of the complex was estimated to be a major contribution only at millimolar concentrations of the complex or at very large ratios of complex to free ion concentration. The true K_m for uptake of Cd²⁺ and Zn²⁺ was estimated at <5 nm, three orders of magnitude smaller than the K_m measured in unbuffered solutions. Published Michaelis constants for plant uptake of Cd and Zn likely strongly overestimate physiological ones and should not be interpreted as an indicator of transporter affinity.Internalization of metals by biota is traditionally described by Michaelis-Menten kinetics (Wilkinson and Buffle, 2004). The K_m corresponds to the concentration in solution at which the uptake is one-half of the maximal uptake, F_max. The Michaelis-Menten equation relates the uptake flux, F, to the free ion concentration at the site of uptake, [M]_s:If diffusion of a metal across a diffusive boundary layer adjacent to the roots is the rate-limiting step for uptake, the concentration at the site of uptake will be lower than that in the bulk solution. As a result, diffusion limitations result in an overestimate of the K_m, if the concentration at the root surface is assumed to be the same as in the bulk solution, as is usually done. This bias in K_m has been discussed in detail by Winne (1973) and has, for instance, been demonstrated experimentally for uptake of Glc in rabbit jejunum (Thomson and Dietschy, 1980) and for uptake of several sugars, amino acids, and bile acids in rat ileum (Wilson and Dietschy, 1974).Models used to predict ion availability and toxicity of metals by plants usually rely on the assumption that uptake is controlled by the free metal ion activity and the activity of competing ions in the bulk solution. For instance, the biotic ligand model (BLM), originally developed to predict metal toxicity to aquatic organisms, assumes that toxicity of an ion is mitigated by the presence of competing ions that bind on the biotic ligand (Paquin et al., 2002). Hough et al. (2005) used a free ion activity model taking into account proton competition effects to predict cadmium (Cd) uptake by soil-grown ryegrass (Lolium perenne). The uptake was reasonably well predicted; however, as the authors pointed out, it was not clear whether the derived constants truly represented physiological affinity constants or were just fitting parameters in a rate-limited uptake process. In case of strong diffusion limitation, ion competition effects on the internalization are expected to have negligible effect on the uptake, as the uptake is controlled by diffusion and not by internalization (Campbell et al., 2002; Degryse and Smolders, 2012).In previous studies, we found strong evidence that uptake of Cd and zinc (Zn) is limited by the diffusive transport of the free metal ion to the root at low free ion concentration. At constant free ion concentration, the uptake of Cd and Zn increased in presence of metal complexes and the contribution of the complex increased with increasing dissociation rate of the complex (Degryse et al., 2006a, 2006c). In unbuffered solutions, i.e. solutions without metal complexes, stirring increased Cd uptake by plants (Degryse and Smolders, 2012). For nickel (Ni), however, contribution of complexes was small or undetectable, and stirring did not increase the uptake (Degryse and Smolders, 2012). Given this evidence that Cd and Zn uptake by plants is limited by diffusion, it is likely that published K_m values for uptake of Cd²⁺ and Zn²⁺ by plants overestimate true physiological values. This bias in the K_m when a diffusive boundary layer is present has been largely ignored in plant-physiological research. Indeed, in numerous studies the K_m value has been interpreted as a characteristic of the carrier-mediated transport process, while in many cases it may reflect mass transfer properties. In addition, these diffusion limitations may mask ion competition effects in the uptake.The aim of this article was (1) to present evidence that K_m values determined for Cd²⁺ and Zn²⁺ uptake by plants in general reflect transport limitations rather than transporter affinity; (2) to derive true K_m values by determining the K_m under conditions where the uptake is not transport limited; (3) to identify the consequence of diffusion limitations on competition effects; and (4) to describe uptake of Cd, Zn, and Ni by plants in a single comprehensive model that combines competitive Michaelis-Menten kinetics with a diffusion equation.

Theoretical Framework

In the following, we qualitatively discuss the bias in the K_m because of diffusion limitations, based on Figure 1. Equations are given in the “Materials and Methods” section. Figure 1A presents a case where the potential internalization flux by the plant at low concentrations is much larger than the maximal rate at which the free ion can be supplied through diffusive transport of the ion to the root surface. In this case, the actual uptake flux by the plant will approach the maximal diffusive flux, and the free ion concentration at the root surface is much smaller than that in the bulk solution. The apparent K_m (K_m*; determined as the concentration where the uptake flux is one-half of the maximal uptake) is much larger than the true K_m value. In Figure 1B, the potential internalization flux at low concentration is of the same order of magnitude as the maximal diffusion flux. In this case, the concentration at the root surface is slightly smaller than that in the bulk solution, and there is only a slight bias in the K_m value.Open in a separate windowFigure 1.Conceptual diagram of the internalization flux (Michaelis-Menten curve; full line), maximal diffusive supply from solution to root (dashed line), and actual uptake flux (dotted line) as a function of free ion concentration for two theoretical cases. Left and right sections show the same curves, on log (left) or linear (right) scale. In A, the potential internalization flux is much larger than the maximal diffusive supply at low concentrations, i.e. the uptake is strongly limited by the transport of the free ion to the root. The plant acts as a near-zero sink, and the actual plant uptake equals the maximal diffusive flux. The K_m* is much larger than the true K_m, and the experimental permeability P (slope of the actual uptake curve) is much smaller than the membrane permeability P_m (slope of the internalization curve). In B, the maximal diffusive flux is larger than the potential internalization flux. The uptake is not limited by diffusive transport, and the K_m* and true K_m are almost equal.Given the evidence that uptake of Cd and Zn by plants is diffusion limited, even in stirred nutrient solutions, we hypothesize that reported K_m values of Cd²⁺ and Zn²⁺ are biased, and that the physiological K_m values are much smaller. To test this hypothesis, we measured the uptake of Cd, Zn, and Ni in solution, in absence or in presence of labile hydrophilic metal complexes. If diffusion limitations prevail, the complexes dissociate within the diffusion layer and thus enhance the diffusion flux and therefore the metal uptake. By adding labile complexes in large amounts, it should be possible to abolish the diffusion barrier completely, in which case the physiological K_m can be determined.In addition, the effect of competitive ions on uptake was tested. The presence of competitive ions decreases the internalization flux. However, if uptake is rate limited by the diffusive transport to the uptake site and not by internalization, competition effects should theoretically not affect the uptake flux.     

Four decades of post-agricultural forest development have caused major redistributions of soil phosphorus fractions

Fertilisation of agricultural land causes an accumulation of nutrients in the top soil layer, among which phosphorus (P) is particularly persistent. Changing land use from farmland to forest affects soil properties, but changes in P pools have rarely been studied despite their importance to forest ecosystem development. Here, we describe the redistributions of the P pools in a four-decadal chronosequence of post-agricultural common oak (Quercus robur L.) forests in Belgium and Denmark. The aim was to assess whether forest age causes a repartitioning of P throughout the various soil P pools (labile P, slowly cycling P and occluded P); in particular, we addressed the time-related alterations in the inorganic versus organic P fractions. In less than 40 years of oak forest development, significant redistributions have occurred between different P fractions. While both the labile and the slowly cycling inorganic P fractions significantly decreased with forest age, the organic fractions significantly increased. The labile P pool (inorganic + organic), which is considered to be the pool of P most likely to contribute to plant-available P, significantly decreased with forest age (from >20 to <10% of total P), except in the 0-5 cm of topsoil, where labile P remained persistently high. The shift from inorganic to organic P and the shifts between the different inorganic P fractions are driven by biological processes and also by physicochemical changes related to forest development. It is concluded that the organic labile P fraction, which is readily mineralisable, should be taken into account when studying the bioavailable P pool in forest ecosystems.     

In situ gross nitrogen transformations differ between temperate deciduous and coniferous forest soils

Despite long-term enhanced nitrogen (N) inputs, forests can retain considerable amounts of N. While rates of N inputs via throughfall and N leaching are increased in coniferous stands relative to deciduous stands at comparable sites, N leaching below coniferous stands is disproportionally enhanced relative to the N input. A better understanding of factors affecting N retention is needed to assess the impact of changing N deposition on N cycling and N loss of forests. Therefore, gross N transformation pathways were quantified in undisturbed well-drained sandy soils of adjacent equal-aged deciduous (pedunculate oak (Quercus robur L.)) and coniferous (Scots pine (Pinus sylvestris L.)) planted forest stands located in a region with high N deposition (north Belgium). In situ inorganic ¹⁵N labelling of the mineral topsoil (0–10?cm) combined with numerical data analysis demonstrated that (i) all gross N transformations differed significantly (p?<?0.05) between the two forest soils, (ii) gross N mineralization in the pine soil was less than half the rate in the oak soil, (iii) meaningful N immobilization was only observed for ammonium, (iv) nitrate production via oxidation of organic N occurred three times faster in the pine soil while ammonium oxidation was similar in both soils, and (v) dissimilatory nitrate reduction to ammonium was detected in both soils but was higher in the oak soil. We conclude that the higher gross nitrification (including oxidation of organic N) in the pine soil compared to the oak soil, combined with negligible nitrate immobilization, is in line with the observed higher nitrate leaching under the pine forest.     

Evolutionary relationships among narrow-headed rats (genus Stenocephalemys, Muridae, Rodentia) inferred from complete cytochrome b gene sequences

Using the data on complete sequences of cytochrome b gene, phylogenetic relationships were studied among the Stenocephalemys s. lat. (Stenocephalemys ssp. + Praomys albipes) murine rodents, inhabiting adjacent altitudinal belts of the isolated Ethiopian mountain massifs, and among the related Praomys s. lat. species. Extremely low resolution of the relationships among the main Praomys s. lat. lineages hampered identification of the nearest sister group for the Stenocephalemys s. lat. "Ethiopian" clade, monophyly of which was strongly supported. Sister relationships between P. albipes and S. griseicauda (implying "accelerated" morphological and chromosomal evolution upon the formation of the former species), as well as between S. albocaudata and the recently described novel chromosomal form of Stenocephalemus sp. A (2n = 50; NFa = 56) were demonstrated. Definite discordance between the rates of their molecular, chromosomal, and morphological evolution was revealed. Based on phylogenetic reconstructions and the estimates of the divergence time, obtained by use of molecular clock method, an attempt to draw a phylogenetic scenario for the group examined was made. The obtained data were compared to those for analogous Sigmodontinae species complexes, distributed across a marked altitudinal gradient on the Andean slopes. It was shown that molecular genetic data on the rodents from mountain tropics did not support the gradient model of diversification, based on the possibility of morphological diversification prior to their achievement of the species status (without interruption of the gene flow between the forms) due to differently directed selection across a strong environmental gradient.     

Limited by the host: Host age hampers establishment of holoparasite Cuscuta epithymum

A good understanding of the relationship between plant establishment and the ecosystem of which they are part of is needed to conserve rare plant species. Introduction experiments offer a direct test of recruitment limitation, but generally only the seed germination and seedling phases are monitored. Thus the relative importance of different establishment stages in the process of recruitment is not considered. This is particularly true for parasitic plants where empirical data are generally missing. During two consecutive growing seasons we examined the effect of heathland management applications, degree of heathland succession (pioneer, building and mature phase) and seed-density on the recruitment and establishment of the endangered holoparasite Cuscuta epithymum. In general, recruitment after two growing seasons was low with 4.79% of the sown seeds that successfully emerged to the seedling stage and a final establishment of 89 flowering adults (i.e. <1.5% of the sown seeds). Although a higher seed-density resulted in a higher number of seedlings, seed-density did not significantly affected relative germination percentages. The management type and subsequent heath succession had no significant effect on seedling emergence; whereas, seedling attachment to the host, establishment and growth to full-grown size were hampered in older heath vegetation (i.e. high, dense, and mature canopy). Establishment was most successful in turf-cut pioneer heathland, characterised by a relatively open and low vegetation of young Calluna vulgaris. The age of C. vulgaris, C. epithymum's main host, proved to be the most limiting factor. These results emphasise the importance of site quality (i.e. successional phase of its host) on recruitment success of C. epithymum, which is directly affected by the management applied to the vegetation. Lack of any heathland management will thus seriously restrict establishment of the endangered parasite.     

Re-expression of alpha skeletal actin as a marker for dedifferentiation in cardiac pathologies

Differentiation of foetal cardiomyocytes is accompanied by sequential actin isoform expression, i.e. down-regulation of the 'embryonic' alpha smooth muscle actin, followed by an up-regulation of alpha skeletal actin (αSKA) and a final predominant expression of alpha cardiac actin (αCA). Our objective was to detect whether re-expression of αSKA occurred during cardiomyocyte dedifferentiation, a phenomenon that has been observed in different pathologies characterized by myocardial dysfunction. Immunohistochemistry of αCA, αSKA and cardiotin was performed on left ventricle biopsies from human patients after coronary bypass surgery. Furthermore, actin isoform expression was investigated in left ventricle samples of rabbit hearts suffering from pressure- and volume-overload and in adult rabbit ventricular cardiomyocytes during dedifferentiation in vitro . Atrial goat samples up to 16 weeks of sustained atrial fibrillation (AF) were studied ultrastructurally and were immunostained for αCA and αSKA. Up-regulation of αSKA was observed in human ventricular cardiomyocytes showing down-regulation of αCA and cardiotin. A patchy re-expression pattern of αSKA was observed in rabbit left ventricular tissue subjected to pressure- and volume-overload. Dedifferentiating cardiomyocytes in vitro revealed a degradation of the contractile apparatus and local re-expression of αSKA. Comparable αSKA staining patterns were found in several areas of atrial goat tissue during 16 weeks of AF together with a progressive glycogen accumulation at the same time intervals. The expression of αSKA in adult dedifferentiating cardiomyocytes, in combination with PAS-positive glycogen and decreased cardiotin expression, offers an additional tool in the evaluation of myocardial dysfunction and indicates major changes in the contractile properties of these cells.