Enhanced bactericidal, antibiofilm and antioxidative response of Lawsonia inermis leaf extract synthesized ZnO NPs loaded with commercial antibiotic.

Globally, antibiotic resistance is a challenging issue in healthcare sector. The emergence of multiple drug-resistant bacteria has forced us to modify existing medicines and or formulate newer medicines that are effective and inexpensive. In this perspective, this study involves the formation of zinc oxide nanoparticles (ZnO NPs) by utilizing the Lawsonia inermis (Li) leaf extract. The prepared L. inermis leaf extract mediated ZnO NPs (Li-ZnO NPs) were bio-physically characterized. The antibacterial and radical scavenging effects of Li-ZnO NPs were evaluated. In addition, ZnO NPs were conjugated with standard antibiotic (ciprofloxacin) and its drug loading efficiency, drug release and antibacterial efficacy were tested and compared with non-drug loaded ZnO NPs. An absorbance peak at 340 nm was noted for Li-ZnO NPs. After conjugation with the drug, two absorbance peaks- one at 242 nm characteristic of ciprofloxacin and the other at 350 nm characteristics of ZnO NPs were observed. The crystallite size was 18.7 nm as determined by XRD. The antibacterial effect was higher on Gram-positive (S. aureus and S. pyogenes) than the Gram-negative pathogens (E. coli and K. pneumoniae). Inhibition of S. aureus and S. pyogenes biofilm at 100 µg mL-1were, respectively, 97.5 and 92.6%. H2O2 free radicals was inhibited to 90% compared to the standard ascorbic acid at 100 µg mL-1. After drug loading, the FTIR spectrum confirmed the existence of ciprofloxacin peaks at 965 cm-1 and Zn-O bond at 492 cm-1. The drug loading capacity of 15 nm sized ZnO NPs was higher (58, 75, 90 and 95% at 1, 2.5, 5 and 10% drug concentrations, respectively) compared to 20 nm. Similarly, the percentage of drug (ciprofloxacin) released from 15 nm ZnO NPs were increased to 90% at 10% drug-loaded samples, respectively. Also, the antibiotic loaded ZnO NPs had significant antibacterial effects against tested bacteria compared to Li-ZnO NPs and ciprofloxacin alone. This revealed that the antibiotic loaded ZnO NPs offer a sustainable route to treat multi-drug-resistant bacterial infections.

The oldest known clones of Salix herbacea growing in the Northern Apennines, Italy are at least 2000 years old.

PREMISE: Dominant in many ecosystems around the world, clonal plants can reach considerable ages and sizes. Due to their modular growth patterns, individual clonal plants (genets) can consist of many subunits (ramets). Since single ramets do not reflect the actual age of genets, the ratio between genet size (radius) and longitudinal annual growth rate (LAGR) of living ramets is often used to approximate the age of clonal plants. However, information on how the LAGR changes along ramets and how LAGR variability may affect age estimates of genets is still limited. METHODS: We assessed the variability of LAGR based on wood-section position along the ramets and on the duration of the growing season on three genetically distinct genets of Salix herbacea growing in the Northern Apennines (Italy). We compared genet ages estimated by dividing genet radius by the LAGRs of its ramets. RESULTS: LAGR increased significantly from the stem apex to the root collar; indicating that ramet growth rate decreased with time. Furthermore, a difference of ca. 2 weeks in the onset of the growing period did not impact LAGR. Considering the high LAGR variability, we estimated that the three genets started to grow between ~2100 and ~7000 years ago, which makes them the oldest known clones of S. herbacea even considering the most conservative age estimate. CONCLUSIONS: Our findings indicate that analyzing ramets at the root collar provides an integrative measurement of their overall LAGR, which is crucial for estimating the age of genets.

Plant and soil nitrogen in oligotrophic boreal forest habitats with varying moss depths: does exclusion of large grazers matter?

The boreal forest consists of drier sunlit and moister-shaded habitats with varying moss abundance. Mosses control vascular plant-soil interactions, yet they all can also be altered by grazers. We determined how 2 decades of reindeer (Rangifer tarandus) exclusion affect feather moss (Pleurozium schreberi) depth, and the accompanying soil N dynamics (total and dissolvable inorganic N, δ15N), plant foliar N, and stable isotopes (δ15N, δ13C) in two contrasting habitats of an oligotrophic Scots pine forest. The study species were pine seedling (Pinus sylvestris L.), bilberry (Vaccinium myrtillus L.), lingonberry (V. vitis-idaea L.), and feather moss. Moss carpet was deeper in shaded than sunlit habitats and increased with grazer exclusion. Humus N content increased in the shade as did humus δ15N, which also increased due to exclusion in the sunlit habitats. Exclusion increased inorganic N concentration in the mineral soil. These soil responses were correlated with moss depth. Foliar chemistry varied due to habitat depending on species identity. Pine seedlings showed higher foliar N content and lower foliar δ15N in the shaded than in the sunlit habitats, while bilberry had both higher foliar N and δ15N in the shade. Thus, foliar δ15N values of co-existing species diverged in the shade indicating enhanced N partitioning. We conclude that despite strong grazing-induced shifts in mosses and subtler shifts in soil N, the N dynamics of vascular vegetation remain unchanged. These indicate that plant-soil interactions are resistant to shifts in grazing intensity, a pattern that appears to be common across boreal oligotrophic forests.

Past the climate optimum: Recruitment is declining at the world's highest juniper shrublines on the Tibetan Plateau.

Alpine biomes are climate change hotspots, and treeline dynamics in particular have received much attention as visible evidence of climate-induced shifts in species distributions. Comparatively little is known, however, about the effects of climate change on alpine shrubline dynamics. Here, we reconstruct decadally resolved shrub recruitment history (age structure) through the combination of field surveys and dendroecology methods at the world's highest juniper (Juniperus pingii var. wilsonii) shrublines on the south-central Tibetan Plateau. A total of 1,899 shrubs were surveyed at 12 plots located in four regions along an east-to-west declining precipitation gradient. We detected synchronous recruitment with 9 out of 12 plots showing a gradual increase from 1600 to 1900, a peak at 1900-1940, and a subsequent decrease from the 1930s onward. Shrub recruitment was significantly and positively correlated with reconstructed summer temperature from 1600 to 1940, whereas it was negatively associated with temperature in recent decades (1930-2000). Recruitment was also positively correlated with precipitation, except in the 1780-1830 period, when a trend toward wetter climate conditions began. Warming-induced drought limitation has likely reduced the recruitment potential of alpine juniper shrubs in recent decades. Ongoing warming without a simultaneous increase in precipitation is expected to further impair recruitment at the world's highest juniper shrublines and alter the dynamics and competitive balance between woody plant species throughout these alpine biomes.

Arctic browning: Impacts of extreme climatic events on heathland ecosystem CO2 fluxes.

Extreme climatic events are among the drivers of recent declines in plant biomass and productivity observed across Arctic ecosystems, known as "Arctic browning." These events can cause landscape-scale vegetation damage and so are likely to have major impacts on ecosystem CO2 balance. However, there is little understanding of the impacts on CO2 fluxes, especially across the growing season. Furthermore, while widespread shoot mortality is commonly observed with browning events, recent observations show that shoot stress responses are also common, and manifest as high levels of persistent anthocyanin pigmentation. Whether or how this response impacts ecosystem CO2 fluxes is not known. To address these research needs, a growing season assessment of browning impacts following frost drought and extreme winter warming (both extreme climatic events) on the key ecosystem CO2 fluxes Net Ecosystem Exchange (NEE), Gross Primary Productivity (GPP), ecosystem respiration (Reco ) and soil respiration (Rsoil ) was carried out in widespread sub-Arctic dwarf shrub heathland, incorporating both mortality and stress responses. Browning (mortality and stress responses combined) caused considerable site-level reductions in GPP and NEE (of up to 44%), with greatest impacts occurring at early and late season. Furthermore, impacts on CO2 fluxes associated with stress often equalled or exceeded those resulting from vegetation mortality. This demonstrates that extreme events can have major impacts on ecosystem CO2 balance, considerably reducing the carbon sink capacity of the ecosystem, even where vegetation is not killed. Structural Equation Modelling and additional measurements, including decomposition rates and leaf respiration, provided further insight into mechanisms underlying impacts of mortality and stress on CO2 fluxes. The scale of reductions in ecosystem CO2 uptake highlights the need for a process-based understanding of Arctic browning in order to predict how vegetation and CO2 balance will respond to continuing climate change.

Increased fire severity alters initial vegetation regeneration across Calluna-dominated ecosystems.

Calluna vulgaris-dominated habitats are valued for ecosystem services such as carbon storage and for their conservation importance. Climate and environmental change are altering their fire regimes. In particular, more frequent summer droughts will result in higher severity wildfires. This could alter the plant community composition of Calluna habitats and thereby influence ecosystem function. To study the effect of fire severity on community composition we used rain-out shelters to simulate drought prior to experimental burns at two Calluna-dominated sites, a raised bog and a heathland. We analysed species abundance in plots surveyed ca. 16 months after fire in relation to burn severity (indicated by fire-induced soil heating). We found that fire severity was an important control on community composition at both sites. Higher fire severity increased the abundance of ericoids, graminoids and acrocarpous mosses, and decreased the abundance of pleurocarpous mosses compared to lower severity fires. At the raised bog, the keystone species Sphagnum capillifolium and Eriophorum vaginatum showed no difference in regeneration with fire severity. Species and plant functional type beta-diversity increased following fire, and was similar in higher compared to lower severity burns. Our results further our understanding of the response of Calluna-dominated habitats to projected changes in fire regimes, and can assist land managers using prescribed fires in selecting burning conditions to achieve management objectives.

Sea-ice induced growth decline in Arctic shrubs.

Measures of increased tundra plant productivity have been associated with the accelerating retreat of the Arctic sea-ice. Emerging studies document opposite effects, advocating for a more complex relationship between the shrinking sea-ice and terrestrial plant productivity. I introduce an autoregressive plant growth model integrating effects of biological and climatic conditions for analysing individual ring-width growth time series. Using 128 specimens of Salix arctica, S. glauca and Betula nana sampled across Greenland to Svalbard, an overall negative effect of the retreating June sea-ice extent was found on the annual growth. The negative effect of the retreating June sea-ice was observed for younger individuals with large annual growth allocations and with little or no trade-off between previous and current year's growth.

Four years of experimental warming do not modify the interaction between subalpine shrub species.

Climate warming can lead to changes in alpine plant species interactions through modifications in environmental conditions, which may ultimately cause drastic changes in plant communities. We explored the effects of 4 years of experimental warming with open-top chambers (OTC) on Vaccinium myrtillus performance and its interaction with neighbouring shrubs at the Pyrenean treeline ecotone. We examined the effects of warming on height, above-ground (AG) and below-ground (BG) biomass and the C and N concentration and isotope composition of V. myrtillus growing in pure stands or in stands mixed with Vaccinium uliginosum or Rhododendron ferrugineum. We also analysed variations in soil N concentrations, rhizosphere C/N ratios and the functional diversity of the microbial community, and evaluated whether warming altered the biomass, C and N concentration and isotope composition of V. uliginosum in mixed plots. Our results showed that warming induced positive changes in the AG growth of V. myrtillus but not BG, while V. uliginosum did not respond to warming. Vaccinium myrtillus performance did not differ between stand types under increased temperatures, suggesting that warming did not induce shifts in the interaction between V. myrtillus and its neighbouring species. These findings contrast with previous studies in which species interactions changed when temperature was modified. Our results show that species interactions can be less responsive to warming in natural plant communities than in removal experiments, highlighting the need for studies involving the natural assembly of plant species and communities when exploring the effect of environmental changes on plant-plant interactions.

Clonality increases with snow depth in the arctic dwarf shrub Empetrum hermaphroditum.

PREMISE OF THE STUDY: Vegetative reproduction and spread through clonal growth plays an important role in arctic-alpine ecosystems with short cool growing seasons. Local variation in winter snow accumulation leads to discrete habitat types that may provide divergent conditions for sexual and vegetative reproduction. Therefore, we studied variation in clonal structure of a dominant, evergreen, dwarf shrub (Empetrum nigrum s.l. with the two taxa E. nigrum L. s.s. and E. hermaphroditum Hagerup) along a snow cover gradient and compared clonal diversity and spatial genetic structure between habitats. METHODS: We studied 374 individual shoots using 105 polymorphic AFLP markers and analyses based on hierarchical clustering, clonal diversity indices, and small-scale spatial genetic structure with pairwise kinship coefficient. We used two approaches to define a threshold of genotypic distance between two samples that are considered the same clone. Clonality was examined among three habitats (exposed ridges, sheltered depressions, birch forest) differing in snow conditions replicated in four study regions in Norway and Sweden. KEY RESULTS: Clonality of E. hermaphroditum differed between habitats with an increase in clonal diversity with decreasing snow depth. Small-scale spatial genetic structure increased with decreasing clonal diversity and increasing clone size. In three study regions, E. hermaphroditum was the only species, whereas in one region E. nigrum also occurred, largely confined to exposed ridges. CONCLUSIONS: Our results demonstrated that snow cover in conjunction with associated habitat conditions plays an important role for the mode of propagation of the dwarf shrub E. hermaphroditum.

Soil warming and CO2 enrichment induce biomass shifts in alpine tree line vegetation.

Responses of alpine tree line ecosystems to increasing atmospheric CO2 concentrations and global warming are poorly understood. We used an experiment at the Swiss tree line to investigate changes in vegetation biomass after 9 years of free air CO2 enrichment (+200 ppm; 2001-2009) and 6 years of soil warming (+4 °C; 2007-2012). The study contained two key tree line species, Larix decidua and Pinus uncinata, both approximately 40 years old, growing in heath vegetation dominated by dwarf shrubs. In 2012, we harvested and measured biomass of all trees (including root systems), above-ground understorey vegetation and fine roots. Overall, soil warming had clearer effects on plant biomass than CO2 enrichment, and there were no interactive effects between treatments. Total plant biomass increased in warmed plots containing Pinus but not in those with Larix. This response was driven by changes in tree mass (+50%), which contributed an average of 84% (5.7 kg m(-2) ) of total plant mass. Pinus coarse root mass was especially enhanced by warming (+100%), yielding an increased root mass fraction. Elevated CO2 led to an increased relative growth rate of Larix stem basal area but no change in the final biomass of either tree species. Total understorey above-ground mass was not altered by soil warming or elevated CO2 . However, Vaccinium myrtillus mass increased with both treatments, graminoid mass declined with warming, and forb and nonvascular plant (moss and lichen) mass decreased with both treatments. Fine roots showed a substantial reduction under soil warming (-40% for all roots <2 mm in diameter at 0-20 cm soil depth) but no change with CO2 enrichment. Our findings suggest that enhanced overall productivity and shifts in biomass allocation will occur at the tree line, particularly with global warming. However, individual species and functional groups will respond differently to these environmental changes, with consequences for ecosystem structure and functioning.

Global warming alters Himalayan alpine shrub growth dynamics and climate sensitivity.

Global climate change is having significant effects on plant growth patterns and mountain plants can be particularly vulnerable to accelerated warming. Rising temperatures are releasing plants from cold limitation, such as at high elevations and latitudes, but can also induce drought limitation, as documented for trees from lower elevations and latitudes. Here we test these predictions using a unique natural experiment with Himalayan alpine shrub Rhododendron anthopogon and its growth responses to changing climate over a large portion of its latitudinal and elevational ranges, including steep precipitation and temperature gradients. We determined growth dynamics during the last three decades, representing period of accelerated warming, using annual radial growth increments for nine populations growing on both wet and warm southern localities and drier and cold northern localities in the Himalayas along elevation gradients encompassing the lower and upper species range limits. A significant growth increase over past decades was observed after controlling for confounding effect of shrub age and microsites. However, the magnitude of increase varied among populations. Particularly, populations situated in the lower elevation of the northernmost (cold and dry) locality exhibited most substantial growth enhancement. The relationship between growth variability and climate varied among populations, with the populations from the coldest location displaying the strongest responsiveness to increasing minimum temperatures during July. Minimum temperatures of April and August were the most important factor limiting the growth across most populations. Potential warming-induced drought limitation had no significant impact on growth variation in any part of the species geographic range. Overall, our findings indicate that plant growth is continuously increasing in recent decades and growth-climate relationships are not consistent across populations, with populations from the coldest and wettest localities showing stronger responses. The observed patterns suggest that dwarf-shrubs benefit from ongoing warming, leading to increased shrubification of high elevation alpine ecosystems.

Emissions of biogenic volatile organic compounds from adjacent boreal fen and bog as impacted by vegetation composition.

Peatland ecosystems emit biogenic volatile organic compounds (BVOC), which have a net cooling impact on the climate. However, the quality and quantity of BVOC emissions, and how they are regulated by vegetation and peatland type remain poorly understood. Here we measured BVOC emissions with dynamic enclosures from two major boreal peatland types, a minerotrophic fen and an ombrotrophic bog situated in Siikaneva, southern Finland and experimentally assessed the role of vegetation by removing vascular vegetation with or without the moss layer. Our measurements from four campaigns during growing seasons in 2017 and 2018 identified emissions of 59 compounds from nine different chemical groups. Isoprene accounted for 81 % of BVOC emissions. Measurements also revealed uptake of dichloromethane. Total BVOC emissions and the emissions of isoprene, monoterpenoids, sesquiterpenes, homoterpenes, and green leaf volatiles were tightly connected to vascular plants. Isoprene and sesquiterpene emissions were associated with sedges, whereas monoterpenoids and homoterpenes were associated with shrubs. Additionally, isoprene and alkane emissions were higher in the fen than in the bog and they significantly contributed to the higher BVOC emissions from intact vegetation in the fen. During an extreme drought event in 2018, emissions of organic halides were absent. Our results indicate that climate change with an increase in shrub cover and increased frequency of extreme weather events may have a negative impact on total BVOC emissions that otherwise are predicted to increase in warmer temperatures. However, these changes also accompanied a change in BVOC emission quality. As different compounds differ in their capacity to form secondary organic aerosols, the ultimate climate impact of peatland BVOC emissions may be altered.

Germination and seedling growth of Calluna vulgaris is sensitive to regional climate, heathland succession, and drought.

The coastal heathlands of Northwest Europe are highly valued cultural landscapes, that are critically endangered due to land use and climatic changes, such as increased frequency and severity of drought events. Our study is the first to assess how the germination and early seedling growth of Calluna vulgaris respond to drought. In a factorial design field experiment, we exposed maternal plants to three in-situ drought treatments (control, 60%, 90% roof coverage), across three successional stages after fire (pioneer, building, mature), and two regions (60°N, 65°N). Seeds from 540 plants within the experiment were, weighed, and exposed to five water potentials, ranging from -0.25 to -1.7 MPa, in a growth chamber experiment. We recorded germination (percentage, rate), seedling growth (above- vs. belowground allocation), and seedling functional traits (specific leaf area [SLA], specific root length [SRL]). Overall variation in germination between regions, successional stages, and maternal drought treatments was largely mediated by variation in seed mass. Plants from the northernmost region had higher seed mass and germination percentages. This is indicative of higher investment in seeds, likely linked to the populations' absence of vegetative root sprouting. Seeds from the mature successional stage germinated to lower final percentages than those from earlier successional stages, especially when the maternal plants had been exposed to drought (60% and 90% roof coverage). Exposure to reduced water availability decreased germination percentage and increased the time to 50% germination. Seedlings fully developed in the range -0.25 to -0.7 MPa, with increased root:shoot and lower SRL during reduced water availability, suggesting a resource-conservative response to drought during the early stages of development. Our results thus suggest a sensitivity to drought during the germination and seedling life-history stages that may reduce Calluna's ability to re-establish from seeds as the incidence and severity of droughts are projected to increase under future climates.

Communities of Putative Ericoid Mycorrhizal Fungi Isolated from Alpine Dwarf Shrubs in Japan: Effects of Host Identity and Microhabitat.

Dwarf shrubs of the family Ericaceae are common in arctic and alpine regions. Many of these plants are associated with ericoid mycorrhizal (ERM) fungi, which allow them to take nutrients and water from the soil under harsh environmental conditions and, thus, affect host plant survival. Despite the importance of ERM fungi to alpine plant communities, limited information is available on the effects of microhabitat and host identity on ERM fungal communities. We investigated the communities of putative ERM fungi isolated from five dwarf shrub species (Arcterica nana, Diapensia lapponica, Empetrum nigrum, Loiseleuria procumbens, and Vaccinium vitis-idaea) that co-occur in an alpine region of Japan, with reference to distinct microhabitats provided by large stone pine (Pinus pumila) shrubs (i.e. bare ground, the edge of stone pine shrubs, and the inside of stone pine shrubs). We obtained 703 fungal isolates from 222 individual plants. These isolates were classified into 55 operational taxonomic units (OTUs) based on the sequencing of internal transcribed spacer regions in ribosomal DNA. These putative ERM fungal communities were dominated by Helotiales fungi for all host species. Cistella and Trimmatostroma species, which have rarely been detected in ERM roots in previous studies, were abundant. ERM fungal communities were significantly different among microhabitats (R2=0.28), while the host effect explained less variance in the fungal communities after excluding the microhabitat effect (R2=0.17). Our results suggest that the host effect on ERM fungal communities is minor and the distributions of hosts and fungal communities may be assessed based on microhabitat conditions.

Effects of Alpine land-use changes: Soil macrofauna community revisited.

Although soil invertebrates play a decisive role in maintaining ecosystem functioning, little is known about their structural composition in Alpine soils and how their abundances are affected by the currently ongoing land-use changes. In this study, we re-assessed the soil macrofauna community structure of managed and abandoned Alpine pastureland, which has already been evaluated 14 years earlier. Our results confirm clear shifts in the community composition after abandonment, in that (1) Chilopoda and Diplopoda were recorded almost exclusively on the abandoned sites, (2) Coleoptera larvae and Diptera larvae were more abundant on the abandoned than on the managed sites, whereas (3) Lumbricidae dominated on the managed sites. By revisiting managed and abandoned sites, we infer community patterns caused by abandonment such as changes in the epigeic earthworm community structure, and we discuss seasonal and sampling effects. Our case study improves the still limited understanding of spatio-temporal biodiversity patterns of Alpine soil communities.

Impact of early and late winter icing events on sub-arctic dwarf shrubs.

Polar regions are predicted to undergo large increases in winter temperature and an increased frequency of freeze-thaw cycles, which can cause ice layers in the snow pack and ice encasement of vegetation. Early or late winter timing of ice encasement could, however, modify the extent of damage caused to plants. To determine impacts of the date of ice encasement, a novel field experiment was established in sub-arctic Sweden, with icing events simulated in January and March 2008 and 2009. In the subsequent summers, reproduction, phenology, growth and mortality, as well as physiological indicators of leaf damage were measured in the three dominant dwarf shrubs: Vaccinium uliginosum, Vaccinium vitis-idaea and Empetrum nigrum. It was hypothesised that January icing would be more damaging compared to March icing due to the longer duration of ice encasement. Following 2 years of icing, E. nigrum berry production was 83% lower in January-iced plots compared to controls, and V. vitis-idaea electrolyte leakage was increased by 69%. Conversely, electrolyte leakage of E. nigrum was 25% lower and leaf emergence of V. vitis-idaea commenced 11 days earlier in March-iced plots compared to control plots in 2009. There was no effect of icing on any of the other parameters measured, indicating that overall these study species have moderate to high tolerance to ice encasement. Even much longer exposure under the January icing treatment does not clearly increase damage.