Exploration of plant growth and development using the European Modular Cultivation System facility on the International Space Station

Space experiments provide a unique opportunity to advance our knowledge of how plants respond to the space environment, and specifically to the absence of gravity. The European Modular Cultivation System (EMCS) has been designed as a dedicated facility to improve and standardise plant growth in the International Space Station (ISS). The EMCS is equipped with two centrifuges to perform experiments in microgravity and with variable gravity levels up to 2.0 g. Seven experiments have been performed since the EMCS was operational on the ISS. The objectives of these experiments aimed to elucidate phototropic responses (experiments TROPI‐1 and ‐2), root gravitropic sensing (GRAVI‐1), circumnutation (MULTIGEN‐1), cell wall dynamics and gravity resistance (Cell wall/Resist wall), proteomic identification of signalling players (GENARA‐A) and mechanism of InsP₃ signalling (Plant signalling). The role of light in cell proliferation and plant development in the absence of gravity is being analysed in an on‐going experiment (Seedling growth). Based on the lessons learned from the acquired experience, three preselected ISS experiments have been merged and implemented as a single project (Plant development) to study early phases of seedling development. A Topical Team initiated by European Space Agency (ESA), involving experienced scientists on Arabidopsis space research experiments, aims at establishing a coordinated, long‐term scientific strategy to understand the role of gravity in Arabidopsis growth and development using already existing or planned new hardware.     

Development of a plant growth support system for experiments on the ISS

The European Modular Cultivation System (EMCS) is one of a wide range of laboratory modules under construction by ESA that will be placed on the International Space Station (ISS). In the present study the development and construction of an important component in the EMCS, the Plant Cultivation Container (PCC), is described. The PCC as a "flower pot" will automatically provide the plants with water and liquid nutrients as needed. The PCC is located inside the plant growth unit, the Experiment Container (EC), on the EMCS and interfaces with the EMCS. The essential parts of the PCC are a Peltier element, a micro valve, a monitoring RH sensor with an integrated platinum RTD temperature sensor, a RH sensor that detects air leaving the PCC and controls the peristaltic pump, a DC-DC board that provides correct current to the Peltier element, and a switch/connector board. The PCC is presently being tested out at ESTEC/ESA.     

Rhizosphere microbiome functional diversity and pathogen invasion resistance build up during plant development

The rhizosphere microbiome is essential for plant growth and health, and numerous studies have attempted to link microbiome functionality to species and trait composition. However, to date little is known about the actual ecological processes shaping community composition, complicating attempts to steer microbiome functionality. Here, we assess the development of microbial life history and community-level species interaction patterns that emerge during plant development. We use microbial phenotyping to experimentally test the development of niche complementarity and life history traits linked to microbiome performance. We show that the rhizosphere microbiome assembles from pioneer assemblages of species with random resource overlap into high-density, functionally complementary climax communities at later stages. During plant growth, fast-growing species were further replaced by antagonistic and stress-tolerant ones. Using synthetic consortia isolated from different plant growth stages, we demonstrate that the high functional diversity of ‘climax’ microbiomes leads to a better resistance to bacterial pathogen invasion. By demonstrating that different life-history strategies prevail at different plant growth stages and that community-level processes may supersede the importance of single species, we provide a new toolbox to understand microbiome assembly and steer its functionality at a community level.     

BIOLAB, EPU and EMCS for cell culture experiments on the ISS

Two ESA facilities are under development for biological research on the International Space Station: BIOLAB as part of the European "Columbus" Laboratory and the European Modular Cultivation System (EMCS), foreseen for accommodation in the US Lab "Destiny". Both facilities have an incubator (18-40 degrees C) and use standard Experiment Containers, mounted on two centrifuge rotors providing either microgravity or variable g-levels from 0.001 x g to 2.0 x g. Standard interface plates supply each container with power and data lines, with gas (controlled CO2, O2 and water vapour concentration; trace gas removal), and--for EMCS only--with water. The degree of automation is higher in BIOLAB: it contains a robotic Handling Mechanism for automatic sampling and handling of liquids, which can be stored at cool or cold temperatures or injected for automatic on-board analysis into a microscope or a spectrophotometer. For analyses on the running centrifuge, small automatic microscopes can be installed in the Experiment Containers. Several designs for supporting cell culture experiments have been studied for BIOLAB and EMCS. BIOLAB has in addition a Bio-Glovebox, which can be sterilised and where new cell cultures may be prepared under 1 x g conditions from deep-frozen samples in the Experiment Preparation Unit (EPU): the cryo-protectant will be removed by automatic washing cycles. Both facilities, EMCS and BIOLAB (with EPU), have also provisions for telescience operations through video, data and command lines, either operated by the crew or by the experimenter on ground.     

Solubilization of inorganic phosphate and plant growth promotion by cold tolerant mutants of Pseudomonasfluorescens

A study for screening and selection of cold tolerant mutants of Pseudomonas fluorescens strains GRS1, PRS9 and ATCC13525 based on 'P' solubilization ability and subsequent effect on plant growth promotion under in vitro and in situ conditions was conducted. Of all the mutants tested, two were selected, as there was a 21-fold increase in CRPF, (GRS, mutant) and a 10-fold decrease in CRPF7 (PRS9 mutant) over their respective wild types. Under in vitro conditions at 10 degrees C, these cold tolerant mutants exhibited increased plant growth indicating their functionality at low temperature. Subsequently, greenhouse trials using soil-plant microcosms were conducted which revealed that CRPF, (high 'P' solubilizer) was a good rhizosphere colonizer showing a significant increase in root (30 and 20%) and shoot length (20 and 24%) of mung bean, both in sterilized and unsterilized soil, respectively. On the contrary, CRPF, (low 'P' solubilizer) did not stimulate plant growth. Furthermore, sand experiments indicated that tricalcium phosphate served as better phosphorus source for CRPF2 treated mung bean seeds.     

A biohybrid artificial lung prototype with active mixing of endothelialized microporous hollow fibers

Acute respiratory distress syndrome (ARDS) affects nearly 150,000 patients per year in the US, and is associated with high mortality (≈40%) and suboptimal options for patient care. Mechanical ventilation and extracorporeal membrane oxygenation are limited to short‐term use due to ventilator‐induced lung injury and poor biocompatibility, respectively. In this report, we describe the development of a biohybrid lung prototype, employing a rotating endothelialized microporous hollow fiber (MHF) bundle to improve blood biocompatibility while MHF mixing could contribute to gas transfer efficiency. MHFs were surface modified with radio frequency glow discharge (RFGD) and protein adsorption to promote endothelial cell (EC) attachment and growth. The MHF bundles were placed in the biohybrid lung prototype and rotated up to 1,500 revolutions per minute (rpm) using speed ramping protocols to condition ECs to remain adherent on the fibers. Oxygen transfer, thrombotic deposition, and EC p‐selectin expression were evaluated as indicators of biohybrid lung functionality and biocompatibility. A fixed aliquot of blood in contact with MHF bundles rotated at either 250 or 750 rpm reached saturating pO₂ levels more quickly with increased rpm, supporting the concept that fiber rotation would positively contribute to oxygen transfer. The presence of ECs had no effect on the rate of oxygen transfer at lower fiber rpm, but did provide some resistance with increased rpm when the overall rate of mass transfer was higher due to active mixing. RFGD followed by fibronectin adsorption on MHFs facilitated near confluent EC coverage with minimal p‐selectin expression under both normoxic and hyperoxic conditions. Indeed, even subconfluent EC coverage on MHFs significantly reduced thrombotic deposition adding further support that endothelialization enhances, blood biocompatibility. Overall these findings demonstrate a proof‐of‐concept that a rotating endothelialized MHF bundle enhances gas transfer and biocompatibility, potentially producing safer, more efficient artificial lungs. Biotechnol. Bioeng. 2010; 106: 490–500. © 2010 Wiley Periodicals, Inc.     

Most soil trophic guilds increase plant growth: a meta‐analytical review

Trophic cascades are important drivers of plant and animal abundances in aquatic and aboveground systems, but in soils trophic cascades have been thought to be of limited importance due to omnivory and other factors. Here we use a meta‐analysis of 215 studies with 1526 experiments that measured plant growth responses to additions or removals of soil organisms to test how different soil trophic levels affect plant growth. Consistent with the trophic cascade hypothesis, we found that herbivores and plant pathogens (henceforth pests) decreased plant growth and that predators of pests increased plant growth. The magnitude of this trophic cascade was similar to that reported for aboveground systems. In contrast, we did not find evidence for trophic cascades in decomposer‐ and symbiont‐based (henceforth mutualist) food chains. In these food chains, mutualists increased plant growth and predators of mutualists also increased plant growth, presumably by increasing nutrient cycling rates. Therefore, mutualists, predators of mutualists and predators of pests all increased plant growth. Further, experiments that added multiple organisms from different trophic levels also increased plant growth. As a result, across the dataset, soil organisms increased plant growth 29% and non‐pest soil organisms increased plant growth 46%. Omnivory has traditionally been thought to confound soil trophic dynamics, but here we suggest that omnivory allows for a simplified perspective of soil food webs – one in which most soil organisms increase plant growth by preying on pests or increasing nutrient cycling rates. An implication of this perspective is that processes that decrease soil organism abundance (e.g. soil tillage) are likely to decrease aboveground productivity. Synthesis Soil foodwebs have resisted generalizations due to their diversity and interconnectedness. Here we use results from a meta‐analysis to inform a simplified perspective of soil foodwebs: one in which most soil trophic guilds increase plant growth. Our review also includes the first widespread support for the presence of trophic cascades in soils.     

Improved Anchorage of Ti6Al4V Orthopaedic Bone Implants through Oligonucleotide Mediated Immobilization of BMP-2 in Osteoporotic Rats

The aim of the present study was to test the biocompatibility and functionality of orthopaedic bone implants with immobilized oligonucleotides serving as anchor stands for rhBMP-2 and rhVEGF-A conjugated with complementary oligonucleotides in an osteoporotic rat model. Al₂O₃-blasted acid etched Ti6Al4V implants, carrying oligonucleotide anchor strands and hybridized with rhBMP-2 or rhVEGF-A through complementary 31-mer oligonucleotide stands were inserted into the proximal tibia of ovariectomized rats. At the time of surgery (15 weeks after ovariectomy) microCT analysis showed significantly lower bone mineral density compared to non-ovariectomized animals. Bone-implant contact (BIC) and pullout-force were not negatively affected by non-hybridized anchor strands. Twelve weeks after surgery, a significantly higher pullout force was found for BMP-2 hybridized to the anchor strands compared to non-hybridized anchor strands or native samples, and on histomorphometric analysis BIC was highest in the BMP group. Thus, we could show the biocompatibility and in vivo functionality of this modular, self-organizing system for immobilization and subsequent release of BMP-2 in vivo.     

Photosynthetic apparatus performance in function of the cytokinins used during the in vitro multiplication of <Emphasis Type="Italic">Aechmea blanchetiana</Emphasis> (Bromeliaceae)

During the in vitro multiplication phase, the employment of cytokinins may be necessary to induce side shoots of many plant species. However, the mechanism by which cytokinins influence the physiology of plants in vitro is not well understood. Therefore, the objective of this study was to assess the influence of two cytokinins in function of concentration on the o photosynthetic apparatus performance and the stomatal functionality of Aechmea blanchetiana during in vitro multiplication. Plants previously established in vitro were transferred to MS culture media supplemented with 6-benzylaminopurine (BAP) or 6-furfurylaminopurine (kinetin—KIN) at concentration of 0, 5, 10, 15 or 20 µM. After 60 days of exposure to the plant growth regulators, the multiplication rate, photosynthetic apparatus performance and stomatal functionality were assessed. The use of KIN did not induce the formation of microshoots. On the other hand, the shoot number increased with rising BAP concentration. There was a reduction of the maximum fluorescence (F_m) and maximum quantum yield (φP₀) as a function of concentration of cytokinins. The most pronounced decrease was observed in the microshoots grown with KIN. The increase in concentration of cytokinins induced greater absorption flux (ABS/RC), trapping flux (TR₀/RC) and dissipation flux (DI₀/RC) of energy per reaction center. The stomatal functionality declined with rising cytokinin concentration. The use of KIN is not recommended for in vitro multiplication of this species. The use of BAP at low concentrations assures a multiplication rate with lower degree of disorders in the photosynthetic apparatus of the formed microshoots.     

Automated culture system experiments hardware: developing test results and design solutions

The experiment proposed by Prof. Ricci University of Milan is funded by ASI with Laben as industrial Prime Contractor. ACS-EH (Automated Culture System-Experiment Hardware) will support the multigenerational experiment on weightlessness with rotifers and nematodes within four Experiment Containers (ECs) located inside the European Modular Cultivation System (EMCS) facility..Actually the Phase B is in progress and a concept design solution has been defined. The most challenging aspects for the design of such hardware are, from biological point of view the provision of an environment which permits animal's survival and to maintain desiccated generations separated and from the technical point of view, the miniaturisation of the hardware itself due to the reduce EC provided volume (160mmx60mmx60mm). The miniaturisation will allow a better use of the available EMCS Facility resources (e.g. volume. power etc.) and to fulfil the experiment requirements. ACS-EH, will be ready to fly in the year 2005 on boar the ISS.     

Inference of allelopathy is complicated by effects of activated carbon on plant growth

Allelopathy can play an important role in structuring plant communities, but allelopathic effects are often difficult to detect because many methods used to test for allelopathy can be confounded by experimental artifacts. The use of activated carbon, a technique for neutralizing allelopathic compounds, is now employed in tests for allelopathy; however, this technique also could produce large experimental artifacts. In three independent experiments, it was shown that adding activated carbon to potting media affected nutrient availability and plant growth. For most species tested, activated carbon increased plant biomass, even in the absence of the potentially allelopathic agent. The increased growth corresponded to increased plant nitrogen content, likely resulting from greater nitrogen availability. Activated carbon also affected nitrogen and other nutrient concentrations in soil media in the absence of plants. The observed effects of activated carbon on plant growth can confound its use to test for allelopathy. The detection of allelopathy relies on the difference between plant growth in medium with carbon and that in medium without carbon in the presence of the potentially allelopathic competitor; however, this difference may be biased if activated carbon alters soil nutrient availability and plant growth even in the absence of the focal allelopathic agent.     

Enhancing the growth of Vicia faba plants by microbial inoculation to improve their phytoremediation potential for oily desert areas

Two experiments were conducted to investigate the effect of inoculating Vicia faba plants (broad beens) raised in clean and oily sand with nodule-forming rhizobia and plant-growth-promoting rhizobacteria (PGPR) on growth of these plants in sand and to test whether this can improve the phytoremediation potential of this crop for oily desert areas. It was found that crude oil in sand at concentrations < 1.0% (w/w) enhanced the plant heights, their fresh and dry weights, the total nodule weights per plant, and the nitrogen contents of shoots and fruits. Similar enhancing effects were recorded when roots of the young plants were inoculated with nodule bacteria alone, PGPR alone, or a mixture of one strain of nodule bacteria and one of the PGPR. Such plant growth effects were associated with a better phytoremediation potential of V. faba plants for oily sand. The total numbers of oil-utilizing bacteria increased in the rhizosphere and more hydrocarbons were eliminated in sand close to the roots. The nodule bacteria tested were two strains of Rhizobium leguminosarum and the PGPR were Pseudomonas aeruginosa and Serratia liquefaciens. The four strains were found to use crude oil, n-octadecane, and phenanthrene as sole sources of carbon and energy. It was concluded that coinoculation of V. faba plant roots in oily sand with nodule bacteria and PGPR enhances the phytoremediation potential of this plant for oily desert sand through improving plant growth and nitrogen fixation.     

Evidence for temperature limitation of nitrogen mineralisation in the Drakensberg Alpine Centre

We made use of pot experiments and soil mineralisation assays to test the effect of temperature on the soil nitrogen (N) economy of the Drakensberg Alpine Centre (‘mountain site’). The approach was enhanced by the inclusion of a contrasting warm, subtropical environment on the east coast of southern Africa (‘coast site’) which presented an opportunity to test plant growth in mountain soil outside of the mountain site's natural climatic envelope. This study was further augmented by two greenhouse experiments that helped isolate the factors responsible for the growth responses in the experiments above. Plant morphology, plant nutrients and soil nutrients were used as the basis for comparing treatment effects. The primary pot experiment showed that plant growth was uniform in the mountain site regardless of whether the test species was grown in intrinsically N-rich mountain soil or intrinsically N-poor coast soil. However, we noted significant growth differences at the coast site using the aforementioned soil nutrient regimes. In terms of the soil mineralisation assay, coast soil, derived from intrinsically N-poor sandstone, predictably mineralised little soil inorganic N at the mean spring temperature of 19 °C. However against expectations, the intrinsically N-rich mountain soil mineralised < 1% of its total soil N budget into inorganic N at 12 °C, most probably because the microbes responsible for the conversion of organic soil N to inorganic soil N were severely inhibited at this mean spring temperature. However, the potential to mineralise far more N in mountain soil was apparent when using an elevated experimental temperature of 30 °C, with 369% more soil N being available under the latter regime. Our results suggest that the cooler temperatures associated with high elevations in the mountain site constrain the activity of soil microbes in mountain soil, resulting in a functionally N-poor soil economy particularly deficient in inorganic N. This also explains the similar growth responses regardless of the soil being intrinsically N-rich or N-poor. We speculate whether or not more soil inorganic N may become available under a regime of warming due to accelerated N mineralisation, to the detriment of plant taxa adapted to low soil N availability.     

In vitro bio-functionality of gallium nitride sensors for radiation biophysics

There is an increasing interest in the integration of hybrid bio-semiconductor systems for the non-invasive evaluation of physiological parameters. High quality gallium nitride and its alloys show promising characteristics to monitor cellular parameters. Nevertheless, such applications not only request appropriate sensing capabilities but also the biocompatibility and especially the biofunctionality of materials. Here we show extensive biocompatibility studies of gallium nitride and, for the first time, a biofunctionality assay using ionizing radiation. Analytical sensor devices are used in medical settings, as well as for cell- and tissue engineering. Within these fields, semiconductor devices have increasingly been applied for online biosensing on a cellular and tissue level. Integration of advanced materials such as gallium nitride into these systems has the potential to increase the range of applicability for a multitude of test devices and greatly enhance sensitivity and functionality. However, for such applications it is necessary to optimize cell-surface interactions and to verify the biocompatibility of the semiconductor. In this work, we present studies of mouse fibroblast cell activity grown on gallium nitride surfaces after applying external noxa. Cell-semiconductor hybrids were irradiated with X-rays at air kerma doses up to 250 mGy and the DNA repair dynamics, cell proliferation, and cell growth dynamics of adherent cells were compared to control samples. The impact of ionizing radiation on DNA, along with the associated cellular repair mechanisms, is well characterized and serves as a reference tool for evaluation of substrate effects. The results indicate that gallium nitride does not require specific surface treatments to ensure biocompatibility and suggest that cell signaling is not affected by micro-environmental alterations arising from gallium nitride-cell interactions. The observation that gallium nitride provides no bio-functional influence on the cellular environment confirms that this material is well suited for future biosensing applications without the need for additional chemical surface modification.     

Reactive polymer multilayers fabricated by covalent layer-by-layer assembly: 1,4-conjugate addition-based approaches to the design of functional biointerfaces

We report on conjugate addition-based approaches to the covalent layer-by-layer assembly of thin films and the post-fabrication functionalization of biointerfaces. Our approach is based on a recently reported approach to the "reactive" assembly of covalently cross-linked polymer multilayers driven by the 1,4-conjugate addition of amine functionality in poly(ethyleneimine) (PEI) to the acrylate groups in a small-molecule pentacrylate species (5-Ac). This process results in films containing degradable β-amino ester cross-links and residual acrylate and amine functionality that can be used as reactive handles for the subsequent immobilization of new functionality. Layer-by-layer growth of films fabricated on silicon substrates occurred in a supra-linear manner to yield films ≈ 750 nm thick after the deposition of 80 PEI/5-Ac layers. Characterization by atomic force microscopy (AFM) suggested a mechanism of growth that involves the reactive deposition of nanometer-scale aggregates of PEI and 5-Ac during assembly. Infrared (IR) spectroscopy studies revealed covalent assembly to occur by 1,4-conjugate addition without formation of amide functionality. Additional experiments demonstrated that acrylate-containing films could be postfunctionalized via conjugate addition reactions with small-molecule amines that influence important biointerfacial properties, including water contact angles and the ability of film-coated surfaces to prevent or promote the attachment of cells in vitro. For example, whereas conjugation of the hydrophobic molecule decylamine resulted in films that supported cell adhesion and growth, films treated with the carbohydrate-based motif D-glucamine resisted cell attachment and growth almost completely for up to 7 days in serum-containing media. We demonstrate that this conjugate addition-based approach also provides a means of immobilizing functionality through labile ester linkages that can be used to promote the long-term, surface-mediated release of conjugated species and promote gradual changes in interfacial properties upon incubation in physiological media (e.g., over a period of at least 1 month). These covalently cross-linked films are relatively stable in biological media for prolonged periods, but they begin to physically disintegrate after ≈ 30 days, suggesting opportunities to use this covalent layer-by-layer approach to design functional biointerfaces that ultimately erode or degrade to facilitate elimination.     

Low root functional dispersion enhances functionality of plant growth by influencing bacterial activities in European forest soils

Current studies show that multispecies forests are beneficial regarding biodiversity and ecosystem functionality. However, there are only little efforts to understand the ecological mechanisms behind these advantages of multispecies forests. Bacteria are among the key plant growth-promoting microorganisms that support tree growth and fitness. Thus, we investigated links between bacterial communities, their functionality and root trait dispersion within four major European forest types comprising multispecies and monospecific plots. Bacterial diversity revealed no major changes across the root functional dispersion gradient. In contrast, predicted gene profiles linked to plant growth activities suggest an increasing bacterial functionality from monospecific to multispecies forest. In multispecies forest plots, the bacterial functionality linked to plant growth activities declined with the increasing functional dispersion of the roots. Our findings indicate that enriched abundant bacterial operational taxonomic units are decoupled from bacterial functionality. We also found direct effects of tree species identity on bacterial community composition but no significant relations with root functional dispersion. Additionally, bacterial network analyses indicated that multispecies forests have a higher complexity in their bacterial communities, which points towards more stable forest systems with greater functionality. We identified a potential of root dispersion to facilitate bacterial interactions and consequently, plant growth activities.     

Physical mechanisms of solid-protein interactions in the interface between amorphous silicon carbide and fibrinogen]

State of the art in biomaterial research and implant design is a compromise between functionality and biocompatibility. Consequently the results often have disadvantages with respect to both aspects. In regard to biocompatibility the activation of the clotting system by alloplastic materials is of great significance, because it necessitates anticoagulant therapy. Further improvements of implant technology require an understanding of the interactions between blood and implants. Therefore a microscopic model of thrombogenesis at alloplastic surfaces will shortly be presented, which relates thrombogenicity of a material to the electronic structure of its surface. The requirements for high hemocompatibility, which result from this model--especially in regard to the density of states and the conductivity at the surface--are fulfilled by an amorphous alloy of silicon and carbon (a-SiC:H). The advantage of amorphous materials is that they do not obey stoichiometric rules. Thus they allow a continuous adjustment of the electronic parameters without fundamental changes of their mechanical and chemical properties. The theoretical results where checked by total internal reflection intrinsic fluorescence spectroscopy (TIRIF) as well as thrombelastography experiments (TEG). In comparison to conventional materials like titanium or LTI carbon the TEG-clotting time of a-SiC:H-coatings is prolonged in excess of 200%. As a consequence a-SiC:H is well suited as a hemocompatible coating material for hybrid structuring of cardiovascular implants.     

Do plant functional types based on leaf dry matter content allow characterizing native grass species and grasslands for herbage growth pattern?

Few studies have focused on vegetation characteristics of importance to feeding domestic herbivores, mainly the seasonal pattern of herbage growth at spring. Our objective is to establish and to evaluate a simple method of ranking grassland communities for these characteristics. We combined approaches at plant species level (comparison of grass species growing in a pure stand) and plant community level (comparison of grasslands differing mainly in their nutrient availability). Firstly, we ask if the ranking of species by leaf dry matter content (LDMC), a functional parameter used to assess the plant strategy for resource acquisition and use, is consistent with a classification of the species using three plant features that determine plant growth pattern at spring (beginning and ending of stem elongation, leaf lifespan). Secondly, for three networks of natural grasslands, we test whether there is consistency when ranking them by their dominant plant functional type (PFT A, B or C) established previously at species level, and by the three agronomic characteristics. For species growing in pure stands, there was a significant effect of PFT for the three plant features. For species having a low LDMC (A and B PFT), there were earlier stem elongation in the season, earlier flowering and shorter leaf lifespan. The opposite was observed for species having a high LDMC (C and D PFT). For grassland communities dominated by A-PFT, the ceiling yield for leaves and stems occurred earlier in spring than for those dominated by C-PFT. Results were consistent at plant and community levels. Scaling up from plant to community was well mediated by PFT. Plant features which characterize species for resource acquisition and use are consistent with herbage growth patterns at plant community level. These results show that herbage growth pattern and composition depend on PFTs and that knowing the PFT dominance is of great importance to plan the use of grasslands. We can expect to use the PFT approach to perform vegetation diagnosis at field level when the objective is to rank grassland communities for their agronomic characteristics. Fernando L. F. de Quadros was supported by CAPES and CNPq (BP-2).     

Nondestructive leaf-area estimation and validation for green pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) grown under different stress conditions

Leaf area of a plant is essential to understand the interaction between plant growth and environment. This useful variable can be determined by using direct (some expensive instruments) and indirect (prediction models) methods. Leaf area of a plant can be predicted by accurate and simple leaf area models without damaging the plant, thus, provide researchers with many advantages in horticultural experiments. Several leaf-area prediction models have been produced for some plant species in optimum conditions, but not for a plant grown under stress conditions. This study was conducted to develop leaf area estimation models by using linear measurements such as lamina length and width by multiple regression analysis for green pepper grown under different stress conditions. For this purpose, two experiments were conducted in a greenhouse. The first experiment focused to determine leaf area of green pepper grown under six different levels of irrigation water salinity (0.65, 2.0, 3.0, 4.0, 5.0, and 7.0 dS m⁻¹) and the other under four different irrigation regime (amount of applied water was 1.43, 1.0, 0.75, and 0.50 times of required water). In addition to general models for each experiment, prediction models of green pepper for each treatment of irrigation water salinity and of irrigation regime experiments were obtained. Validations of the models for both experiments were realized by using the measurements belong to leaf samples allocated for validation purposes. As a result, the determined equations can simply and readily be used in prediction of leaf area of green pepper grown under salinity and water stress conditions. The use of such models enable researchers to measure leaf area on the same plants during the plant growth period and, at the same time, may reduce variability in experiments.     

Assessment of essential oils activity used in liquid or volatile phase against biofilm cultures of Staphylococcus aureus and Candida albicans. The methodological study

Biofilm formation is a significant factor in chronic infections with fungal and bacterial pathogens. Due to the high drug resistance of biofilm populations and the frequent failures of chemotherapy i such infections, it seems necessary to take recourse to unconventional treatment methods involving e.g. the use of some phytocompounds such as essential oils or their components. In order to evaluate the effect of their action on the microbial biomass a variety of techniques are used. However, there is still a need to develop new tests or modifications of these known, for the biofilm viability assessment. They should be adapted to the physico-chemical nature of the tested compounds and should decrease the risk ofbiofilm damage during staining procedure. We described a test assessing the effect of essential oils on bacterial and fungal biofilm formed on the membrane of cell culture inserts. The proposed model provides a minimal violation of the biofilm integrity during the test. It allows easily explore the activity of essential oil volatile fraction and is useful in determination of the kinetics of their action. Using this test it is also easy to examine the relationship between antimicrobial activity and the cytotoxic effect, known as the biocompatibility index (BI, biocompatibility index). Moreover, it allows qualitative and quantitative analysis of metabolic products, released into the growth medium from biofilm's cells. In successively repeated experiments high reproducibility of results has been obtained, thus the developed methodology seems to be useful in our future studies in this field.