COMPETITION AMONG EUCALYPTUS TREES DEPENDS ON GENETIC VARIATION AND RESOURCE SUPPLY

Genetic variation and environmental heterogeneity fundamentally shape the interactions between plants of the same species. According to the resource partitioning hypothesis, competition between neighbors intensifies as their similarity increases. Such competition may change in response to increasing supplies of limiting resources. We tested the resource partitioning hypothesis in stands of genetically identical (clone-origin) and genetically diverse (seed-origin) Eucalyptus trees with different water and nutrient supplies, using individual-based tree growth models. We found that genetic variation greatly reduced competitive interactions between neighboring trees, supporting the resource partitioning hypothesis. The importance of genetic variation for Eucalyptus growth patterns depended strongly on local stand structure and focal tree size. This suggests that spatial and temporal variation in the strength of species interactions leads to reversals in the growth rank of seed-origin and clone-origin trees. This study is one of the first to experimentally test the resource partitioning hypothesis for intergenotypic vs. intragenotypic interactions in trees. We provide evidence that variation at the level of genes, and not just species, is functionally important for driving individual and community-level processes in forested ecosystems.

Why don`t our stands grow even faster? Control of production and carbon cycling in eucalypt plantations

The growth of Eucalyptus stands varies several fold across sites, under the influence of resource availability, stand age and stand structure. We describe a series of related studies that aim to understand the mechanisms that drive this great range in stand growth rates. In a seven-year study in Hawaii of Eucalyptus saligna at a site that was not water limited, we showed that nutrient availability differences led to a two-fold difference in stand wood production. Increasing nutrient supply in mid-rotation raised productivity to the level attained in continuously fertilised plots. Fertility affected the age-related decline in wood and foliage production; production in the intensive fertility treatments declined more slowly than in the minimal fertility treatments. The decline in stem production was driven largely by a decline in canopy photosynthesis. Over time, the fraction of canopy photosynthesis partitioned to below-ground allocation increased, as did foliar respiration, further reducing wood production. The reason for the decline in photosynthesis was uncertain, but it was not caused by nutrient limitation, a decline in leaf area or in photosynthetic capacity, or by hydraulic limitation. Most of the increase in carbon stored from conversion of the sugarcane plantation to Eucalyptus plantation was in the above-ground woody biomass. Soil carbon showed no net change. This study and other studies on carbon allocation showed that resource availability changes the fraction of annual photosynthesis used below-ground and for wood production. High resources (nutrition or water) decrease the partitioning below-ground and increase partitioning to wood production. Annual foliage and wood respiration and foliage production as a fraction of annual photosynthesis was remarkably constant across a wide range of fertility treatments and forest age. In the Brazil Eucalyptus Productivity Project, stand structure was manipulated by planting clonal Eucalyptus all at once or in three groups at three-monthly intervals, producing a stand where trees did not segregate into dominants and one that had strong dominance. The uneven stand structure reduced production 10-15% throughout the rotation.

Production and carbon allocation in a clonal Eucalyptus plantation with water and nutrient manipulations

We examined resource limitations on growth and carbon allocation in a fast-growing, clonal plantation of Eucalyptus grandis x urophylla in Brazil by characterizing responses to annual rainfall, and response to irrigation and fertililization for 2 years. Productivity measures included gross primary production (GPP), total belowground carbon allocation (TBCA), bole growth, and net ecosystem production (NEP). Replicate plots within a single plantation were established at the midpoint of the rotation (end of year 3), with treatments of no additional fertilization or irrigation, heavy fertilization (to remove any nutrient limitation), irrigation (to remove any water limitation), and irrigation plus fertilization. Rainfall was unusually high in the first year (1769mm) of the experiment, and control plots had high rates of GPP (6.64 kg C m(-2) year(-1)), TBCA (2.14 kg C m(-2) year(-1)), and bole growth (1.81 kg C m(-2) year). Irrigation increased each of these rates by 15-17%. The second year of the experiment had average rainfall (1210 mm), and lower rainfall decreased production in control plots by 46% (GPP), 52% (TBCA), and 40% (bole growth). Fertilization treatments had neglible effects. The response to irrigation was much greater in the drier year, with irrigated plots exceeding the production in control plots by 83% (GPP), 239% (TBCA), and 24% (bole growth). Even though the rate of irrigation ensured no water limitation to tree growth, the high rainfall year showed higher production in irrigated plots for both GPP (38% greater than in drier year) and bole growth (23% greater). Varying humidity and supplies of water led to a range in NEP of 0.8-2.7 kg C m(-2) year. This difference between control and irrigated treatments, combined with differences between drier and wetter years, indicated a strong response of these Eucalyptus trees to both water supply and atmospheric humidity during the dry season. The efficiency of converting light energy into fixed carbon ranged from a low of 0.027 mol C to a high of 0.060 mol C per mol of absorbed photosynthetically active radiation (APAR), and the efficiency of bolewood production ranged from 0.78 to 1.98 g wood per MJ of APAR. Irrigation increased the efficiency of wood production per unit of water used from 2.55 kg wood m(-3) in the rainfed plot to 3.51 kg m(-3) in irrigated plots. Detailed information on the response of C budgets to environmental conditions and resource supplies will be necessary for accurate predictions of plantation yields across years and landscapes. (V) 2007 Elsevier B.V. All rights reserved.

Isolation and characterization of microsatellite loci in Colletotrichum acutatum, the causal agent of postbloom fruit drop on citrus

From a genomic enriched library, we developed 27 primer pairs from microsatellite flanking sequences for Colletotrichum acutatum, associated to postbloom fruit drop disease on citrus. Loci were characterized using 40 monosporic C. acutatum isolates. Nine primer pairs successfully amplified polymorphic microsatellite regions, with 3-6 alleles per locus, and mean heterozygosities ranging 0.093-0.590 across loci. The suitability of these primers was investigated in four Colletotrichum species as well. These microsatellite markers will be useful for genetic analyses and epidemiological studies of C. acutatum.

Volatile organic compounds produced by Saccharomyces cerevisiae inhibit the in vitro development of Guignardia citricarpa, the causal agent of citrus black spot

Due to the low chemical control effectiveness of citrus black spot, caused by the fungus Guignardia citricarpa at postharvest, and to the search for alternative control methods, this study aimed to evaluate the in vitro effect of volatile organic compounds (VOCs), produced by yeast Saccharomyces cerevisiae, on G. citricarpa. It was observed that the yeast strains evaluated acted as antagonists by VOC production, whose maximum inhibitory capacity was as high as 87.2%. The presence of fermentable carbon sources in the medium was essential for the bioactive VOC production by the yeast. The analysis of VOCs produced in PDA medium by SPME-GC-MS indicated the presence of high quantities of alcohols as well as esters. An artificial VOC mixture prepared on the basis of the composition of the VOCs mimicked the inhibitory effects of the natural VOCs released by S. cerevisiae. Thus, the VOCs produced by the yeast or the artificial mixtures can be a promising control method for citrus black spot or others postharvest diseases.

Colletotrichum gloeosporioides, a new causal agent of citrus post-bloom fruit drop

Citrus post-bloom fruit drop (caused by Colletotrichum acutatum) frequently occurs in the southwestern region of So Paulo State, Brazil. A survey of Colletotrichum isolates associated with symptoms of post-bloom fruit drop in So Paulo State showed C. gloeosporioides in addition to C. acutatum. The objectives of this study were to confirm the identification of C. gloeosporioides isolated from symptomatic citrus flowers, to test the pathogenicity of C. gloeosporioides isolates, to compare the development of disease caused by C. gloeosporioides and C. acutatum, and to determine the frequency of C. gloeosporioides in a sample of isolates obtained from symptomatic flowers in different regions of So Paulo State. Through the use of species-specific primers by PCR, 17.3% of 139 isolates were C. gloeosporioides, and the remaining 82.7% were C. acutatum. The pathogenicity tests, carried out in 3-year old potted plants of sweet oranges indicated that both species caused typical symptoms of the disease including blossom blight and persistent calyces. Incubation periods (3.5 and 3.9 days, respectively, for C. acutatum and C. gloeosporioides) and fruit sets (6.7 and 8.5%, respectively for C. acutatum and C. gloeosporioides) were similar for both species. The incidences of blossom blight and persistent calyces were higher on plants inoculated with C. acutatum than in those inoculated with C. gloeosporioides. Conidial germination was similar for both species under different temperatures and wetness periods. Under optimal conditions, appressorium formation and melanisation were higher for C. gloeosporioides than for C. acutatum. These results indicated that Colletotrichum gloeosporioides is a new causal agent of post-bloom fruit drop.

A split-pot experiment with sorghum to test a root water uptake partitioning model

Correct modeling of root water uptake partitioning over depth is an important issue in hydrological and crop growth models. Recently a physically based model to describe root water uptake was developed at single root scale and upscaled to the root system scale considering a homogeneous distribution of roots per soil layer. Root water uptake partitioning is calculated over soil layers or compartments as a function of respective soil hydraulic conditions, specifically the soil matric flux potential, root characteristics and a root system efficiency factor to compensate for within-layer root system heterogeneities. The performance of this model was tested in an experiment performed in two-compartment split-pot lysimeters with sorghum plants. The compartments were submitted to different irrigation cycles resulting in contrasting water contents over time. The root system efficiency factor was determined to be about 0.05. Release of water from roots to soil was predicted and observed on several occasions during the experiment; however, model predictions suggested root water release to occur more often and at a higher rate than observed. This may be due to not considering internal root system resistances, thus overestimating the ease with which roots can act as conductors of water. Excluding these erroneous predictions from the dataset, statistical indices show model performance to be of good quality.

Mycovirus in Pseudocercospora griseola, the causal agent of angular leaf spot in common bean

Pseudocercospora griseola (Sacc.) Crous &. Braun is a widespread fungal phytopathogen that is responsible for angular leaf spot in the common bean (Phaseolus vulgaris L.). A number of fungal phytopathogens have been shown to harbour mycoviruses, and this possibility was investigated in populations of Pseudocercospora griseola. The total nucleic acid extracts of 61 fungal isolates were subjected to agarose gel electrophoresis. Small fragments (800-4800 bp) could be identified in 42 of the samples. The presence of dsRNA in isolate Ig838 was confirmed by treatment of total nucleic acid with DNase, RNase A, and nuclease S I. Transmission electron microscopy revealed the presence of viral-like particles 40 nm in diameter in the mycelia of 2 fungal isolates, namely 29-3 and Ig838. The transmission of dsRNA by means of conidia was 100% for isolate 29-3, but there was loss of 1-6 fragments of dsRNA in monosporic colonies of isolate Ig848. Cycloheximide treatment failed to inhibit the mycovirus in isolate 29-3, but proved efficient in the elimination of the 2.2, 2.0, 1.8, 1.2 and 1.0 kb fragments in 2 colonies of isolate Ig848. The occurrence of a mycovirus in Pseudocercospora griseola was demonstrated for the first time in the present study.

Separation and partitioning of Green Fluorescent Protein from Escherichia coli homogenate in poly(ethylene glycol)/sodium-poly(acrylate) aqueous two-phase systems

The partitioning of Green Fluorescent Protein (GFP) in poly(ethylene glycol)/Na-poly(acrylate) aqueous two-phase systems (PEG/NaPA-ATPS) has been investigated. The aqueous two-phase systems are formed by mixing the polymers with a salt and a protein solution. The protein partitioning in the two-phase system was investigated at 25 degrees C. The concentration of the GFP was measured by fluorimetry. It was found that the partitioning of GFP depends on the salt type, pH and concentration of PEG. The data indicates that GFP partitions more strongly to the PEG phase in presence of Na2SO4 relative to NaCl. Furthermore, the GFP partitions more to the PEG phase at higher pH. The partition to the PEG phase is strongly favoured in systems with larger tie-line lengths (i.e. systems with higher polymer concentrations). The molecular weight of PEG is important since the partition coefficient (K) of GFP gradually decreases with increasing PEG size, from K ca. 300-400 for PEG 400 to K equal to 1.19 for PEG 8000. A separation process was developed where GFP was separated from a homogenate in two extraction steps: the GFP is first partitioned to the PEG phase in a PEG 3000/NaPA 8000 system containing 3 wt% Na2SO4, where the K value of GFP was 8. The GFP is then re-extracted to a salt phase formed by mixing the previous top-phase with a Na2SO4 solution. The K-value of GFP in this back-extraction was 0.22. The total recovery based on the start material was 74%. (c) 2008 Elsevier B.V. All rights reserved.

Protein partitioning in poly(ethylene glycol)/sodium polyacrylate aqueous two-phase systems

The partition of hemoglobin, lysozyme and glucose-6-phospate dehydrogenase (G6PDH) in a novel inexpensive aqueous two-phase system (ATPS) composed by poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA) has been studied. The effect of NaCl and Na2SO4, pH and PEG molecular size on the partitioning has been studied. At high pH (above 9), hemoglobin partitions strongly to the PEG-phase. Although some precipitation of hemoglobin occurs, high recovery values are obtained particularly for lysozyme and G6PDH. The partitioning forces are dominated by the hydrophobic and electrochemical (salt) effects, since the positively charged lysozyme and negatively charged G6PDH partitions to the non-charged PEG and the strongly negatively charged polyacrylate enriched phase, respectively. (c) 2007 Elsevier B.V. All rights reserved.

Liquid-liquid extraction of commercial and biosynthesized nisin by aqueous two-phase micellar systems

Nisin is a natural additive for conservation of food, and can also be used as a therapeutic agent. Nisin inhibits the outgrowth of spores, the growth of a variety of Gram-positive and Grain-negative bacteria. In this paper we present a potentially scalable and cost-effective way to purify commercial and biosynthesized in bioreactor nisin, including simultaneously removal of impurities and contaminants, increasing nisin activity. Aqueous two-phase micellar systems (ATPMS) are considered promising for bioseparation and purification purposes. Triton X-114 was chosen as the as phase-forming surfactant because it is relatively mild to proteins and it also forms two coexisting phases within a convenient temperature range. Nisin activity was determined by the agar diffusion assay utilizing Lactobacillus sake as a sensitive indicator microorganism. Results indicated that nisin partitions preferentially to the micelle rich-phase, despite the surfactant concentration tested, and its antimicrobial activity increases. The successful implementation of this peptide partitioning, from a suspension containing other compounds, represents an important step towards developing a separation method for nisin, and more generally, for other biomolecules of interest. (C) 2007 Elsevier Inc. All rights reserved.

The anti-cancer agent guttiferone-A permeabilizes mitochondrial membrane: Ensuing energetic and oxidative stress implications

Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with cytotoxic action in vitro and anti-tumor action in rodent models. We addressed a potential involvement of mitochondria in GA toxicity (1-25 mu M) toward cancer cells by employing both hepatic carcinoma (HepG2) cells and succinate-energized mitochondria, isolated from rat liver. In HepG2 cells GA decreased viability, dissipated mitochondrial membrane potential, depleted ATP and increased reactive oxygen species (ROS) levels. In isolated rat-liver mitochondria GA promoted membrane fluidity increase, cyclosporine A/EGTA-insensitive membrane permeabilization, uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca(2+) efflux, ATP depletion, NAD(P)H depletion/oxidation and ROS levels increase. All effects in cells, except mitochondrial membrane potential dissipation, as well as NADPH depletion/oxidation and permeabilization in isolated mitochondria, were partly prevented by the a NAD(P)H regenerating substrate isocitrate. The results suggest the following sequence of events: 1) GA interaction with mitochondrial membrane promoting its permeabilization; 2) mitochondrial membrane potential dissipation; 3) NAD(P)H oxidation/depletion due to inability of membrane potential-sensitive NADP(+) transhydrogenase of sustaining its reduced state; 4) ROS accumulation inside mitochondria and cells; 5) additional mitochondrial membrane permeabilization due to ROS; and 6) ATP depletion. These GA actions are potentially implicated in the well-documented anti-cancer property of GA/structure related compounds. (C) 2011 Elsevier Inc. All rights reserved.

The anti-cancer agent nemorosone is a new potent protonophoric mitochondrial uncoupler

Nemorosone, a natural-occurring polycyclic polyprenylated acylphloroglucinol, has received increasing attention due to its strong in vitro anti-cancer action. Here, we have demonstrated the toxic effect of nemorosone (1-25 mu M) on HepG2 cells by means of the MTT assay, as well as early mitochondrial membrane potential dissipation and ATP depletion in this cancer cell line. In mitochondria isolated from rat liver, nemorosone (50-500 nM) displayed a protonophoric uncoupling activity, showing potency comparable to the classic protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Nemorosone enhanced the succinate-supported state 4 respiration rate, dissipated mitochondrial membrane potential, released Ca(2+) from Ca(2+)-loaded mitochondria, decreased Ca(2+) uptake and depleted ATP. The protonophoric property of nemorosone was attested by the induction of mitochondrial swelling in hyposmotic K(+)-acetate medium in the presence of valinomycin. In addition, uncoupling concentrations of nemorosone in the presence of Ca(2+) plus ruthenium red induced the mitochondrial permeability transition process. Therefore, nemorosone is a new potent protonophoric mitochondrial uncoupler and this property is potentially involved in its toxicity on cancer cells. (C) 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

HPLC separation, NMR and QTOF/MS/MS structure elucidation of a prominent nitric oxide donor agent based on an isomeric composition of a nitrosyl ruthenium complex

A new and promising nitrosyl ruthenium complex, [Ru(NO)(bdqi-COOH)(terpy)](PF(6))(3), bdqi-COOH is 3,4-diiminebenzoic acid and terpy is 2,2`-terpyridine, has been synthesized as a NO donor agent. The procedure used for [Ru(NO)(bdqi-COOH)(terpy)](PF(6))(3) synthesis has, apparently, yielded the formation of two isomers in which the ligand bdqi-COOH appears to be coordinated in its reduced form (bdcat-COOH), which could have differences in their pharmacological properties. Therefore, it was intended to separate the two possible isomers by high-performance liquid chromatography (HPLC) and to characterize them by high resolution mass spectrometry (QTOF MS) and by magnetic nuclear resonance spectroscopy (NMR). The results obtained by MS showed that the ESI-MS mass spectra of both HPLC column fractions, e.g. peak 1 and peak 2, are essentially equal, showing that both isomers display nearly identical gas-phase behavior with clusters of isotopologue ions centered at m/z 573, m/z 543 and m/z 513. Regarding the NMR analysis, the results showed that the positional isomerism is located in the bdqi-COOH ligand. From the observed results it can be concluded that the synthesis procedure that has been used results in the formation of two [Ru(terpy)(bdqi-COOH)NO](PF(6))(3) isomers. (c) 2009 Elsevier B.V. All rights reserved.

Chitosan as a Bioadhesive Agent Between Porphyrins and Phospholipids in a Biomembrane Model

Porphyrins are currently used in photodynamic therapy as photosensitizers. In this paper we studied the interaction of two charged porphyrins, 5, 10, 15, 20-mesotetrakis(N-metyl-4-pyridyl) porphyrin, (TMPyP/chloride salt) cationic, and 5, 10, 15, 20-meso-tetrakis(sulfonatophenyl) porphyrin, (TPPS(4)/sodium salt) anionic, nanoassembled in phospholipid Langmuir monolayers and Langmuir-Blodgett films. Furthermore, we used chitosan to mediate the interaction between the porphyrins and the model membrane, aiming to understand the role of the polysaccharide in a molecular level. The effect of the interaction of the photosensitizers on the fluidity of the lipid monolayer was investigated by using dilatational surface elasticity. We also used photoluminescence (PL) spectroscopy to identify the porphyrins adsorbed in the phospholipid films. We observed an expansion of the monolayer promoted by the adsorption of the porphyrins into the lipid-air interface which was more pronounced in the case of TMPyP, as a consequence of a strong electrostatic interaction with the anionic monolayer. The chitosan promoted a higher adsorption of the porphyrins on the phospholipid monolayers and enabled the porphyrin to stay in its monomeric form (as confirmed by PL spectroscopy), thus demonstrating that chitosan can be pointed out as a potential photosensitizer delivery system in photodynamic therapy.