Maize and soybeans production in integrated system under no-tillage with different pasture combinations and animal categories

The adoption of no-till system (NTS) combined with crop-livestock integration (CLI) has been a strategy promoted in Brazil, aiming to maximize areas yield and increase agribusiness profitability. This study aimed to evaluate grains yield and phytotechnical attributes from maize and soybean culture by CLI system under NTS after winter annual pure and diversified pastures with the absence or presence of grazing animals. The experiment was installed in Castro (Parana State, Brazil) on in a dystrophic Humic Rhodic Hapludox with a clay texture, using experimental design of randomized complete blocks in 4 x 2 factorial scheme with three replications. Treatments included four pasture combinations (diversified or pure) and animal categories (light and heavy) subjected or not to grazing animals during the winter. During 2008/09 and 2009/10 summers, the area was cultivated with soybeans and maize, respectively, with yield assessment of grains and phytotechnical attributes. Treatments did not alter the yield and weight of a thousand seeds (WTS) of soybeans. In maize culture, the grazing animal during the winter increased the plant population and grains yield, but gave slight decrease in WTS. Pasture combinations (diversified or pure) and animal categories (light and heavy) did not interfere in soybean culture, but benefited the maize crop.

Parasites in the neighbourhood: Interactions of the mistletoe Phoradendron affine (Viscaceae) with its dispersers and hosts in urban areas of Brazil

Mistletoes constitute an important food resource for animals in many ecosystems. However, these plants are considered pests in urban areas because of deleterious effects they have on the host trees. Studies in urban areas were mostly focused on listing host species or procedures to control the "pest". In this sense, broader studies including several aspects of mistletoes ecology in urban ecosystems are still missing. We studied the interaction of the mistletoe, Phoradendron affine, with its dispersers and hosts in two urban sites in Uberlandia, Brazil. Phoradendron affine fruits were consumed almost exclusively by Euphonia chlorotica, which was crucial for seed germination. Parasitism was recorded in five hosts, two native (Handroanthus chrysotrichus and Tabebuia roseoalba) and three exotic species (Spathodea campanulata, Ligustrum lucidum and Melia azedarach). Mistletoes were found parasitizing larger host trees, a trend commonly reported for mistletoe-host interaction. Mistletoe seed germination was not affected by the trees species, whether host or non-host, but the radicle of germinated seeds could not penetrate the bark and seedlings invariably died in non-host species. We found a high prevalence of parasitism in our study, in comparison to what previous studies reported for natural areas. The spatial distribution of the hosts and high light incidence on isolated host trees may lead to this high prevalence in urban areas. Rather than eradicated, mistletoes in urban areas should be ecologically managed and their importance for bird species conservation must be considered. More studies to determine which bird species are favoured by mistletoe presence in urban areas will be essential for, this purpose. (C) 2012 Elsevier GmbH. All rights reserved.

Within-plant isoprene oxidation confirmed by direct emissions of oxidation products methyl vinyl ketone and methacrolein

Isoprene is emitted from many terrestrial plants at high rates, accounting for an estimated 1/3 of annual global volatile organic compound emissions from all anthropogenic and biogenic sources combined. Through rapid photooxidation reactions in the atmosphere, isoprene is converted to a variety of oxidized hydrocarbons, providing higher order reactants for the production of organic nitrates and tropospheric ozone, reducing the availability of oxidants for the breakdown of radiatively active trace gases such as methane, and potentially producing hygroscopic particles that act as effective cloud condensation nuclei. However, the functional basis for plant production of isoprene remains elusive. It has been hypothesized that in the cell isoprene mitigates oxidative damage during the stress-induced accumulation of reactive oxygen species (ROS), but the products of isoprene-ROS reactions in plants have not been detected. Using pyruvate-2-13C leaf and branch feeding and individual branch and whole mesocosm flux studies, we present evidence that isoprene (i) is oxidized to methyl vinyl ketone and methacrolein (iox) in leaves and that iox/i emission ratios increase with temperature, possibly due to an increase in ROS production under high temperature and light stress. In a primary rainforest in Amazonia, we inferred significant in plant isoprene oxidation (despite the strong masking effect of simultaneous atmospheric oxidation), from its influence on the vertical distribution of iox uptake fluxes, which were shifted to low isoprene emitting regions of the canopy. These observations suggest that carbon investment in isoprene production is larger than that inferred from emissions alone and that models of tropospheric chemistry and biotachemistryclimate interactions should incorporate isoprene oxidation within both the biosphere and the atmosphere with potential implications for better understanding both the oxidizing power of the troposphere and forest response to climate change.

Mutualism from the inside: coordinated development of plant and insect in an active pollinating fig wasp

Recent studies on the obligate interaction between fig trees and their pollinating agaonid wasps have focused on population aspects and wasp-seed exploitation at the level of the inflorescence. Detailed studies on larval and gall development are required to more fully understand how resources are exploited and adaptations fine-tuned by each partner in nursery pollination mutualisms. We studied the larval development of the active pollinating fig wasp, Pegoscapus sp., and the galling process of individual flowers within the figs of its monoecious host, Ficus citrifolia, in Brazil. The pollinator development is strongly dependent on flower pollination. Figs entered by pollen-free wasps were in general more likely to abort. Retained, unpollinated figs had both higher larval mortality and a lower number of wasps. Pegoscapus sp. larvae are adapted to plant development, with two contrasting larval feeding strategies proceeding alongside gall development. The first two larval stages behave as ovary parasites. Later larval stages feed on hypertrophied endosperm. This indicates that a successful galling process relies on endosperm, and also reveals why pollination would be a prerequisite for the production of high-quality galls for this Pegoscapus species.

CLEANING ASSOCIATIONS BETWEEN BIRDS AND HERBIVOROUS MAMMALS IN BRAZIL: STRUCTURE AND COMPLEXITY

Birds that remove ectoparasites and other food material from their hosts are iconic illustrations of mutualistic-commensalistic cleaning associations. To assess the complex pattern of food resource use embedded in cleaning interactions of an assemblage of birds and their herbivorous mammal hosts in open habitats in Brazil, we used a network approach that characterized their patterns of association. Cleaning interactions showed a distinctly nested pattern, related to the number of interactions of cleaners and hosts and to the range of food types that each host species provided. Hosts that provided a wide range of food types (flies, ticks, tissue and blood, and organic debris) were attended by more species of cleaners and formed the core of the web. On the other hand, core cleaner species did not exploit the full range of available food resources, but used a variety of host species to exploit these resources instead. The structure that we found indicates that cleaners rely on cleaning interactions to obtain food types that would not be available otherwise (e.g., blood-engorged ticks or horseflies, wounded tissue). Additionally, a nested organization for the cleaner bird mammalian herbivore association means that both generalist and selective species take part in the interactions and that partners of selective species form an ordered subset of the partners of generalist species. The availability of predictable protein-rich food sources for birds provided by cleaning interactions may lead to an evolutionary pathway favoring their increased use by birds that forage opportunistically. Received 30 June 2011, accepted 10 November 2011.

Endophytic and rhizospheric enterobacteria isolated from sugar cane have different potentials for producing plant growth-promoting substances

Background and aims Endophytic and rhizospheric environments differ in many respects, leading to the presence of different bacterial communities at each site. However, microorganisms such as enterobacteria can be found both within plants and in the surrounding soil. Bacteria must present differences in the traits that affect such environments in order to successfully colonise them. The present study compared the plant growth-promoting potential of diazotrophic enterobacteria isolated from the rhizosphere and from within surface-disinfected plants. Methods A total of 46 diazotrophic enterobacterial strains (21 rhizospheric and 25 putatively endophytic) belonging to the Klebsiella and Enterobacter genera, which are prevalent in sugar cane plantations, were isolated from the rhizosphere and from surface-disinfected plants. Their ability to synthesise amino acids using combined nitrogen obtained from nitrogen fixation, and their ability to synthesise indole-3-acetic acid (IAA) were determined by high performance liquid chromatography. Endogenous ethylene production by the bacteria was measured using gas chromatography, and biocontrol of phytopathogenic fungi was determined qualitatively using a dual culture technique. Results The putative endophytes released significantly higher amounts of amino acids than the rhizospheric bacteria, whilst the latter produced higher quantities of ethylene and were more actively antagonistic to fungi. Both types of bacteria released similar amounts of IAA. Conclusion Endophytic and rhizospheric bacteria differ in their capacity to release plant growth-promoting substances, which may be a reflection of their adaptations and an indication of their potential impact on their natural environment.

The importance of plant life form on spatial associations along a subtropical coastal dune gradient

Questions Does the spatial association between isolated adult trees and understorey plants change along a gradient of sand dunes? Does this association depend on the life form of the understorey plant? Location Coastal sand dunes, southeast Brazil. Methods We recorded the occurrence of understorey plant species in 100 paired 0.25 m2 plots under adult trees and in adjacent treeless sites along an environmental gradient from beach to inland. Occurrence probabilities were modelled as a function of the fixed variables of the presence of a neighbour, distance from the seashore and life form, and a random variable, the block (i.e. the pair of plots). Generalized linear mixed models (GLMM) were fitted in a backward step-wise procedure using Akaike's information criterion (AIC) for model selection. Results The occurrence of understorey plants was affected by the presence of an adult tree neighbour, but the effect varied with the life form of the understorey species. Positive spatial association was found between isolated adult neighbour and young trees, whereas a negative association was found for shrubs. Moreover, a neutral association was found for lianas, whereas for herbs the effect of the presence of an adult neighbour ranged from neutral to negative, depended on the subgroup considered. The strength of the negative association with forbs increased with distance from the seashore. However, for the other life forms, the associational pattern with adult trees did not change along the gradient. Conclusions For most of the understorey life forms there is no evidence that the spatial association between isolated adult trees and understorey plants changes with the distance from the seashore, as predicted by the stress gradient hypothesis, a common hypothesis in the literature about facilitation in plant communities. Furthermore, the positive spatial association between isolated adult trees and young trees identified along the entire gradient studied indicates a positive feedback that explains the transition from open vegetation to forest in subtropical coastal dune environments.

SmTRC1, a novel Schistosoma mansoni DNA transposon, discloses new families of animal and fungi transposons belonging to the CACTA superfamily

Abstract Background The CACTA (also called En/Spm) superfamily of DNA-only transposons contain the core sequence CACTA in their Terminal Inverted Repeats (TIRs) and so far have only been described in plants. Large transcriptome and genome sequence data have recently become publicly available for Schistosoma mansoni, a digenetic blood fluke that is a major causative agent of schistosomiasis in humans, and have provided a comprehensive repository for the discovery of novel genes and repetitive elements. Despite the extensive description of retroelements in S. mansoni, just a single DNA-only transposon belonging to the Merlin family has so far been reported in this organism. Results We describe a novel S. mansoni transposon named SmTRC1, for S. mansoni Transposon Related to CACTA 1, an element that shares several characteristics with plant CACTA transposons. Southern blotting indicates approximately 30–300 copies of SmTRC1 in the S. mansoni genome. Using genomic PCR followed by cloning and sequencing, we amplified and characterized a full-length and a truncated copy of this element. RT-PCR using S. mansoni mRNA followed by cloning and sequencing revealed several alternatively spliced transcripts of this transposon, resulting in distinct ORFs coding for different proteins. Interestingly, a survey of complete genomes from animals and fungi revealed several other novel TRC elements, indicating new families of DNA transposons belonging to the CACTA superfamily that have not previously been reported in these kingdoms. The first three bases in the S. mansoni TIR are CCC and they are identical to those in the TIRs of the insects Aedes aegypti and Tribolium castaneum, suggesting that animal TRCs may display a CCC core sequence. Conclusion The DNA-only transposable element SmTRC1 from S. mansoni exhibits various characteristics, such as generation of multiple alternatively-spliced transcripts, the presence of terminal inverted repeats at the extremities of the elements flanked by direct repeats and the presence of a Transposase_21 domain, that suggest a distant relationship to CACTA transposons from Magnoliophyta. Several sequences from other Metazoa and Fungi code for proteins similar to those encoded by SmTRC1, suggesting that such elements have a common ancestry, and indicating inheritance through vertical transmission before separation of the Eumetazoa, Fungi and Plants.

Interactions between calcium and heavy metals in Norway spruce : Accumulation and binding of metals in wood and bark

Waste products from the forest industry are to be spread in forests in Sweden to counteract nutrient depletion due to whole tree harvesting. This may increase the bioavailability of calcium (Ca) and heavy metals, such as cadmium (Cd), copper (Cu) and zinc (Zn) in forest soils. Heavy metals, like Cd, have already been enriched in forest soils in Sweden, due to deposition of air pollutions, and acidification of forest soils has increased the bioavailability of toxic metals for plant uptake. Changes in the bioavailability of metals may be reflected in altered accumulation of Ca and heavy metals in forest trees, changes in tree growth, including wood formation, and altered tree species composition. This thesis aims at examining: A) if inter- or intra- specific differences in sensitivity to Cd occur in the most common tree species of Sweden, and if so, to study if these can be explained by the uptake and distribution of Cd within the plant: B) how elevated levels of Ca, Cd, Cu and Zn affect the accumulation and attachment of metals in bark and wood, and growth of young Norway spruce (Picea abies): C) how waste products from the forest industry, such as wood ash, influence the contents of Ca, Cd, Cu and Zn in wood and bark of young Norway spruce. Sensitivity to Cd, and its uptake and distribution, in seedlings of Picea abies, Pinus sylvestris and Betula pendula from three regions (southern, central and northern parts) of Sweden, treated with varying concentrations of Cd, were compared. Differences in root sensitivity to Cd both among and within woody species were found and the differences could to some extent be explained by differences in uptake and translocation of Cd. The root sensitivity assays revealed that birch was the least, and spruce the most, sensitive species, both to the external and to tissue levels of Cd. The central ecotype of the species tested tended to be most Cd resistant. The radial distribution, accumulation and attachment of, and interactions between Ca and heavy metals in stems of two-year-old Norway spruce trees treated with elevated levels of Cd, Cu, Zn and/or Ca, were investigated. Further, the influence of these metals on growth, and on root metal content, was examined. Accumulation of the metals was enhanced in wood, bark and/or roots at elevated levels of the metal in question. Even at low levels of the metals, similar to after application of wood ash, an enhanced accumulation was apparent in wood and/or bark, except for Cd. The increased accumulation of Zn and Cu in the stem did not affect the growth. However, Cu decreased the accumulation of Ca in wood. Higher levels of Cu and Cd reduced the stem diameter and the toxic effect was associated with a reduced Ca content in wood. Copper and Cd also decreased the accumulation of Zn in the stem. On the other hand, elevated levels of Ca increased the stem diameter and reduced the accumulation of Cd, Cu, Zn and Mn in wood and/or bark. When metals interacted with each other the firmly bound fraction of the metal reduced was in almost all cases not affected. As an exception, Cd decreased the firmly bound fraction of Zn in the stem. The influence of pellets of wood ash (ash) or a mixture of wood ash and green liquor dregs (ash+GLD), in the amount of 3000 kg ha-1, on the contents of Ca, Cd, Cu and Zn in wood and bark of young Norway spruce in the field was examined. The effect of the treatments on the metal content of bark and wood was larger after 3 years than after 6 years. Treatment with ash+GLD had less effect on the heavy metal content of bark and wood than treatment with ash alone. The ash treatment increased the Cu and Zn content in bark and wood, respectively, after 3 years, and decreased the Ca content of the wood after 6 years. The ash+GLD treatment increased the Ca content of the bark and decreased the Zn content of bark and wood after 3 years. Both treatments reduced, or tended to decrease, the Cd content in wood and bark at both times. To conclude, small changes in the bioavailability of Ca, Cu, Cd and Zn in forest soils, such as after spreading pellets of wood ash or a mixture of wood ash and green liquor dregs from the forest industry, will be reflected in an altered accumulation of metals in wood and bark of Norway spruce. It will not only be reflected in changed accumulation of those metals in which bioavailability in the soil has been enhanced, but also of other metals, probably partly due to interactions between metals. When metals interact the exchangeable bound fraction of the metal reduced is suggested to be the main fraction affected. The small alterations in accumulation of metals should not affect the growth of Norway spruce, especially since the changes in accumulation of metals are low, and further since these decrease over time. However, as an exception, one positive and maybe persistent effect of the waste products is that these may decrease the accumulation of Cd in Norway spruce, which partly may be explained by competition with Ca for uptake, translocation and binding. A decreased accumulation of Cd in Norway spruce will probably affect the trees positively, since Norway spruce is one of the most sensitive species to Cd of the forest trees in Sweden. Thus, spreading of waste products from the forest industry may be a solution to decrease the accumulation of Cd in Norway spruce. In a longer perspective, this will decrease the risk of Cd altering the tree species composition of the forest ecosystem. An elevated bioavailability of Ca in forest soils will, in addition to Cd, probably also decrease the accumulation of other less competitive heavy metals, like Zn and Mn, in the stem.

Global trade, food production and ecosystem support : Making the interactions visible

Modern food production is a complex, globalized system in which what we eat and how it is produced are increasingly disconnected. This thesis examines some of the ways in which global trade has changed the mix of inputs to food and feed, and how this affects food security and our perceptions of sustainability. One useful indicator of the ecological impact of trade in food and feed products is the Appropriated Ecosystem Areas (ArEAs), which estimates the terrestrial and aquatic areas needed to produce all the inputs to particular products. The method is introduced in Paper I and used to calculate and track changes in imported subsidies to Swedish agriculture over the period 1962-1994. In 1994, Swedish consumers needed agricultural areas outside their national borders to satisfy more than a third of their food consumption needs. The method is then applied to Swedish meat production in Paper II to show that the term “Made in Sweden” is often a misnomer. In 1999, almost 80% of manufactured feed for Swedish pigs, cattle and chickens was dependent on imported inputs, mainly from Europe, Southeast Asia and South America. Paper III examines ecosystem subsidies to intensive aquaculture in two nations: shrimp production in Thailand and salmon production in Norway. In both countries, aquaculture was shown to rely increasingly on imported subsidies. The rapid expansion of aquaculture turned these countries from fishmeal net exporters to fishmeal net importers, increasingly using inputs from the Southeastern Pacific Ocean. As the examined agricultural and aquacultural production systems became globalized, levels of dependence on other nations’ ecosystems, the number of external supply sources, and the distance to these sources steadily increased. Dependence on other nations is not problematic, as long as we are able to acknowledge these links and sustainably manage resources both at home and abroad. However, ecosystem subsidies are seldom recognized or made explicit in national policy or economic accounts. Economic systems are generally not designed to receive feedbacks when the status of remote ecosystems changes, much less to respond in an ecologically sensitive manner. Papers IV and V discuss the problem of “masking” of the true environmental costs of production for trade. One of our conclusions is that, while the ArEAs approach is a useful tool for illuminating environmentally-based subsidies in the policy arena, it does not reflect all of the costs. Current agricultural and aquacultural production methods have generated substantial increases in production levels, but if policy continues to support the focus on yield and production increases alone, taking the work of ecosystems for granted, vulnerability can result. Thus, a challenge is to develop a set of complementary tools that can be used in economic accounting at national and international scales that address ecosystem support and performance. We conclude that future resilience in food production systems will require more explicit links between consumers and the work of supporting ecosystems, locally and in other regions of the world, and that food security planning will require active management of the capacity of all involved ecosystems to sustain food production.

Effetti del chitosano su composti polifenolici in colture cellulari di vite: aspetti molecolari e metabolici

Polyphenols, including flavonoids and stilbenes, are an essential part of human diet and constitute one of the most abundant and ubiquitous group of plant secondary metabolites. The level of these compounds is inducible by stress or fungal attack, so attempts are being made to identify likely biotic and abiotic elicitors and to better understand the underlying mechanism. Resveratrol (3,5,4’-trihydroxystilbene), which belongs to the stilbene family, is a naturally occurring polyphenol, found in several fruits, vegetables and beverages including red wine. It is one of the most important plant polyphenols with proved benefic activity on animal health. In the last two decades, the potential protective effects of resveratrol against cardiovascular and neurodegenerative diseases, as well as the chemopreventive properties against cancer, have been largely investigated. The most important source of polyphenols and in particular resveratrol for human diet is grape (Vitis vinifera). Since stilbenes and flavonoids play a very important role in plant defence responses and enviromental interactions, and their effects on human health seem promising, the aim of the research of this Thesis was to study at different levels the activation and the regulation of their biosynthetic pathways after chitosan treatment. Moreover, the polyphenol production in grape cells and the optimisation of cultural conditions bioreactor scale-up, were also investigated. Cell suspensions were obtained from cv. Barbera (Vitis vinifera L.) petioles and were treated with a biotic elicitor, chitosan (50 μg/mL, dissolved in acetic acid) to promote phenylpropanoid metabolism. Chitosan is a D-glucosamine polymer from fungi cell wall and therefore mimes fungal pathogen attack. Liquid cultures have been monitored for 15 days, measuring cell number, cell viability, pH and grams of fresh weight. The endogenous and released amounts of 7 stilbenes (trans and cis isomers of resveratrol, piceid and resveratroloside, and piceatannol), gallic acid, 6 hydroxycinnamic acids (trans-cinnamic, p-coumaric, caffeic, ferulic, sinapic and chlorogenic acids), 5 catechines (catechin, epicatechin, epigallocatechin-gallate (EGCG), epigallocatechin and epicatechin-gallate) and other 5 flavonoids (chalcon, naringenin, kaempferol, quercetin and rutin) in cells and cultural medium, were measured by HPLC-DAD analysis and total anthocyanins were quantified by spectrophotometric analysis. Chitosan was effective in stimulating trans-resveratrol endogenous accumulation with a sharp peak at day 4 (exceeding acetic acid and water controls by 36% and 63%, respectively), while it did not influence the production of the cis-isomer. Compared to both water and acetic acid controls, chitosan decreased the release of both trans- and cis-resveratrol respect to controls. No effect was shown on the accumulation of single resveratrol mono-glucoside isomers, but considering their total amount, normalized for the relative water control, it was possible to evidence an increase in both accumulation and release of those compounds, in chitosan-treated cells, throughout the culture period and particularly during the second week. Many of the analysed flavonoids and hydroxycinnamic acids were not present or detectable in trace amounts. Catechin, epicatechin and epigallocatechin-gallate (EGCG) were detectable both inside the cells and in the culture media, but chitosan did not affect their amounts. On the contrary, total anthocyanins have been stimulated by chitosan and their level, from day 4 to 14, was about 2-fold higher than in both controls, confirming macroscopic observations that treated suspensions showed an intense brown-red color, from day 3 onwards. These elicitation results suggest that chitosan selectively up-regulates specific biosynthetic pathways, without modifying the general accumulation pattern of other flavonoids. Proteins have been extracted from cells at day 4 of culture (corresponding to the production peak of trans-resveratrol) and separated by bidimensional electrophoresis. The 73 proteins that showed a consistently changed amount between untreated, chitosan and acetic acid (chitosan solvent) treated cells, have been identified by mass spectrometry. Chitosan induced an increase in stilbene synthase (STS, the resveratrol biosynthetic enzyme), chalcone-flavanone isomerase (CHI, that switches the pathway from chalcones to flavones and anthocyanins), pathogenesis-related proteins 10 (PRs10, a large family of defence proteins), and a decrease in many proteins belonging to primary metabolisms. A train of six distinct spots of STS encoded by the same gene and increased by chitosan, was detected on the 2-D gels, and related to the different phosphorylation degree of STS spots. Northern blot analyses have been performed on RNA extracted from cells treated with chitosan and relative controls, using probes for STS, PAL (phenylalanine ammonia lyase, the first enzyme of the biosynthetic pathway), CHS (chalcone synthase, that shares with STS the same precursors), CHI and PR-10. The up-regulation of PAL, CHS and CHI transcript expression levels correlated with the accumulation of anthocyanins. The strong increase of different molecular weight PR-10 mRNAs, correlated with the 11 PR-10 protein spots identified in proteomic analyses. The sudden decrease in trans-resveratrol endogenous accumulation after day 4 of culture, could be simply explained by the diminished resveratrol biosynthetic activity due to the lower amount of biosynthetic enzymes. This might be indirectly demonstrated by northern blot expression analyses, that showed lower levels of phenylalanine ammonia lyase (PAL) and stilbene synthase (STS) mRNAs starting from day 4. Other possible explanations could be a resveratrol oxidation process and/or the formation of other different mono-, di-glucosides and resveratrol oligomers such as viniferins. Immunolocalisation experiments performed on grape protoplasts and the subsequent analyses by confocal microscope, showed that STS, and therefore the resveratrol synthetic site, is mostly associated to intracellular membranes close to the cytosolic side of plasma membrane and in a smaller amount is localized in the cytosol. STS seemed not to be present inside vacuole and nucleus. There were no differences in the STS intracellular localisation between the different treatments. Since it was shown that stilbenes are largely released in the culture medium and that STS is a soluble protein, a possible interaction of STS with a plasma membrane transporter responsible for the extrusion of stilbenes in the culture medium, might be hypothesized. Proteomic analyses performed on subcellular fractions identified in the microsomial fraction 5 proteins taking part in channel complexes or associated with channels, that significantly changed their amount after chitosan treatment. In soluble and membrane fractions respectively 3 and 4 STS and 6 and 3 PR-10 have been identified. Proteomic results obtained from subcellular fractions substantially confirmed previous result obtained from total cell protein extracts and added more information about protein localisation and co-localisation. The interesting results obtained on Barbera cell cultures with the aim to increase polyphenol (especially stilbenes) production, have encouraged scale up tests in 1 litre bioreactors. The first trial fermentation was performed in parallel with a normal time-course in 20 mL flasks, showing that the scale-up (bigger volume and different conditions) process influenced in a very relevant way stilbenes production. In order to optimise culture parameters such as medium sucrose amount, fermentation length and inoculum cell concentration, few other fermentations were performed. Chitosan treatments were also performed. The modification of each parameter brought relevant variations in stilbenes and catechins levels, so that the production of a certain compound (or class of compounds) could be hypothetically promoted by modulating one or more culture parameters. For example the catechin yield could be improved by increasing sucrose content and the time of fermentation. The best results in stilbene yield were obtained in a 800 mL fermentation inoculated with 10.8 grams of cells and supplemented with chitosan. The culture was fed with MS medium added with 30 g/L sucrose, 25 μg/mL rifampicin and 50 μg/mL of chitosan, and was maintained at 24°C, stirred by marine impeller at 100 rpm and supplied of air at 0.16 L/min rate. Resveratroloside was the stilbene present in the larger amount, 3-5 times more than resveratrol. Because resveratrol glucosides are similarly active and more stable than free resveratrol, their production using a bioreactor could be a great advantage in an hypothetical industrial process. In my bioreactor tests, stilbenes were mainly released in the culture medium (60-80% of the total) and this fact could be another advantage for industrial applications, because it allows recovering the products directly from the culture medium without stopping the fermentation and/or killing the cells. In my best cultural conditions, it was possible to obtain 3.95 mg/L of stilbenes at day 4 (maximum resveratrol accumulation) and 5.13 mg/L at day 14 (maximum resveratroloside production). In conclusion, chitosan effect in inducing Vitis vinifera defense mechanisms can be related to its ability to increase the intracellular content of a large spectrum of antioxidants, and in particular of resveratrol, its derivates and anthocyanins. Its effect can be observed at transcriptional, proteomic (variation of soluble and membrane protein amounts) and metabolic (polyphenols production) level. The chitosan ability to elicit specific plant matabolisms can be useful to produce large quantities of antioxidant compounds from cell culture in bioreactor.

Changes in plant metabolism induced by dioxygenase inhibitors and their effect on the epiphytic microbial community and fire blight (Erwinia amylovora) control

Fire blight, caused by the gram negative bacterium Erwinia amylovora, is one of the most destructive bacterial diseases of Pomaceous plants. Therefore, the development of reliable methods to control this disease is desperately needed. This research investigated the possibility to interfere, by altering plant metabolism, on the interactions occurring between Erwinia amylovora, the host plant and the epiphytic microbial community in order to obtain a more effective control of fire blight. Prohexadione-calcium and trinexapac-ethyl, two dioxygenase inhibitors, were chosen as a chemical tool to influence plant metabolism. These compounds inhibit the 2-oxoglutarate-dependent dioxygenases and, therefore, they greatly influence plant metabolism. Moreover, dioxygenase inhibitors were found to enhance plant resistance to a wide range of pathogens. In particular, dioxygenase inhibitors application seems a promising method to control fire blight. From cited literature, it is assumed that these compounds increase plant defence mainly by a transient alteration of flavonoids metabolism. We tried to demonstrate, that the reduction of susceptibility to disease could be partially due to an indirect influence on the microbial community established on plant surface. The possibility to influence the interactions occurring in the epiphytic microbial community is particularly interesting, in fact, the relationships among different bacterial populations on plant surface is a key factor for a more effective biological control of plant diseases. Furthermore, we evaluated the possibility to combine the application of dioxygenase inhibitors with biological control in order to develop an integrate strategy for control of fire blight. The first step for this study was the isolation of a pathogenic strain of E. amylovora. In addition, we isolated different epiphytic bacteria, which respond to general requirements for biological control agents. Successively, the effect of dioxygenase inhibitors treatment on microbial community was investigated on different plant organs (stigmas, nectaries and leaves). An increase in epiphytic microbial population was found. Further experiments were performed with aim to explain this effect. In particular, changes in sugar content of nectar were observed. These changes, decreasing the osmotic potential of nectar, might allow a more consistent growth of epiphytic bacteria on blossoms. On leaves were found similar differences as well. As far as the interactions between E. amylovora and host plant, they were deeply investigated by advanced microscopical analysis. The influence of dioxygenase inhibitors and SAR inducers application on the infection process and migration of pathogen inside different plant tissues was studied. These microscopical techniques, combined with the use of gpf-labelled E. amylovora, allowed the development of a bioassay method for resistance inducers efficacy screening. The final part of the work demonstrated that the reduction of disease susceptibility observed in plants treated with prohexadione-calcium is mainly due to the accumulation of a novel phytoalexins: luteoforol. This 3-deoxyflavonoid was proven to have a strong antimicrobial activity.

Characterization of primary biogenic aerosol particles by DNA analysis: Diversity of airborne Ascomycota and Basidiomycota

Primary biogenic aerosol (PBA) particles account for large proportions of air particulate matter, and they can influence the hydrological cycle and climate as nuclei for water droplets and ice crystals in clouds, fog, and precipitation. Moreover, they can cause or enhance human, animal, and plant diseases. The actual abundance and properties of PBA particles and components in the atmosphere are, however, still poorly understood and quantified. rnIn this study, the identity, diversity, and frequency of occurrence of PBA particles were investigated by DNA analysis. Methods for the extraction, amplification, and analysis of DNA from aerosol filter samples were developed and optimized for different types of organisms, including fungi, bacteria, and plants. The investigations were focused on fungal DNA, and over 2500 sequences were obtained from air samples collected at different locations and climatic zones around the world (tropical, mid-latitude, sub-polar; continental, marine). rnNearly all fungal DNA sequences could be attributed to the phyla of Ascomycota and Basidiomycota. With regard to species richness, the ratio of Basidiomycota to Ascomycota was much higher in continental air samples (~60:40) than in marine air samples (~30:70). Pronounced differences in the relative abundance and seasonal cycles of various groups of fungi were detected in coarse and fine particulate matter from continental air, with more plant pathogens in the coarse and more human pathogens and allergens in the respirable fine particle fraction (<3 µm). The results of this study provide new information and insights into the sources of PBA particles and the interactions of the biosphere with the atmosphere, climate, and public health. rn

Use of Biotechnology to increase the content of bioactive compounds in fermented foods of plant origin

The objectives of this PhD research were: i) to evaluate the use of bread making process to increase the content of β-glucans, resistant starch, fructans, dietary fibers and phenolic compounds of kamut khorasan and wheat breads made with flours obtained from kernels at different maturation stage (at milky stage and fully ripe) and ii) to study the impact of whole grains consumption in the human gut. The fermentation and the stages of kernel development or maturation had a great impact on the amount of resistant starch, fructans and β-glucans as well as their interactions resulted highly statistically significant. The amount of fructans was high in kamut bread (2.1g/100g) at the fully ripe stage compared to wheat during industrial fermentation (baker’s yeast). The sourdough increases the content of polyphenols more than industrial fermentation especially in bread made by flour at milky stage. From the analysis of volatile compounds it resulted that the sensors of electronic nose perceived more aromatic compound in kamut products, as well as the SPME-GC-MS, thus we can assume that kamut is more aromatic than wheat, so using it in sourdough process can be a successful approach to improve the bread taste and flavor. The determination of whole grain biormakers such as alkylresorcinols and others using FIE-MS AND GC-tof-MS is a valuable alternative for further metabolic investigations. The decrease of N-acetyl-glucosamine and 3-methyl-hexanedioic acid in kamut faecal samples suggests that kamut can have a role in modulating mucus production/degradation or even gut inflammation. This work gives a new approach to the innovation strategies in bakery functional foods, that can help to choose the right or best combination between stages of kernel maturation-fermentation process and baking temperature.

Wind plant interaction with series-compensated power systems

Wind power based generation has been rapidly growing world-wide during the recent past. In order to transmit large amounts of wind power over long distances, system planners may often add series compensation to existing transmission lines owing to several benefits such as improved steady-state power transfer limit, improved transient stability, and efficient utilization of transmission infrastructure. Application of series capacitors has posed resonant interaction concerns such as through subsynchronous resonance (SSR) with conventional turbine-generators. Wind turbine-generators may also be susceptible to such resonant interactions. However, not much information is available in literature and even engineering standards are yet to address these issues. The motivation problem for this research is based on an actual system switching event that resulted in undamped oscillations in a 345-kV series-compensated, typical ring-bus power system configuration. Based on time-domain ATP (Alternative Transients Program) modeling, simulations and analysis of system event records, the occurrence of subsynchronous interactions within the existing 345-kV series-compensated power system has been investigated. Effects of various small-signal and large-signal power system disturbances with both identical and non-identical wind turbine parameters (such as with a statistical-spread) has been evaluated. Effect of parameter variations on subsynchronous oscillations has been quantified using 3D-DFT plots and the oscillations have been identified as due to electrical self-excitation effects, rather than torsional interaction. Further, the generator no-load reactance and the rotor-side converter inner-loop controller gains have been identified as bearing maximum sensitivity to either damping or exacerbating the self-excited oscillations. A higher-order spectral analysis method based on modified Prony estimation has been successfully applied to the field records identifying dominant 9.79 Hz subsynchronous oscillations. Recommendations have been made for exploring countermeasures.