The perspective of the After-Cu project will focus at demonstrating the anti-infective properties of innovative peptide molecules against plant pathogenic bacteria. These molecules are specifically acting against virulence mechanisms universally present in pathogenic bacteria and, more importantly, without having any bactericide activity. The goal of the After-Cu project will be the demonstration of environmentally friendly and sustainable strategies for the control of plant bacterial diseases, in a view to replace traditional copper compounds used in conventional and organic agriculture. Accordingly, recent restrictions established within the EU countries concerning copper compounds for plant protection will be met (Directive 2009/37/EC; Council Directive 91/414/EEC). The aim of the project will be pursued specifically by demonstrating the efficacy and reliability of antivirulence oligopeptide based molecules, to control the early steps of the interaction between a phytopathogenic bacterium and a plant. These molecules exhibit unique anti-infective properties towards pathogenic bacteria, targeting those highly conserved mechanisms, through which the fate of the initial interaction between a pathogen and a plant comes to disease development. In preliminary studies carried out by the applicant working team, a 93% reduction of symptoms caused by Pseudomonas savastanoi on Olive and Oleander plants was observed following the treatments with the oligopeptides proposed in this project and in absence of any copper application. The control of bacterial diseases of plants is largely based on the use of copper salts, which are some of the few chemicals still allowed also in organic agriculture. Unfortunately, copper derivatives cannot be degraded or destroyed in the environment, and thus treatments with copper derivatives used fungicides and bactericides contribute to its accumulation in soils more than any other agricultural activity. Cu tendency to accumulate in soil and water poses a serious threat to a wide range of organisms and microorganisms, and to their ecosystems. Accordingly, recent restrictions have been established within the EU countries concerning copper compounds used in plant protection and their maximum residue levels in food and feed (Directive 2009/37/EC; Regulation 396/2005/EC; Council Directive 91/414/EEC; Commission Regulation 149/2008/EC). To better focus the environmental importance of this problem, the input of copper into European agricultural soils, included Italy and Spain, is around 504 tonnes per year (included that coming from manure and sludge), with a net input of about 400 tonnes per year, when considering output through crops (Scher, 2009). In this frame, it has absolutely also to be considered that climate changes are highly increasing potential for infection from several highly damaging plant pathogens, both fungi and bacteria. While for phytopathogenic fungi some promising alternatives to the use of copper derivatives are under study, in order to meet the needs related both to the protection of the environment and to the productivity of the agro-industry, no substainable and alternative options have ever been investigated for plant pathogenic bacteria. Furthermore, applications of copper salts are even more strictly regulated and temporally limited within the EU countries when used against phytopathogenic bacteria (Commission Regulations 396/2005/EC, 149/2008/EC): this poses huge difficulties in controlling plant diseases on perennial plants caused by destructive phytopathogenic Gram negative bacteria, such as for example Pseudomonas syringae pv. actinidiae, that in 2010 caused economic losses reaching 2 million Euros just in Italy, and that now is spreading in France, Spain, Portugal, Switzerland and other kiwifruit producing European countries. Moreover, copper-contaminated agricultural soils were proved containing high percentages of antibiotic resistant bacteria compared to non-contaminated soils. Therefore copper-contaminated soils have to be considered a dangerous reservoir of genes for antibiotic resistance, easily transmitted to pathogenic bacteria infecting animals and humans, with a dramatic impact on their health. The added value of the project is the care and protection of the environment through the replacement of copper compounds used in agriculture to control bacterial diseases of vegetable and fruit crops. Furthermore, the project will also contribute to the resolution of the most pressing health problems for scientists and the public on a worldwide scale: that is the increasing and alarming spread of antibiotic resistant bacteria, which quite often originate within natural environments. The significant environmental milestones of the project will be reached through: – The demonstration of the use of synthetic peptides to possess anti-infective activity against Pseudomonas savastanoi pv. savastanoi, P. syringae pv. actinidiae, and P. syringae pv. syringae. These bacteria are the causal agents of Olive knot disease, of the emerging and highly destructive bacterial canker of kiwifruit, and of black pit of many Citrus species, respectively. These bacteria will be used as models. They cause heavy damages in all European countries of the Mediterranean basin and currently, application of copper salts is still the only remedy to try to control the spread of these bacteria. – The demonstration of the compliance of synthetic processes for the anti-infective peptides with the current EU legislation (REACH 1907/2006), which regulates the production and import of chemical substances within the European Union. – The demonstration of the biotechnological synthesis of the anti-infective peptides to lower the costs at industrial production scale. – The demonstration of the use of these peptide molecules for control of bacterial disease of plants to replace and reduce copper compounds used in conventional and organic agriculture, in accordance with EC Regulation No 396/2005 of the European Parliament and of the Council on Maximum Residue Levels of pesticides in products of plant and animal origin, which fully harmonised the rules for pesticide residues in EU Member States. – The demonstration of the use of peptide molecules specifically targeted towards bacterial virulence mechanisms, having no antibiotic-like activity, in order to avoid the spread of antibiotic resistance in environmental microbiota, with dramatic consequences on animal and human health. – The demonstration of the use of an eco-friendly biotechnological approach for the control of bacterial diseases of plants, which could be easily transferred to human and animal Gram negative pathogenic bacteria as well, being the target of the anti-virulence peptides highly conserved. Copper is an essential microelement whose presence into the soil is variable, according to the specific characteristics of the different soils. These parameters combine with physical and biological processes occurring in the terrestrial environments, such as leaching, runoff and uptake by microrganisms and plants. Moreover, these factors strongly influence copper bioavailability, which is defined as the portion of copper in the soil that is available for uptake by soil microrganisms/organisms and plants. In the soil copper is almost exclusively in the ionic divalent form Cu2+, and it is naturally attracted by negatively charged clay minerals, anionic salts and organic matter producing several metal-coordination compounds: the complete decomposition and mineralization of organic matter is indirectly prevented by copper, causing severe ecological imbalances in nutrient recycling. Copper is the third most abundant trace element in the human body, ranging from 1.4 to 2.1 mg/Kg of body weight in adults. The suggested average daily uptake of copper is about 0.4 mg for children and 1.2 mg for adults; its shortage in the human body frequently leads to an increase in several diseases. Copper is a cofactor for more than 13 enzymes, involved in energy production, prevention of anemia and bone diseases, and the reduction of cell damage. Moreover it is necessary in fetal and infant development and even for maintenance of connective tissue and pigmentation of hair and skin. On the other hand high concentrations of copper salts dangerously affect physiological and biochemical processes in microrganisms and higher organisms. In humans dramatic toxic reactions are observed in cases of excessively elevated intake, such as concentrations higher than 11.0 mg/kg body weight. The main target organs are liver, kidneys, non-striated and heart muscle tissues, and the brain. Therefore the World Health Organisation and the Food and Agricultural Administration established that the daily mean intake of copper should not exceed 0.15 mg/Kg body weight. In this frame a great concern is given by the residual copper in vegetables and fruits for human consumption.