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Eimeria spp. infections in broiler still cause huge economic losses. An experiment was conducted to evaluate the efficacy of a combination of drugs in the control of multiple infections of these parasitic protozoans in chickens experimentally infected. Five groups of 20 Ross308 broilers at 21st day of life were infected per os with 1 ml of a suspension containing 5000 oocists of Eimeria tenella, 5000 of E. maxima and 10.000 of E. acervulina per liter. At 35th day, the therapy with the association of 200 mg di Sulfadimethoxine and 40 mg of Trimethoprim per ml was subdivided in 4 groups: “A” control; “B” with 0.5 ml/L in drinking water for 5 days; “C” with 1 ml/L for the first day and 0.5 ml/L for 4 days; “D” with 1 ml/L for 5 days, group “E” 2 ml/L for 1 day and 1 ml/L for 4 days. All dead specimens were necropsied and intestinal lesion score was evaluated using Johnson et al. (1970) method. Every day for each group oocyst number were counted on a pool of feces by McMaster method as well as direct observation and enumeration of oocyst were performed on the subjects who died. Clinical signs attributable to Coccidiosis have appeared at 6th day after inoculation and first mortality was observed after 14 days. In the same day the treatments started and mortality go further for 2 days only in the control group. The lesion scores show how the inoculation causes macroscopic lesions referable to E. acervulina and E. tenella, but less obvious for E. maxima, in the relative portions of the gut. The groups treated with different dosages had a highly significative reduction (p0.05) were found between constant dosage (B and D) or variable (C and E). The treatment has seen a good efficacy in reducing mortality, opening a good perspective for the control of these parasites.

Pseudomonas syringae pv. actinidiae (Psa),the causative agent of bacterial canker of kiwi, was firstly isolated in 1992, but the second outbreak dating back to 2008 is now in charge of a large pandemic that has led to major economic losses worldwide. Despite the high economic impact of Pseudomonas syringae pv. actinidiae (Psa) on kiwifruit industry, and the many research efforts, the molecular mechanisms involved in pathogenesis are still not fully characterized. In particular, the precise mechanism of recognition/communication between Psa and its host plant has not been yet elucidated. Our findings shows that plant crude extracts or xylem juices of Actinidia deliciosa and A. chinensis were able to increase Psa virulent in comparison with artificial growing media such as Luria Broth or Nutrient agar. The study of gene expression in response to plant-based or artificial growing medium was performed by QPCR and showed a differential expression of many virulence related genes. Also genes related with biofilm formation, motility and inter cell signaling (e.g. quorum sensing in the broad sense) were differencially expressed according to the type of growing media. Interestringly, many of the studied gene expression responded differentially depending both to plant extracts and Psa cell density. The molecular data were corroborated by phenological studies aiming to test the effect of plant based medium on biofilm formation, motility and symptom development in microcuttings of Actinidia spp. The effect of other plant extracts such as tobacco, tomato, tea, hazelnut and A. arguta were also studied to determine whether the activation of genes and phenotypes observed is related to a non specific plant molecules. To determine which plant molecules are specifically recognized by PSA, the putative bacterial receptors have been cloned, expressed heterologously, purified and immobilized in chromatographic columns in order to capture their ligands present in those plant extracts specifically activating Psa genes. The presented study demonstrates the existence of a not yet identified, host-specific group of molecules able to active the pathogenesis machinery of Psa. Moreover these molecules could led to different physiological and molecular behavior depending on Psa population density. Finally, it was also demonstrated that these molecules were differentially present in leaves and xylem sap collected by different Actinidia species.

The quorum sensing (QS) is a complex bacterial intra and inter communication system which is regulated by signalling molecules produced by cell density dependent mechanisms. The QS may regulate different phenotypes such as virulence, motility and biofilm production. The aim of the research was to investigate the existence of a QS system in Pseudomonas syringae pv. actinidiae (Psa). Psa lacks of the canonical QS system based on signal-synthase gene. However, at least three signal receptors (PsaR1, PsaR2, PsaR3) were recently identified. The presented study was conducted in order to clarify the effects of different cell densities on the intraspecific communication in Psa cultures. Therefore, interspecific interaction between Psa and other bacteria associated with the plant host (i.e. in the phyllosphere) or known to have an active QS system were also investigated. A bioinformatic research was conducted for the identification of genes putatively involved in Psa QS system. About 30 genes were selected due to their involvement in influencing specific phenotypes such as virulence, density perception, biofilm formation and motility. The regulation of these genes was analysed by QPCR in Psa samples derived from different culture conditions (i.e. density; co-cultures). Therefore, after a nucleotide sequences blast analysis of several bacterial signal synthase promoters that respond to all Psa signal receptors, homology with the promoter region in front of a transcriptional regulator was found. We suppose that this regulator may be involved in the QS signal transduction. Motility and biofilm formation crucial phenotypes in the early stages of host colonization, are under control of QS circuitries. The experiments conducted demonstrated Psa ability of producing biofilms in LB liquid media in a population density manner. Psa motility resulted particularly enhanced on QS-bacteria supernatants (such as Pseudomonas fluorescens, or an overproducing QS-signals strain of Pseudomonas putida). Quantitative expression of QS-related genes was analysed by QPCR. The expression fold change significantly varied in Psa populations depending on the cell density (from 105 up to 108 cfu/ml). Therefore, the gene expression profiles also changed when Psa cultures were grown in bacterial co-cultured media, remarking the importance of interspecific communication on Psa QS-system and potentially on Psa virulence. In conclusion, from our preliminary studies we observed that QS-phenotypes seem to be influenced not only by the cell density, but also by the microbial community that may share with Psa the same environment. For now, these findings are leading to the hypothesis that Psa has evolved to eavesdrop signals coming from the environment to regulate the complexity of its QS mechanisms. Presumably only by considering a multiple model system, where Psa interacts both with the plant host and other bacterial species, the functionality of Psa QS-system will be fully elucidated.

The bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae, is considered one of the most severe diseases affecting several cultivated Actinidia species, including A. chinensis and A. deliciosa. Kiwifruits have been always considered fruit with an high intrinsic quality due to the strong nutraceutical value, but also to the absence of contaminants such as pesticide residues. With the emergence of this devastating disease, the use of pesticides rapidly increased and therefore the kiwifruit quality can be maintained only by ensuring agricultural practices that reduce the need of toxic xenobiotic compounds. The aim of this study was to provide an in-depth understating of the influence of agricultural practices on disease development and spread and to harmonise the agricultural practices with the current disease control inputs. Therefore, the role of fertilization, irrigation, use of bio-regulators, rootstock, training system and pruning on the incidence and epidemiology of PSA were examined. Nitrogen fertilization had a direct effect on the pathogen's endophytic growth. Furthermore, the depletion of some micronutrients, such as iron, increased the disease. The water stress consequent to a reduced irrigation resulted in higher symptoms. Concerning pruning, the open cuts remains a possible entry point for more than 30 days. In addition, the pruning performed in late season resulted more risk for infection. To test curative pruning, preliminary data were collected on the migration rate of Psa inside the different plant organs. Among the different training system evaluated in this study, the gender double curtain the allowed a more efficient penetration of the phytosanitary treatments that may increase the disease control. However, no differences on disease incidence were observed. Concerning the use of bioregulators, synthetic gibberellin, such as forchlorfenuron, reduced in controlled condition both the disease incidence and severity. Synthetic auxins, on the other hand, showed a detrimental effect with higher symptomatology. In field conditions, none of the used bioregulators showed any effect on the disease incidence and development. Several species were tested for their susceptibility to Psa in order to develop a tolerant rootstock. Arguta spp. resulted the most tolerant to the disease and further experiments are need to test its possible exploitation as a commercial rootstock. Finally, the a preliminary guideline harmonizing the best cultural practices with the most widely used control methods (i.e resistance inducers and copper) is provided.

Following the discovery of Pseudomonas syringae pv. actinidiae (Psa ) in New Zealand, a massive screening programme on young kiwifruit plants in a glasshouse was conducted in the hope of finding new compounds for the control of this pathogen. Unfortunately, control of plant pathogenic bacteria is difficult, and Psa is no exception. Some of the difficulties with controlling Psa come from its ability to survive and to colonise tissues without causing symptoms, and its ability to resist inhibitory compounds by forming a biofilms on and within plant surfaces. The screening programme was not only directed at compounds already known to kill or inhibit plant pathogenic bacteria, such as antibiotics or heavy metals (mostly copper), but it was also directed at biological control agents, bacteriophages, antimicrobial peptides, novel chemicals and elicitors. Very few of the products trialled were successful in reducing the incidence of Psa in our experimental conditions. However, from this screening programme streptomycin, kasugamycin, copper-containing compounds and the elicitor Actigard™ (also known as Bion™), which consistently reduced significantly disease incidence, were registered in New Zealand for the control of Psa. Characteristics of some of those compounds, including their potential and limits for control of Psa, will be discussed. One of the interesting results found during this programme was the differential reaction of the green-fleshed Actinidia deliciosa and the yellow-fleshed A. chinensis to some elicitors. To understand the molecular mechanisms involved in the elicitation of resistance by kiwifruit plants, gene expression is being studied in A. chinensis ‘Zesy002’ (commonly known as Gold3) treated with different elicitor compounds before inoculation with Psa.