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Background: Uncertainty exists about the optimal schedule of adjuvant treatment of breast cancer with aromatase inhibitors and, to our knowledge, no trial has directly compared the three aromatase inhibitors anastrozole, exemestane, and letrozole. We investigated the schedule and type of aromatase inhibitors to be used as adjuvant treatment for hormone receptor-positive early breast cancer. Methods: FATA-GIM3 is a multicentre, open-label, randomised, phase 3 trial of six different treatments in postmenopausal women with hormone receptor-positive early breast cancer. Eligible patients had histologically confirmed invasive hormone receptor-positive breast cancer that had been completely removed by surgery, any pathological tumour size, and axillary nodal status. Key exclusion criteria were hormone replacement therapy, recurrent or metastatic disease, previous treatment with tamoxifen, and another malignancy in the previous 10 years. Patients were randomly assigned in an equal ratio to one of six treatment groups: oral anastrozole (1 mg per day), exemestane (25 mg per day), or letrozole (2·5 mg per day) tablets upfront for 5 years (upfront strategy) or oral tamoxifen (20 mg per day) for 2 years followed by oral administration of one of the three aromatase inhibitors for 3 years (switch strategy). Randomisation was done by a computerised minimisation procedure stratified for oestrogen receptor, progesterone receptor, and HER2 status; previous chemotherapy; and pathological nodal status. Neither the patients nor the physicians were masked to treatment allocation. The primary endpoint was disease-free survival. The minimum cutoff to declare superiority of the upfront strategy over the switch strategy was assumed to be a 2% difference in disease-free survival at 5 years. Primary efficacy analyses were done by intention to treat; safety analyses included all patients for whom at least one safety case report form had been completed. Follow-up is ongoing. This trial is registered with the European Clinical Trials Database, number 2006-004018-42, and ClinicalTrials.gov, number NCT00541086. Findings: Between March 9, 2007, and July 31, 2012, 3697 patients were enrolled into the study. After a median follow-up of 60 months (IQR 46–72), 401 disease-free survival events were reported, including 211 (11%) of 1850 patients allocated to the switch strategy and 190 (10%) of 1847 patients allocated to upfront treatment. 5-year disease-free survival was 88·5% (95% CI 86·7–90·0) with the switch strategy and 89·8% (88·2–91·2) with upfront treatment (hazard ratio 0·89, 95% CI 0·73–1·08; p=0·23). 5-year disease-free survival was 90·0% (95% CI 87·9–91·7) with anastrozole (124 events), 88·0% (85·8–89·9) with exemestane (148 events), and 89·4% (87·3 to 91·1) with letrozole (129 events; p=0·24). No unexpected serious adverse reactions or treatment-related deaths occurred. Musculoskeletal side-effects were the most frequent grade 3–4 events, reported in 130 (7%) of 1761 patients who received the switch strategy and 128 (7%) of 1766 patients who received upfront treatment. Grade 1 musculoskeletal events were more frequent with the upfront schedule than with the switch schedule (924 [52%] of 1766 patients vs 745 [42%] of 1761 patients). All other grade 3–4 adverse events occurred in less than 2% of patients in either group. Interpretation: 5 years of treatment with aromatase inhibitors was not superior to 2 years of tamoxifen followed by 3 years of aromatase inhibitors. None of the three aromatase inhibitors was superior to the others in terms of efficacy. Therefore, patient preference, tolerability, and financial constraints should be considered when deciding the optimal treatment approach in this setting. Funding: Italian Drug Agency.

Two years after its official start, the national vegetation database VegItaly, a collaborative project supported by the Italian scientific community and developed by a large group of scientists, is presented. This article offers a concise overview of the content of the database, currently consisting of 31,100 vegetation plot, including published and unpublished data. Some basic statistics are analysed; for example, data distribution in space and time, represented vegetation types expressed as physiognomic categories. Although rather young and still in progress, VegItaly already contains data from all the Italian regions and stands as an optimal candidate for the development of an Italian national vegetation database. Its main goals,theoretical basis, technical features, functionalities and recent progresses are outlined, showing glimpses of future prospects.

The present work is aimed at presenting the data, methods and criteria that are being used for the assessment of ecosystem collapse risk in Italy.

The essential oils (EOs) of Origanum compactum and Satureja montana chemotyped (CT) at carvacrol, two Thymus vulgaris CT at thujanol and thymol, and Hydrolates (Hys) of S. montana and Citrus aurantium var. amara were chosen for studying their bactericidal efficacy against few phytobacterial pathogens. The Minimal Inhibitory Concentration (MIC) and Bactericidal Concentration (MBC) were found by microdilution assay. The essential oils of O. compactum (MBC 0.06% v/v), T. vulgaris CT thymol (MBC 0.06% v/v), and Hy of C. aurantium (MBC 6.25% v/v) resulted in being the most effective against Erwinia amylovora; thus, they were used as starting concentrations for ex vivo assays. Despite the great in vitro effectiveness, the disease incidence and the population dynamic ex vivo assays showed no significant results. On the other hand, EO of O. compactum and Hy of C. aurantium (at 0.03% and 4.5% v/v, respectively) showed resistance induction in tomato plants against Xanthomonas vesicatoria infections; both treatments resulted in approximately 50% protection. In conclusion, EOs and Hys could be promising tools for agricultural defense, but further studies will be necessary to stabilize the EOs emulsions, while Hys application could be an effective method to prevent bacterial diseases when used as resistance inducer by pre-transplantation treatment at roots.

The main purpose of the 92/43/EEC Habitats Directive is to contribute to the conservation of biodiversity, understood as habitat types and species of the flora and fauna of the European Union. To achieve this goal, natural and semi-natural biodiversity as a whole must be recognized and included in its annexes. As for the conservation of biotopes, named habitat types, Italy is unfortunately lacking as it the Annex I does not include important ecosystems that are typical of its territory, rare for biogeographical reasons or threatened. Therefore, the opportunity to identify a first list of significant habitats for central Italy is discussed here. For each of the new proposed types (new habitats or new subtypes) a sheet has been prepared to highlight their salient characteristics. The new proposals concern seven habitat types and one subtype: sedge and reeds formations (Freshwater large sedge and reed beds), willow shrublands (Shrubby willow formations of river banks and fens), Apennine garrigues (Apennine hilly and montane garrigues), a new subtype of Annex I Habitat 6130 (Communities of herbaceous and dwarf shrub-suffrutescent plants of Italian ultramafic substrates), ancient olive groves ("Centuries-old olive groves" with evergreen Quercus spp. and arborescent matorral), secondary meadows (Italian submontane and montane pastured meadows dominated by Cynosurus cristatus), badlands (Pioneer halophilous and sub-halophilous communities of “calanchi” and “biancane” badlands) and hop-hornbeam woods (Italian-Balkan hop-hornbeam woods).

Leone Piccioni e la sua passione per le forme brevi e aneddotiche nei volumi "Cardarelliana", "Ungarettiana" e "Memoriette"

Pseudomonas syringae pv. actinidiae is an important pathogen of kiwifruit (Actinidia deliciosa), and bacterial canker of this host is managed by monitoring and chemical control strategies. The efficacy of the bio-pesticides Amylo-X (R) (based on Bacillus amyloliquefaciens subsp. plantarum strain D747) and Serenade Max (R) (strain QST713 of B. subtilis) was evaluated by in vitro and in vivo experiments. Both antagonists inhibited different biovars of the pathogen in in vitro assays; QST713 was more efficient than D747. The two Bacillus strains also colonized A. deliciosa flowers (c. 10(5-7) cfu per flower) up to 96 h after inoculation. 1)747 persisted on leaves (c. 10(4-6) cfu cm(-2)) up to 4 weeks after inoculation, during 2 years in Emilia Romagna and Latium regions of Italy. On flowers, the antagonists reduced pathogen populations, compared to untreated (control) flowers. On A. deliciosa and A. chinensis plants under controlled conditions, Amylo-X (R) reduced severity of bacterial canker, providing ca. 50% relative protection on A. deliciosa and 70% on A. chinensis. Serenade Max (R) was less effective, giving 0% relative protection on A. deliciosa and 40% on A. chinensis. In a field trial, on A. deliciosa plants, Amylo-X (R) reduced the severity of bacterial canker on leaves, providing ca. 40% relative protection. The sensitivity of both antagonistic strains to streptomycin sulphate was confirmed by testing the most used concentration where antibiotics are approved for management of bacterial pathogens.

A study was conducted on 14 accessions of Actinidia spp. to evaluate their susceptibility to the bacterial canker. Experimental inoculations were carried out, under controlled conditions, on young leaves of kiwifruit plants grown in pots by spraying a calibrated water suspension of an Italian, virulent strain (isolated in 2013) of Pseudomonas syringae pv. actinidiae. Actinidia chinensis ‘Belen’ was used as positive control, being a known susceptible genotype. Plants inoculated with sterile distilled water and non-inoculated ones were used as negative control. The disease assessments were performed 21 days after inoculation with the pathogen. The disease severity (mean of leaf spots per treatment) allowed to detect one A. chinensis selection showing low susceptibility, comparable to that of selections belonging to A. arguta, A. hemsleyana and A. eriantha species, that showed a very low susceptibility. No selection was found more susceptible than ‘Belen’, but among all selections, one resulted comparable with it. Eight selections were found to be moderately susceptible, and three of them (A. chinensis selections) were less susceptible than A. deliciosa ‘Hayward’, that is known having an intermediate susceptibility.

Plant bacterial diseases are routinely managed with scheduled treatments based on heavy metal compounds or on antibiotics; to reduce the negative environmental impact due to the use of such chemical compounds, as pollution or selection of antibiotic resistant pathogens, the integrated control management is required. In the frame of a sustainable agriculture the use of bacterial antagonists, biological agents, plant defence response elicitors or resistant host plant genotypes are the most effective approaches. In this work, cold atmospheric pressure plasma (CAP) was applied to sterile distilled water, inducing the production of a hydrogen peroxide, nitrite and nitrate, and a pH reduction. In particular, an atmospheric pressure dielectric barrier discharge (DBD) has been used to produce plasma activated water (PAW), that was firstly assayed in in vitro experiments and then in planta through application at the root apparatus of tomato plants, against Xanthomonas vesicatoria (Xv), the etiological agent of bacterial leaf spot. Moreover, the transcription abundance of five genes related to the plant defense was investigated in response to PAW treatment. PAW did not show direct antimicrobial activity against Xv in in vitro experiments, but it enhanced the tomato plants defenses. It was effective in reducing the disease severity by giving relative protections of ca. 61, 51 and 38% when applied 1 h, 24 h and 6 days before the experimental inoculation, respectively. In addition, the experiments highlighted the pal gene involvement in response to the PAW treatments and against the pathogen; its transcription levels resulted significantly high from 1 to 48 h until their decrease 192 h after PAW application.

Knowing and recognizing the cases in which structural elements and components of building systems of our built heritage are subject to pathologies and forms of degradation is the first step towards a culture of prevention of catastrophes and risks to which citizens and housing would be subjected. Specifically, in the wake of the recent events that occurred not only in our country, due to negligence of professionals and technical underestimations, they undergo a preliminary study of diagnosis and treatment of reinforced concrete structures, starting from the material matrix. We focus on pathologies that are recognized in the corrosion of the reinforcements and in the consequent cracks, of which a variegated system of causes and origins has been found. Knowing them, in the pre-design phase, involves a correct realization of the structures, from the implementation to the creative, environmental and functional choices. The work is based on the study of specific pathologies, such as corrosion by carbonation, by attacks of chloride ions and those by sulphates, and the relative behaviors of the concrete subjected to them, so as to prevent discomfort, prohibit its enduring over time and stop threatening contaminations.

Structured AbstractObjectives To investigate possible changes and/or device-related impairments in phonetic habits produced by rapid maxillary expansion (RME). Materials and Methods Thirty-five patients scheduled for RME were divided into two groups: Group A (banded two-arm Hyrax) and Group B (banded four-arm Hyrax). Speech samples were collected at six time points, before, during and after RME removal. Acoustical analysis was performed using PRAAT and BioVoice analysis tools. Ten volunteers completed a questionnaire on the acceptability of patient's speech. Maxillary dimensions and palatal volume were measured on dental casts before and after expansion using a digital gauge. Results Voice analysis showed an increase in the peak frequency of fricative consonants (/s/,/ʃ/) after expansion, whereas there was no change of formant frequencies of palatal consonants (/ɲ/,/ʎ/). Vowel /i/ displayed a lowering of the first formant frequency, and an increase in the second and third formant frequencies. After bonding, Group B showed both a greater reduction in the peak frequency of fricatives and a greater increase in the formant frequencies of palatal consonants than Group A. Conclusion Rapid maxillary expansion causes a slight phonetic change in the acoustical parameters of both consonants and vowels. The two-arm Hyrax caused less speech impairment than the four-arm Hyrax during the treatment.

In Italia, Pseudomonas syringae pv. actinidiae (PSA), agente causale del cancro batterico dell’actinidia (Fig. 1), a partire dal 2008 (Balestra et al., 2008; 2009) è stato facilitato nella sua rapida diffusione soprattutto a causa del commercio di materiale di propagazione privo di adeguate verifiche, sia per le ridotte conoscenze relative alla sua biologica/epidemiologia e, conseguentemente, alle opportune pratiche agronomico-colturali e fitosanitarie da adottare. In questi anni le strategie di difesa adottate nei confronti di questo batterio spesso non sono state soddisfacenti; solo da alcuni anni si è preso coscienza dell’importanza e della necessità di un impiego corretto, costante e preventivo dei sali di rame, ma, in assenza di ciò, anche in concomitanza di situazioni climatiche particolarmente avverse, come le gelate della scorsa primavera, quest’anno è stata registrata una recrudescenza dei danni causati da PSA.

La utilización de agua activada con plasma frío (plasma activated water, PAW) ha sido reportada en diversas áreas vinculadas con salud, agroindustria y agricultura. Diversas investigaciones han demostrado que PAW puede inactivar microorganismos como hongos y bacterias. Los objetivos de este estudio fueron: 1) evaluar eficacia de PAW in vitro (difusión y dilución) contra Xanthomonas vesicatoria (Xv), 2) determinar efecto inductor de defensa en plantas de tomate inoculadas con Xv, 3) cuantificar la inducción de transcriptos asociados a la defensa en plantas de tomate. PAW fue producido tratando agua desionizada estéril con plasma frío generado por un reactor de descarga de barrera dieléctrica (DBD) a voltaje de 19 kV (10 min) y frecuencia de repetición de impulsos de 1000 Hz. Los resultados in vitro no demostraron eficacia directa de PAW contra Xv, mientras que in planta hubo disminución significativa de mancha foliar por Xv (ca. 35% respecto al tratamiento testigo). Este efecto inductor, cuantificado por RT-qPCR, fue asociado principalmente a una mayor abundancia relativa del transcripto del gen pal (fenilalanina amonio liasa). La terapia de enfermedades bacterianas es basada principalmente en el uso de bactericidas en base a cobre; al respecto, el uso de PAW constituiría una promisoria opción de profilaxis contribuyendo a la producción de alimentos más inocuos y en la agricultura sostenible

Tomato bacterial spot caused by Xanthomonas vesicatoria (Xv) represent a constant threat to tomato commercial production. Bacterial plant disease control is nowadays limited to copper- or streptomycin-based bactericides, but occurrence of pathogenic strains tolerant to these compounds significantly reduces their effectiveness [1]. As an alternative to copper- or streptomycin-based compounds, few resistance inducers and biocontrol agents are presently used to manage these plant diseases, while prophylaxis remains the most important control measure. In this study, the efficacy of plasma activated water (PAW) as resistance inducer in the control of plant diseases caused by bacterial pathogens was investigated. To assess the PAW efficacy as resistance inducer, tomato plants experimentally inoculated with Xv were used as model. Sterile distilled water (SDW) have been treated with plasma by means of a nanosecond pulsed dielectric barrier discharge (DBD) source, operating at atmospheric pressure and ambient air [2]. PAW was then assayed in greenhouse trials to evaluate its ability to induce resistance in tomato plants against the pathogen. Plants of the tomato cultivars Moneymaker and VF-10, were grown in pots under greenhouse condition until they reached the 3rd - 4th leaf stage. The roots of tomato plants were soaked for 10 min in 500 ml of PAW, SDW (negative control) and acibenzolar-S methyl (a known resistance inducer, used as positive control) six-days before the inoculation with the pathogen. Experimental inoculation with Xv strain IPV-BO 2684 was carried out by spraying the pathogen aqueous suspension (ca. 107 CFU/mL) on tomato leaves. The phytopathometric assessments were carried out 21 days after pathogen inoculation by counting the number of spots on tomato leaves. The plants pretreated with PAW showed a significantly lower number of leaf spots compared to those pretreated with SDW. The relative protection of PAW with respect to the negative control was estimated in the range of 30%-40%, depending on the tomato cultivar. No phytotoxicity effect was observed. These results highlighted that the preventive application of PAW at the root apparatus was able to reduce the disease severity (number of leaf spots) implying the activation of plant defence responses. Further studies on kinetics of defense related genes expression confirmed the ability of PAW as elicitor of the plant immune system as pal, loxf and erf1 genes resulted significantly upregulated after PAW application.

Specific VOC emissions from infected plants allow their recognition and elicit defence responses in neighboring plants, which are, however, insufficient to induce resistance. A wide range of volatile organic compounds (VOCs) is released during plant–pathogen interactions both by the pathogens and the hosts. Some of these VOCs are specific for the different diseases and are known to play a role in the pathogenicity or in plant defence responses. Besides, disease-specific VOCs may serve as markers for diagnostic protocols, which allow a non-destructive and rapid screening of bulk samples of plant material. This work aimed to verify the feasibility of a VOC-based diagnosis and to investigate the possible biological role of VOCs released during the plant–pathogen interactions. The volatile emissions from Pseudomonas syringae pv. actinidiae in axenic cultures and from inoculated in vitro kiwifruit plants were characterized by gas chromatography–mass spectrometry (GC–MS) and proton transfer reaction–time-of-flight-mass spectrometry (PTR–ToF-MS). By GC–MS, several putative biochemical markers, such as 1-undecene, were identified. PTR–ToF-MS resulted highly effective in screening the plant material for latent infections. To develop a more user-friendly, portable and less expensive diagnosis system, two different electronic nose models were tested for the early diagnosis of P. syringae pv. actinidiae in asymptomatic plant material. Our experiments demonstrated the feasibility of the electronic nose-based screening of infected plant material. Concerning the biological role of the VOCs released during the plant–pathogen interactions, the exposure of healthy plants to VOCs from infected ones influences the plant growth and induces the stimulation of protective responses. However, after the infection, P. syringae pv. actinidiae is able to selectively inactivate the induced plant defences.

Erwinia amylovora (Ea), the causal agent of fire blight, is one of the most destructive pathogens of pome fruits worldwide. Biological approach in the disease control might be potential solution for poor efficacy of available bactericides. Therefore, we isolated bacteriophages specific to this bacterium and studied their characteristics and diversity. Eleven bacteriophage strains specific to Ea were isolated from rhizosphere, irrigation water or symptomless pear and apple leaves. Based on their lytic activity, eight strains were chosen for further characterization by studying their host specificity, plaque and virion morphology and genome properties using PCR, RAPD-PCR and RFLP analysis. All phages lysed 70 Ea strains originating from Serbia, isolated from apple, pear, quince, Japanese pear, medlar, Cotoneaster sp., and Sorbus sp. They produced clear or moderately clear plaques on bacterial lawn of different Ea strains after 24 h of incubation. Examination by transmission electron microscopy indicated that all eight phages belong to the order Caudovirales, the tailed phages, including the members of the families Myoviridae (6 strains) and Siphoviridae (2 strains). Based on phage DNA digestion using seven restriction endonucleases and RAPD-PCR (primers A9, R13, A10) phages were differentiated into two groups. In PCR assay using primers for Ea phages detection (Pun45), specific fragment of three phages was amplified. Although all studied phages showed the same host range, they have been differentiated based on virion morphology and genetic properties, indicating diversity within studied population

Italy is one of the largest producers of Kiwi [Actinidia deliciosa (Chev.) Liang & Ferguson] and cv. Hayward is the most widespread. Over the past 10 years, especially in Northeastern and Central Italy, there was an increase in the cultivation of yellow kiwi (Actinidia chinensis Planchon) in which symptoms of bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) have been reported in cultivations in the Lazio region. In Japan, China and Korea, major economic losses were caused by this bacterial disease that seriously damages all vegetative organs of the plant. The development of reliable diagnostic methods for asymptomatic materials is crucial for the activity of Plant Protection Services in order to confirm the presence of the pathogen in the territory and in marketing materials. Bacteria isolation from the fruits surface (31 samples cv. Hayward from Emilia Romagna region) and from their asymptomatic columellae to detect the presence of Psa was performed. As for the bacteria identification the following tests were carried out: fluorescent pigment production, HR in tobacco leaves, oxidase activity, acid production from sugars and molecular analysis (PCR-RFLP, REP-, BOX-PCR and Bio-PCR). The identification assays of the isolates obtained from the surface and columella fruits of all samples were negative for Psa. In order to discriminate Psa from the other kiwifruit pathogenic bacteria (i.e. Pseudomonas syringae pv. syringae and Pseudomonas viridiflava) and to confirm the isolates identification, the Psa pathogenicity was tested in fruits and kiwifruit plantlets. Moreover, Psa lives in the phyllosphere together with non-pathogenic bacteria that may interfere in the diagnosis of the bacterial canker by means of false negative or positive results. Therefore, from leaves and shoots of asymptomatic plants of Actinidia chinensis Hort 16A and Actinidia deliciosa cv. Hayward, collected from the Emilia Romagna region, epiphytic bacteria were isolated and studied.

Aims To analyse genetic diversity and epidemiological relationships among 54 strains of Allorhizobium vitis isolated in Europe during an 8-year period and to assess the relative contribution of mutation and recombination in shaping their diversity. Methods and Results By using random amplified polymorphic DNA (RAPD) PCR, strains studied were distributed into 12 genetic groups. Sequence analysis of dnaK, gyrB and recA housekeeping genes was employed to characterize a representative subcollection of 28 strains. A total of 15 different haplotypes were found. Nucleotide sequence analysis suggested the presence of recombination events in A. vitis, particularly affecting dnaK locus. Although prevalence of mutation over recombination was found, impact of recombination was about two times greater than mutation in the evolution of the housekeeping genes analysed. Conclusions The RAPD analysis indicated high degree of genetic diversity among the strains. However, the most abundant RAPD group was composed of 35 strains, which could lead to the conclusion that they share a common origin and were distributed by the movement of infected grapevine planting material as a most common way of crossing long distances. Furthermore, it seems that recombination is acting as an important driving force in the evolution of A. vitis. As no substantial evidence of recombination was detected within recA gene fragment, this phylogenetic marker could be reliable to characterize phylogenetic relationships among A. vitis strains. Significance and Impact of the Study We demonstrated clear epidemiological relationship between majority of strains studied, suggesting a need for more stringent phytosanitary measures in international trade. Moreover, this is the first study to report recombination in A. vitis.

En este artículo los autores señalan la necesidad de desarrollar un modelo de prevención de PSA que reduzca la diseminación del cancro bacteriano. Así mismo explica un modelo de detección desarrollado en Italia y experimentado en Chile que se basa en el muestreo y análisis del “lloro primaveral” del kiwi, es decir, del flujo xilemático que se libera por los cortes de poda de las plantas.

A commercial electronic nose (e-nose) was used to discriminate healthy grapevine tissue from crown galled ones. Samples were collected from outdoor trials and from a nursery. Experimental samples were obtained by grapevine inoculation with Agrobacterium vitis suspensions: samples were collected after 9 months (young rootstocks) and 4 years (aged rootstocks). Field samples (1-year old shoots) were provided by Emilia Romagna Phytosanitary Service (Italy). E-nose analysis was carried out on rootstock and shoot segments. Samples were distributed in the principal component analysis score plot in two areas: on the left, healthy samples and those with small tumors were grouped, whereas high disease severity samples were localized on the right. Linear discriminant analysis correctly classified the samples with a total recognition percentage of 79%. The data obtained allowed building a database in which different sizes, organs, and disease severity of samples are taken into consideration.

The use of Plasma Activated Water (PAW) on plants has been reported as a promising tool for the improvement of plants growth [1]; moreover, plasma treatment of water is known to induce the formation of nitrates, nitrites and peroxides and a change in pH, producing PAW with antimicrobial properties [2]. Indeed, conventional management of plant diseases caused by bacteria has been mainly focused on the use of copper compounds and a few biofungicides or, in the case of phytoplasmas, on insect vector chemical control and on infected plant rouging. Treatment of infected plants with PAW represents an innovative alternative in the control of plant diseases caused by phytopathogens. Fundamental investigations are needed to understand the antimicrobial activity of PAW on plants and the possible phytotoxicity induced by such a treatment. In this work, the antibacterial properties of PAW have been investigated with in vivo tests. Experiments were carried out using Sterile Distilled Water (SDW) treated for 10 minutes by means of a dielectric barrier discharge driven by nanosecond pulses with a peak voltage of 19 kV and a pulse repetition frequency of 1 kHz. PAW antibacterial effects were tested on two vegetal models: experimentally infected tomato plants and periwinkles micropropagated shoots. In the case of tomato plants, the roots were soaked for 10 min into PAW or into SDW (positive control); then, the plants were experimentally inoculated by spraying a water suspension of Xanthomonas vesicatoria (Xv) on the leaves and the disease severity was assessed 21 days after. A reduction of the number of leaf spots caused by Xv was observed in the tomatoes plants soaked in PAW compared to positive control. Moreover, no phytotoxicity effect has been detected on treated plants. In the second model, periwinkle micropropagated shoots, maintained in tubes containing MS agar-solidified medium [3] infected with ‘Candidatus phytoplasma asteris’ were treated adding 1 ml of PAW to the surface of the agar in the micropropagation tubes. Preliminary results indicate that treatment by PAW does not induce shoot toxicity and allows an improved detection of phytoplasma presence in shoots and also in liquid phytoplasma isolation medium [4], indeed, after direct PCR, amplicons of the expected lengths were obtained from all the treated shoot, while the use of nested-PCR was required to obtain specific primers amplification from untreated shoots. The reduced symptomatology and the lack of bacterial endophytic colony formation from liquid isolation medium inoculated with treated shoots, induce to speculate that PAW interacts with phytoplasmas and/or with endophytes viability.

In the frame of a CRPV project, a study was conducted on 14 genotype selections of Actinidia spp. to evaluate their susceptibility to the bacterial canker. Under controlled conditions experimental inoculations were carried out on young leaves of kiwifruit plants grown in pots, by spraying a calibrated water suspension of a local, virulent strain (isolated in 2013) of Pseudomonas syringae pv. actinidiae. Actinidia chinensis 'Belen' was used as positive control as the most susceptible genotype and non-inoculated plants were used as negative control. The disease assessments were performed 21 days after the inoculation with the pathogen. The disease severity (mean of leaf spots per treatment) allowed to detect one A. chinensis selection showing low a susceptibility that is comparable to that of selections belonging to A. arguta, A. hemsleyana and A. eriantha. No selections were found more susceptible than 'Belen', but among all selections, one resulted significantly comparable with it. Eight selections were found to be moderately susceptible, and three of them (A. chinensis selections) were apparently less susceptible than A. deliciosa 'Hayward', known having intermediate susceptibility.

Crown gall caused by Agrobacterium spp. is a widespread bacterial disease that occurs on various agricultural crops and may cause significant economic losses. Although the disease and its causal agent are present on many cultivated plants in Serbia, Agrobacterium species have not been studied extensively for more than 30 years. We therefore investigated etiology of crown gall on particular plant hosts in Serbia and characterized the causal agent of the disease. This research was largely based on networking between our laboratory and major phytobacteriological laboratories throughout Europe within COST Actions 873 and FA0807. As a result of this collaboration, bacteria associated with crown gall symptoms on apricot trees were isolated and characterized. Based on PCR analysis, it is determined that six selected strains harbor Ti plasmids, classified as nopaline type. Tumorigenicity of the strains was confirmed in pathogenicity assay on several test plants. Using physiological and biochemical tests, multiplex PCR assay targeting 23S rRNA gene sequences and sequence analysis of the 165 rRNA gene, five strains were assigned as A. rhizogeneslbiovar 2 and the remaining one as A. tumefacienslbiovar 1. Furthermore, the joint research program led to the first report of A. vitis as the causal agent of grapevine crown gall in Serbia. The strains isolated from tumor tissue of grapevine were determined as tumorigenic and identified as A. vrlls by using classical bacteriological and molecular methods. Overall, these results provide a starting point for studying distribution, diversity and economic significance of Agrobacterium species/biovars in stone fruit and grapevine production in Serbia. This is a prerequisite for prevention of further disease spreading and successful protection. Moreover, the expertise gained during this collaboration was implemented in the research of one national project (11146008, Ministry of Education, Science and Technological Development of

Le analisi microbiologiche e molecolari effettuate su quasi 100 campioni di «pianto primaverile» di actinidia hanno permesso di confermare che il metodo diagnostico può avere utili prospettive nel contenimento del cancro batterico. La possibilità di individuare anticipatamente le piante con infezioni conclamate e/o latenti, prima della ripresa vegetativa, è di notevole importanza allo scopo di evitare un’ulteriore disseminazione del patogeno

The bacterial plant disease control is nowadays limited to copper compounds, few resistance inducers and biocontrol agents, but prophylaxis still remains the most important control measure that is poorly explored. Experiments were carried out using sterile distilled water (SDW) treated by a nanopulsed dielectric barrier discharge. The electrodes of the plasma source consisted of aluminum foils, covered by 1 mm thick polystyrene as dielectric; one of the polystyrene dielectrics was shaped with edges in order to contain water to be treated Ten minutes treatment with a peak voltage of 19 kV and a pulse repetition frequency of 1,000 Hz induced in plasma activated water (PAW) the presence of nitrates, nitrites and peroxides, and a pH decrease. After the treatment, PAW was firstly assayed in in vitro experiments, using diffusion and dilution methods, to evaluate its direct efficacy against selected bacterial pathogens such as Erwinia amylovora, Pseudomonas syringae pv. actinidiae and Xanthomonas arboricola pv. pruni. PAW was then assayed in greenhouse trial to evaluate its ability to induce resistance in tomato plants against X. vesicatoria (Xv). PAW was applied at the root apparatus five days before and two days after Xv experimental inoculation; negative and positive controls were Bion®, streptomycin sulphate and SDW, respectively. In in vitro experiments, PAW did not show bacterial inhibition neither using diffusion nor dilution method. In greenhouse no phytotoxicity was detected on tomato plants, disease severity (DS) assessments highlighted a significant disease inhibition and a relative protection of approximately 35%.

DNA-based protocols are the golden standard for the diagnosis of infected plant material. Nevertheless, these methods are time-consuming and require trained personnel, with an efficacy depending on the sampling procedure. Given that plant responses induced by pathogens also include changes in emission of volatiles, the electronic-nose may represent a sensitive, accurate and operator-friendly alternative for rapid and reliable screening of asymptomatic plant material. In comparison with DNA-based diagnosis, which is hampered by the sampling procedure of the plant material, volatiles-based diagnosis can be performed on the whole plant. In the present study, the analysis of volatile compounds is used for the discrimination of two apple diseases: fire blight (Erwinia amylovora) and blossom blight (Pseudomonas syringae pv. syringae) on propagation material. The profile of volatiles emission was initially performed by gas chromatography-mass spectroscopy (GC-MS), proton-transfer time of flight mass spectroscopy (PTR-TOF-MS) to identify possible marker compounds. In addition, two different electronic noses, EOS507 (Sacmi Scrl, Imola, Italy) and PEN3 (Airsense Analytics GmbH, Schwering, Germany), both based on metal oxide semiconductors, were used for diagnosis. After the method validation on in vitro micropropagated plants, a sampling protocol was developed to scale up the diagnosis to real nursery conditions on cold-stored, asymptomatic dormant scions. Electronic-nose can be successfully used in practical conditions, such as nurseries or customs, to screen large quantity of asymptomatic plant material in order precisely steer molecular diagnosis

The rapid spreading of the disease during last few years highlighted the need of a quick, sensitive and reliable method for Pseudomonas syringae pv. actinidiae (Psa) detection, to find possible inoculum sources and limit the pathogen spreading. A PCR method, using new primers designed on the gene encoding a putative outer membrane protein P1, was developed to detect Psa in symptomatic and asymptomatic tissue; a nested-PCR was also applied. Bleeding sap samples, collected in early spring from orchards with symptomatic and asymptomatic trees, were used both for PCR assays and for pathogen isolation and identification. The PCR and nested PCR methods were able to detect Psa presence at very low concentration from plant and pollen extracts; RFLP analyses with BclI on PCR and nested PCR amplicons confirmed the assay specificity, while the digestion with BfmI and AluI allowed to discriminate Psa strains isolated before 2008 from those isolated after 2008. Furthermore, the PCR and nested PCR on crude bleeding sap samples detected the presence of the pathogen in 3 and 5 of the 15 assayed samples, respectively. Direct isolation from the same samples and bacterial identification confirmed the results of molecular analysis.

The strain D747 of Bacillus amyloliquefaciens (Amylo-X®) was assayed to evaluate its efficacy against Pseudomonas syringae pv. actinidiae (Psa), the causal agent of the bacterial canker of kiwifruit which assumed significant relevance after the heavy epidemics that have been occurring in central-northern Italy since 2008. The ability of strain D747 to inhibit the growth of two different Psa strains in vitro, its ability to survive and reduce populations of a rifampicin resistant pathogenic strain (CRA-FRU 3.1 Rifr) on female flowers of Actinidia chinensis, and its population dynamic on leaves of A. deliciosa were investigated. The microbial control agent was effective against Psa in vitro, and was able to survive on kiwifruit female flowers, reaching a population of approx. 106 CFU/flower 96 hours after its application. Moreover, it reduced the Psa population on A. chinensis flowers by more than one order of magnitude 48 hours after pathogen application. Furthermore, strain D747 of B. amyloliquefaciens survived on kiwifruit leaves for 14 days reaching a population of approx. 106 CFU/ml. These results suggest that strain D747 might be a promising tool for biological control of bacterial canker of kiwifruit.

The bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae (Psa), became considerably important during last five years, after severe epidemics occurring from 2008/09 in central and northern Italy. The rapid spreading of the disease during last few years highlighted the need of a fast and sensitive method for the Psa detection in symptomless trees, to find possible inoculum sources and therefore limit the pathogen spreading. In this study we analyzed 51 samples of bleeding sap from asymptomatic trees of Actinidia deliciosa and A. chinensis collected in early spring 2012. The bleeding sap samples were used both for direct PCR assays and for bacteria isolations on NSA medium. The recovered Psa-like isolates were purified and characterized by the following biochemical tests: levan production, presence of oxidase, soft rot activity on potato disks, presence of arginine dehydrolase, hypersensitivity reaction in tobacco leaves (LOPAT) and fluorescence on King’s medium B; isolates were also assayed by PCR. Direct PCR assays on crude bleeding sap samples were able to detect the presence of the pathogen in three of the 51 tested samples, while the direct isolations detected the pathogen in six samples. Although more samples should be analyzed to evaluate the robustness of the method, analysis of kiwifruit bleeding sap may be a useful, rapid and sensitive tool for screening Psa-infected plants and/or to monitor the spreading of the pathogen in the field

Le prove hanno confermato l’effi cacia del biopreparato Serenade a base di Bacillus subtilis nella profi lassi biologica al colpo di fuoco batterico prospettando un interessante impiego anche in strategie di lotta integrata

This work was carried out to confirm and/or evaluate the efficacy of three Pseudomonas spp. strains against fire blight (Erwinia amylovora), southern bacterial wilt of tomato (Ralstonia solanacearum), bacterial spot/canker of stone fruits (Xanthomonas arboricola pv. pruni) and grapevine crown gall (Agrobacterium vitis). In the laboratory, there was a trend towards a higher colonisation of apple and pear flowers when Na-alginate (0.5%) was added to suspensions of freeze-dried cells of wild-types and rifampicin-resistant mutants of IPV.-BO G19 (P. putida) and IPV.-BO 4027C (a non-fluorescent Pseudomonas sp.) strains; E. amylovora populations on apple flowers were significantly reduced by strain IPV.-BO 4027C. In a field experiment, performed on actively growing shoots of pear scions ‘Abbé Fétel’ during Spring-Summer 2005, different treatments were also tested, including copper compounds the resistance inducers prohexadione-Ca (Regalis) and acibenzolar-S-methyl (Bion), and Serenade and BS-F4 (both based on Bacillus subtilis) etc. The strain IPV.-BO G19 plus Na-alginate was the most effective in reducing shoot blight incidence by 65% six weeks after wound inoculation with E. amylovora. A virulent R. solanacearum bv. 2, race 3 strain was inhibited by both these antagonists in vitro. When the pathogen was inoculated 48 h after their application (approx. 108 cfu/ml) to the root apparatus of tomato plants grown in a climatic chamber, bacterial wilt progression rate was clearly reduced; in particular, the strain IPV.-BO G19 was able to delay symptom expression and to reduce disease incidence by 100% and 37%, 9 and 21 days after inoculation, respectively. Under glasshouse conditions, BS-F4, Bion and IPV.-BO G19 reduced relative incidence of grapevine crown gall by 58%, 54% and 48%, respectively. The IPV.-BO G19 strain was also effective against X. arboricola pv. pruni: when sprayed on plum fruitlets it reduced disease incidence by 90% eight days after experimental inoculation with the pathogen.

Studies were carried out in 2002 and 2003 to evaluate the suitability of the highly efficient pollinator of pear Osmia cornuta (Hymenoptera Megachilidae) as a carrier of a rifampicin resistant mutant of the Bacillus subtilis strain BS-F4 (BS-F4rif) on pear flowers cultiver 'Abbé Fétel' for the biological control of fire blight, and its capacity was compared with that of Apis mellifera. A dispenser for the spread of bacteria by O. cornuta was successfully developed. Females exited and entered the dispenser through the proper pathways 81.4% and 97.7% of the attempts, respectively, and the number of BS-F4rif reisolated from the body of O. cornuta exiting the dispenser ranged from 104 to 107 CFU/insect. Individuals of A. mellifera carried an average population of 104 CFU/insect. In net screened tunnel conditions, the efficiency of O. cornuta to carry and deposit the BS-F4rif on pear flowers during a single visit was higher than that of A. mellifera. Both in 2002 and 2003, the dispersal ability of bees was assessed in a 7 ha pear orchard. Trees were labelled at 2, 10, 50, 110 m from the nesting shelter, in the direction of the four cardinal points. Flowers on branches covered with fine netting acted as controls. Mason bees distributed 30 g of the biocontrol agent (BCA). Fifty flowers from both exposed and netted branches were sampled at 3, 7 and 10 days (the latter only in 2003) after the beginning of distribution. BS-F4rif was not recovered from any of the flowers on net-covered branches, whereas the bacterium was consistently recovered on the flowers of non-screened plants. Variation in distribution and population density of the BCA among flowers were observed, with greater colonisation in 2002 than 2003. We conclude that O. cornuta may serve as a carrier for the dispersal of powdered BCA formulations to pear flowers.