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In Xanthomonas genus, which includes Gram-negative bacteria mainly phytopathogenic against various host plants, the Xanthomonas arboricola species has a high genetic variability and, among the current 9 pathovars, pv. corylina causes the halzenut bacterial necrosis which is the main disease present in all hazelnut cultivation areas. In this study, 19 X. arboricola and 21 X. arboricola pv. corylina strains isolated in Chile, during 2018, from Corylus avellana symptomatic plants were characterised by Rep-PCR analysis (ERIC, BOX, REP). The genetic profiles of all strains were transformed into binary matrices which were processed by UPGMA method in single or concatenated way using Jaccard similarity coefficient. Thirteen X. arboricola reference strains belonging to the pathovars celebensis, pruni, juglandis and populi were used as controls, while the DISTAL 9081 strain of X. axonopodis pv. vitians was used as outgroup. The UPGMA cluster analysis of Rep-PCR fingerprints divided the 51 X. arboricola strains into 5 statistically significant groups corresponding to the relative pathovars: pruni, corylina, juglandis, celebensis and populi. The X. arboricola pv. corylina strains showed a high genetic variability (approx. 45%). The Rep-PCR fingerprints of the X. arboricola strains isolated from symptomatic hazelnut shoots and leaves gave similar results with a similarity value of approx. 50% among strains which were included in the corylina pathovar

Ralstonia solanacearum (Rs), the causal agent of tomato southern bacterial wilt and of potato brown rot, affects more than 250 plant species and it is included in the EPPO list of quarantine pathogens (QP). On tomato, the disease is characterized by a general wilting of the plant due to the colonization of its vascular system. Rs is a species complex with a high genetic variability and, based on the host plants, is currently divided into 5 races. In Emilia-Romagna Region (Northern Italy), during the last 15 years, Rs presence has been detected in potatoes, tomatoes and in superficial waters in nearby cultivated areas. 27 Rs strains (belonging to races 1 and 3) were characterised by Rep-PCR (BOX, ERIC, REP). The binary matrices from PCR profiles were used for UPGMA analysis in single or concatenated way. PD 2762 Rs and DISTAL 9081 Xanthomonas axonopodis pv. vitians strains were used as race 3 reference and as outgroup, respectively. From the concatenated profiles, the 27 Rs strains were divided in the two macro-groups of races 3 and 1 with a 15% similarity. The 6 strains race 3 were subdivided in 2 groups with a 50% similarity and 5 of them were more similar to each other than the PD 2762 strain. The 21 strains race 1 were subdivided into 4 groups with more than 60% similarity. The high degree of genetic variability particularly among the strains of race 1 (isolated in Parma and Ferrara provinces) suggests the presence of different haplotypes probably due to the movement of non-symptomatic infected material.

Background Allorhizobium vitis (formerly named Agrobacterium vitis or Agrobacterium biovar 3) is the primary causative agent of crown gall disease of grapevine worldwide. We obtained and analyzed whole-genome sequences of diverse All. vitis strains to get insights into their diversification and taxonomy. Results Pairwise genome comparisons and phylogenomic analysis of various All. vitis strains clearly indicated that All. vitis is not a single species, but represents a species complex composed of several genomic species. Thus, we emended the description of All. vitis, which now refers to a restricted group of strains within the All. vitis species complex (i.e. All. vitis sensu stricto) and proposed a description of a novel species, All. ampelinum sp. nov. The type strain of All. vitis sensu stricto remains the current type strain of All. vitis, K309T. The type strain of All. ampelinum sp. nov. is S4T. We also identified sets of gene clusters specific to the All. vitis species complex, All. vitis sensu stricto and All. ampelinum, respectively, for which we predicted the biological function and infer the role in ecological diversification of these clades, including some we could experimentally validate. All. vitis species complex-specific genes confer tolerance to different stresses, including exposure to aromatic compounds. Similarly, All. vitis sensu stricto-specific genes confer the ability to degrade 4-hydroxyphenylacetate and a putative compound related to gentisic acid. All. ampelinum-specific genes have putative functions related to polyamine metabolism and nickel assimilation. Congruently with the genome-based classification, All. vitis sensu stricto and All. ampelinum were clearly delineated by MALDI-TOF MS analysis. Moreover, our genome-based analysis indicated that Allorhizobium is clearly separated from other genera of the family Rhizobiaceae. Conclusions Comparative genomics and phylogenomic analysis provided novel insights into the diversification and taxonomy of Allorhizobium vitis species complex, supporting our redefinition of All. vitis sensu stricto and description of All. ampelinum. Our pan-genome analyses suggest that these species have differentiated ecologies, each relying on specialized nutrient consumption or toxic compound degradation to adapt to their respective niche.

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.

BackgroundAllorhizobium vitis (formerly named Agrobacterium vitis or Agrobacterium biovar 3) is the primary causative agent of crown gall disease of grapevine worldwide. We obtained and analyzed whole-genome sequences of diverse All. vitis strains to get insights into their diversification and taxonomy.ResultsPairwise genome comparisons and phylogenomic analysis of various All. vitis strains clearly indicated that All. vitis is not a single species, but represents a species complex composed of several genomic species. Thus, we emended the description of All. vitis, which now refers to a restricted group of strains within the All. vitis species complex (i.e. All. vitis sensu stricto) and proposed a description of a novel species, All. ampelinum sp. nov. The type strain of All. vitis sensu stricto remains the current type strain of All. vitis, K309T. The type strain of All. ampelinum sp. nov. is S4T. We also identified sets of gene clusters specific to the All. vitis species complex, All. vitis sensu stricto and All. ampelinum, respectively, for which we predicted the biological function and infer the role in ecological diversification of these clades, including some we could experimentally validate. All. vitis species complex-specific genes confer tolerance to different stresses, including exposure to aromatic compounds. Similarly, All. vitis sensu stricto-specific genes confer the ability to degrade 4-hydroxyphenylacetate and a putative compound related to gentisic acid. All. ampelinum-specific genes have putative functions related to polyamine metabolism and nickel assimilation. Congruently with the genome-based classification, All. vitis sensu stricto and All. ampelinum were clearly delineated by MALDI-TOF MS analysis. Moreover, our genome-based analysis indicated that Allorhizobium is clearly separated from other genera of the family Rhizobiaceae.ConclusionsComparative genomics and phylogenomic analysis provided novel insights into the diversification and taxonomy of Allorhizobium vitis species complex, supporting our redefinition of All. vitis sensu stricto and description of All. ampelinum. Our pan-genome analyses suggest that these species have differentiated ecologies, each relying on specialized nutrient consumption or toxic compound degradation to adapt to their respective niche.

Kiwifruit bleeding sap samples, collected in Italian and Chilean orchards from symptomatic and asymptomatic plants, were evaluated for the presence of Pseudomonas syringae pv. actinidiae, the causal agent of bacterial canker. The saps were sampled during the spring in both hemispheres, before the bud sprouting, during the optimal time window for the collection of an adequate volume of sample for the early detection of the pathogen, preliminarily by molecular assays, and then through its direct isolation and identification. The results of molecular analyses showed more effectiveness in the P. syringae pv. actinidiae detection when compared with those of microbiological analyses through the pathogen isolation on the nutritive and semiselective media selected. The bleeding sap analyses allowed the isolation and identification of two hypersensitive response (HR) negative and hypovirulent P. syringae pv. actinidiae strains from different regions in Italy. Moreover, multilocus sequence analysis (MLSA) and whole genome sequence (WGS) were carried out on selected Italian and Chilean P. syringae pv. actinidiae virulent strains to verify the presence of genetic variability compared with the HR negative strains and to compare the variability of selected gene clusters between strains isolated in both countries. All the strains showed the lack of argK and coronatine gene clusters as reported for the biovar 3 P. syringae pv. actinidiae strains. Despite the biologic differences obtained in the tobacco bioassays and in pathogenicity assays, the MLSA and WGS analyses did not show significant differences between the WGS of the HR negative and HR positive strains; the difference, on the other hand, between PAC_ICE sequences of Italian and Chilean P. syringae pv. actinidiae strains was confirmed. The inability of the hypovirulent strains IPV-BO 8893 and IPV-BO 9286 to provoke HR in tobacco and the low virulence shown in this host could not be associated with mutations or recombinations in T3SS island.

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.

Ralstonia solanacearum (Rs) and Clavibacter michiganensis subsp. sepedonicus (Cms) are the bacterial causal agents of potato brown and ring rot, respectively, and are included in the A2 list of quarantine pathogens in Europe. Identification by GC-MS analysis of volatile organic compounds from Rs or Cms cultured on different nutrient media was performed. GC-MS and PTR-MS analysis were carried out also on unwounded potato tubers infected with the same pathogens. Infected tubers were produced by experimental inoculations of the plants. In in vitro experiments, Rs or Cms emitted volatile compounds, part of which were specific disease markers of potato (2-propanol and 3-methylbutanoic acid), mainly originating from bacterial metabolism (i.e., amino acid degradation, carbohydrate and fatty acid oxidation). In potato tubers, pathogen metabolism modified the volatile compound pattern emitted from healthy samples. Both bacteria seem to accelerate metabolic processes ongoing in potatoes and, in the case of Rs, disease markers (1-hepten-3-ol, 3,6-dimethyl-3-octanone, 3-ethyl-3-methylpentane, 1-chloroctane, and benzothiazole) were identified.

DNA-based protocols are the standard methods 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. In comparison, recognition methods based on volatile compounds emissions are less precise, but allow a non-destructive mass screening of bulk samples, and may be implemented to steer molecular diagnosis. In this study, the analysis of volatile compounds is used for the discrimination of fire blight (Erwinia amylovora) and blossom blight (Pseudomonas syringae pv. syringae) on apple propagation material. Possible marker compounds were identified by gas chromatography–mass spectroscopy (GC-MS) and proton transfer reaction-time of flight-mass spectroscopy (PTR-ToF-MS). In addition, two commercial electronic noses were used for diagnosis. After a preliminary validation in vitro, a diagnostic protocol was successfully developed to scale up to real nursery conditions on cold stored, asymptomatic dormant plants.

In this work, an atmospheric pressure dielectric barrier discharge was used to produce plasma activated water (PAW). In order to evaluate its effectiveness as an alternative method to control plant diseases, three different pathosystems (i.e. plants infected by phytopathogens) were treated. The results on reduction of disease severity and gene transcription suggest that PAW acts as a plant resistance inducer.

The impact of disease outbreaks on the phytosanitary quality of seeds was investigated for two pathosystems: tomato–Xanthomonas vesicatoria and pepper–Xanthomonas euvesicatoria. This study, which was performed in Italy and Serbia, aimed to evaluate the season‐to‐season transmission of phytopathogenic regulated bacteria associated with phytosanitary risks posed by seeds produced in areas where bacterial infections are possible. For each pathosystem, field plots were experimentally inoculated to simulate an initial infection rate of 1%, 5% and 15%. The area under the disease progress curve (AUDPC) was calculated for each field plot, the seeds produced were analysed to determine the contamination level and rate, and the plant‐to‐seed transmission was evaluated by a seedling grow‐out (SGO) assay. To investigate transmission under field conditions, a second‐year experiment was performed, wherein seeds collected from the first year were used to establish new field plots. During the first growing season, AUDPC values were positively correlated with the percentages of initial infection for each pathosystem. Seed contamination levels in pepper ranged from 34 to 100 CFU g⁻¹, and the contamination rate ranged from 1.50% up to 3.17% for X. euvesicatoria, whereas processing and fresh market tomato seeds produced both in Italy and Serbia were not infected by X. vesicatoria. During SGO assays and the second cropping year, no symptoms were observed in either tomato or pepper plants. Therefore, the calculated pepper seed contamination rate for X. euvesicatoria appeared to be less than the threshold necessary to initiate a disease outbreak. Finally, all seeds obtained during the second cropping year were uninfected.

Electronic nose technology has recently been applied to the detection of several plant diseases and pests, with promising results. However, in spite of its numerous advantages, including operational simplicity, non-destructivity, and bulk sampling, drawbacks include a low sensitivity and specificity in comparison with microbiological and molecular methods. A critical review of the use of an electronic nose for plant disease diagnosis and pest detection is presented, describing the instrumental and procedural advances of sensorial analysis, for the improvement of discrimination between healthy and infected or infested plants. In conclusion, the use of electronic nose technology is suggested to assist, direct, and optimise traditionally adopted diagnostic techniques.

A commercial electronic nose (e-nose) equipped with a metal oxide sensor array was trained to recognize volatile compounds emitted by potatoes experimentally infected with Ralstonia solanacearum or Clavibacter michiganensis subsp. sepedonicus, which are bacterial agents of potato brown and ring rot, respectively. Two sampling procedures for volatile compounds were tested on pooled tubers sealed in 0.5-1 L jars at room temperature (laboratory conditions): an enrichment unit containing different adsorbent materials (namely, Tenax(®) TA, Carbotrap, Tenax(®) GR, and Carboxen 569) directly coupled with the e-nose (active sampling) and a Radiello(™) cartridge (passive sampling) containing a generic Carbograph fiber. Tenax(®) TA resulted the most suitable adsorbent material for active sampling. Linear discriminant analysis (LDA) correctly classified 57.4 and 81.3% total samples as healthy or diseased, when using active and passive sampling, respectively. These results suggested the use of passive sampling to discriminate healthy from diseased tubers under intermediate and real scale conditions. 80 and 90% total samples were correctly classified by LDA under intermediate (100 tubers stored at 4°C in net bag passively sampled) and real scale conditions (tubers stored at 4°C in 1.25 t bags passively sampled). Principal component analysis (PCA) of sensorial analysis data under laboratory conditions highlighted a strict relationship between the disease severity and the responses of the e-nose sensors, whose sensitivity threshold was linked to the presence of at least one tuber per sample showing medium disease symptoms. At intermediate and real scale conditions, data distribution agreed with disease incidence (percentage of diseased tubers), owing to the low storage temperature and volatile compounds unconfinement conditions adopted.

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.

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.

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.

For the first time, a portable electronic nose was used to discriminate between healthy and galled grapevines, experimentally inoculated with two tumourigenic strains of Agrobacterium vitis. The volatile profile of target cutting samples was analysed by headspace solid phase microextraction coupled with gas chromatography-mass spectrometry. Spectra from tumoured samples revealed the presence of styrene which is compatible with decarboxylation of cinnamic acid involved in secondary metabolism of plants. Principal Component Analysis confirmed the difference in volatile profiles of infected vines and their healthy controls. Linear Discriminant Analysis allowed the correct discrimination between healthy and galled grapevines (83.3%, cross-validation). Although a larger number of samples should be analysed to create a more robust model, our results give novel interesting clues to go further with research on the diagnostic potential of this innovative system associated with multi-dimensional chemometric techniques.

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.

Bioagents and chemicals were applied to one-year old grafted vines (Ancelotta/420A) in glasshouse and field experiments set up at the Vivai Cooperativi Rauscedo (VCR, Pordenone, Italy). In the glasshouse, holes were drilled in vines on the rootstock and the holes were charged with suspensions of different strains of Pseudomonas spp., and with the biofungicides BS-F4 and Serenade, both based on Bacillus subtilis, before inoculation with a vitopine Agrobacterium vitis strain. The growth retardant Regalis and the resistance inducer Bion were applied to the vines two weeks before inoculation with the pathogen. Six months after inoculation, disease incidence was lowest when BS-F4 had been applied. In the field trial, the vines were wounded by making a cut in the crown, after which they were dipped into the antagonist suspensions just before inoculation with the pathogen. In the two weeks before inoculation, the root systems of the vines were dipped into Regalis and Bion solutions at 7 day intervals. Only these resistance inducers and BS-F4 significantly reduced disease severity. The results indicate that a potential for defence against A. vitis may exist even in susceptible grapevine cultivars, and that this potential can be activated by diverse elicitors.

Pseudomonas viridiflava is identified for the first time as a pathogen of soilless sweet basil seedlings in Italy.

Plasma Activated Water (PAW) represents a promising tool for the improvement of plants growth [1]; moreover, the antimicrobial activity of PAW has been reported and correlated to the production of nitrates, nitrites and hydrogen peroxides and the reduction of pH [2]. These reactive species have an important role in plant defense responses, involving either hypersensitive reaction and systemic acquired resistance (SAR) and also, being bactericidal agents. Plant treatment with PAW might represent an innovative alternative in the control of plant diseases due to bacterial pathogens [3]. This study was aimed to assess the PAW efficacy in planta as resistance inducer using tomato-Xanthomonas vesicatoria (Xv) model. Sterile distilled water have been treated with plasma by means of a nanopulsed dielectric barrier discharge (DBD). The electrodes of the plasma source consist of alluminum foils, covered by 1 mm thick polystyrene as dielectric; one of the polystyrene dielectrics is shaped with edges in order to contain the water to be treated. The plasma forms in the 1 mm region between the liquid surface and the upper dielectric layer. Ten minutes treatment time with a peak voltage of 19 kV and a pulse repetition frequency of 1,000 Hz induced in plasma treated water the presence of nitrates, nitrites and peroxides and a change of pH. Under greenhouse condition, plants of two tomato cultivars, “Moneymaker” and “VF-10”, were grown in pots containing a mixture of peat and vermiculite as substrate until they reached the 3rd - 4th leaf stage. They were then soaked from the roots for 10 min in PAW, SDW (positive control) and in a solution of acibenzolar-S methyl (resistance inducer, as negative 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. 1107 CFU/mL) on tomato leaves. The phytopathometric assessments were conducted 21 days after pathogen inoculation. The tomato plants showed a statistically significantly lower number of leaf spots with respect to those pretreated with SDW. The relative protection of PAW related to negative control was calculated as approx. 30% to 40%, depending on tomato cultivar assayed. No phytotoxicity effect has been observed. These results highlighted an apparent ability of PAW to induce a plant defense response; in fact, the preventive application at the root apparatus, provoked a reduction of the disease severity (number of leaf spots) implying the activation of plant defence responses.

Plasma Activated Water (PAW) represents a promising tool for the improvement of plants growth, probably as a consequence of the presence of reactive oxygen and nitrogen species produced during plasma treatment. These reactive species might also have an important role in plant defence responses, involving both hypersensitive reaction and systemic acquired resistance; thus plant treatment with PAW could represent an innovative alternative in the control of plant diseases caused by bacterial pathogens and phytoplasmas (bacteria lacking the cell wall). In this study, the efficacy of PAW was tested in vivo on i) tomato plants, experimentally inoculated with Xanthomonas vesicatoria (Xv) under greenhouse conditions, and ii) phytoplasma infected grapevine plants in vineyards (grapevine yellows diseases, associated to the presence of phytoplasmas, are an economically relevant diseases affecting vineyards in the European continent). Sterile deionized water was treated by means of a nanosecond pulsed dielectric barrier discharge, operating in ambient air; 10 min treatment with a peak voltage of 19 kV and a pulse repetition frequency of 1 kHz induced in PAW the presence of nitrates, nitrites and peroxides, and a pH decrease. On tomato plants, roots were soaked in PAW before pathogen inoculation; as a consequence, a reduction of Xv disease severity was observed (relative protection of ca. 35%); moreover, PAW did not exert any phytotoxicity on tomato plants. Focusing on the grapevine plants, during 2015 preliminary trials using PAW were carried out in five Glera and one Chardonnay vineyards; nested PCR/RFLP assays were performed in October 2014 on symptomatic and asymptomatic plants to reveal the presence and identity of phytoplasmas. Experiments were performed, inoculating in three different times of the year (on April, June and July 2015) 10-20 ml of PAW or sterile distilled water (SDW, as control) on each selected phytoplasma-infected and phytoplasma-free (to investigate any possible phytotoxicity caused by PAW treatment,) plant; the remaining plants of the vineyards were used as untreated control to verify the natural behaviour of the disease. The liquids were injected into the plant vascular tissue by using specially adapted syringes, since with commercially available devices the injection time would have been too long and would have reduced PAW activity; injections were carried out at different daytimes to evaluate the grapevine absorbance ability. In a relevant number of Glera cases, the PAW treated symptomatic plants showed reduction of symptoms and a production increase, while the SDW and untreated plants did not show any symptom reduction. PCR/RFLP analyses carried out in September 2015 on leaves of PAW treated plants showed a reduction of phytoplasma positive plants (62%); it is worth noticing that a reduction (33%) of phytoplasma positive plants was observed also for plants treated with SDW. No phytotoxicity was observed in the phytoplasma-free plants treated with PAW. At this stage of the research results are very promising and have prompted us to enlarge the trials increasing the number of tested grapevine plants, in order to achieve an increase of statistically valuable data.

Plasma activated water is produced treating sterile distilled water by means of an atmospheric pressure dielectric barrier discharge. Tomato plants, belonging to different cultivars and experimentally inoculated with Xanthomonas vesicatoria under greenhouse conditions, and phytoplasma infected grapevine plants in vineyards were treated with plasma activated water in order to evaluate its effectiveness as an alternative means to control plant diseases.

Bacteria and phytoplasmas (insect-transmitted cell wall lacking bacteria), are involved in severe diseases affecting agronomic relevant crops. Management of the diseases due to these agents has mainly focused on the use of copper compound or antibiotics for bacteria, on insect vector chemical control and infected plant rouging for phytoplasmas, but all of these methods presents some drawbacks. Indeed, many efforts are devoted to find effective and environmental friendly control strategies. The use of plasma activated water (PAW), produced by a nanosecond pulsed dielectric barrier discharge, as innovative and alternative possible management tool to control plant diseases due to bacteria and phytoplasmas was experimentally exploited. The exposure of sterile distilled water (SDW) to a non-equilibrium atmospheric pressure plasma induces a reduction of pH and the production of reactive oxygen and nitrogen species (RONS), such as hydrogen peroxide, nitrates and nitrites [1] that might induce plant defense responses, involving both hypersensitive reaction and systemic acquired resistance. The effects of PAW applications were tested on three different pathosystems: tomato plants experimentally inoculated with Xanthomonas vesicatoria (Xv), phytoplasma infected periwinkle micropropagated shoots and grapevine infected plants in vineyards. Quantitative RT-PCR analyses allowed to determine the transcription level of genes involved in the plant defense response (phenylalanine ammonia-lyase, pal) and in the phytoalexin metabolism of PAW treated materials. The number of leaf spots caused by Xv in tomato plants and the number of symptomatic grapevine plants in in vineyards were significantly reduced by the treatments. Moreover, the transcriptomics results highlighted the pal gene and the genes involved in the phytoalexin production increased expression. The PAW ability to enhance some of the plant defense mechanisms improving the health status of the treated plants was experimentally demonstrated.

The bacterial plant disease control is nowadays limited to copper compounds, few resistance inducers and biocontrol agents, while prophylaxis still remains the most important management tool. In this study, the efficacy of plasma activated water (PAW) as innovative alternative in the control of plant diseases caused by bacterial pathogens is investigated. To assess the PAW efficacy as resistance inducer, tomato plants experimentally inoculated with Xanthomonas vesicatoria (Xv) were used as model. 80 ml of 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 [1]. The electrodes of the plasma source consist of aluminum foils, covered by 1 mm thick polystyrene as dielectric; one of the polystyrene dielectrics is shaped with edges in order to contain the water to be treated. The plasma forms in the 1 mm gap region between the liquid surface and the upper dielectric layer. Ten minutes treatment time with a peak voltage of 19 kV and a pulse repetition frequency of 1,000 Hz generates in plasma treated water nitrates, nitrites and peroxides and induces a reduction of pH. Plants of 'Moneymaker' and 'VF-10' tomato cultivars, were grown in pots under greenhouse condition until they reached the 3rd – 4th leaf stage. The roots of tomato plants were then soaked for 10 min in PAW, SDW (positive control) and in a solution of acibenzolar-S methyl (a resistance inducer, as negative 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. 1×107 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 low number of leaf spots with respect to those pretreated with SDW. The relative protection of PAW related to negative control was estimated in the range 30%-40%, according with the tomato cultivar tested. No phytotoxicity effect has been 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.

Antibacterial properties of plasma activated water (PAW) were investigated in two plant pathosystems: Xanthomonas vesicatoria (Xv) infected tomato plants and ‘Candidatus Phytoplasma asteris’ infected periwinkle micropropagated shoots. Bacterial leaf spot severity caused by Xv was reduced and a reduced symptomatology and lack of bacterial colony formation in PAW treated periwinkle micropropagated shoots was observed, indicating the presence of a possible interaction between PAW and these pathogens.