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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.

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.

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.

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.

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.

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.

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