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According to recent statistics, in Italy a significant spread of the bacterial canker of Actinidia caused by Pseudomonas syringae pv. actinidiae (Psa) is recorded with peaks of 70 and 100%, and lower percentages ranging around 50%. The control of the Psa is a priority especially considering the hazard represented by the endophytic bacterial presence in nursery material. To evaluate the risk that could be associated with micropropagated shoots when using asymptomatic mother plants, Psa survival was studied in micro-propagated shoots of A. deliciosa 'Hayward' inoculated with different concentrations of a virulent Psa gfp-expressing/rifampicin-resistant strain (Psagfp-Rif(res)). Microbiological and molecular analysis were carried out at each in vitro transfer and on rooted plantlets in greenhouse. The Psagfp-Rif(res) reisolation on selective media confirmed by PCR analysis and by the ability to induce symptoms in Actinidia and HR in tobacco plants, was achieved in both the micropropagated materials and in the plants analyzed in toto after their re-establishment. A reliable and efficient method for Psa detection in nursery material is proposed to verify the presence Psa infection before symptom development.

Monarda spp. are promising Lamiaceae due to essential oil (EO) composition and antimicrobial activity. To investigate these properties, Monarda didyma and M. fistulosa were grown for two years in two Italian sites for EO extraction (distillation), analysis (GC and GC/MS), and antimicrobial activity (microplate diffusion broth method). EO yield greatly increased from first to second year of cultivation (0.27 and 0.51% in 2013 and 2014, respectively), showing minor differences between M. didyma and M. fistulosa. Conversely, EO composition significantly varied between the two species and years. Of the fifteen major compounds identified, eight were shared by the two species. However, the amounts differed, especially in thymol (62 vs. 31% in M. didyma vs. M. fistulosa) opposed to α- and β-phellandrene (their average, 1% in M. didyma and 16% in M. fistulosa). An increase of thymol was observed between 2013 and 2014 in both species (average, +11%), reflecting juvenile plants showing only vegetative organs (2013) vs. fully flowered plants (2014). Despite such differences, EO’s from the two Monarda species exhibited a similar activity against pathogenic and non-pathogenic strains of fungi and bacteria from human, animal and plant source. In general, Monarda EO’s showed a lower minimum inhibitory concentration, i.e. a stronger activity, against pathogenic (Escherichia coli, Erwinia amylovora and Candida albicans) than beneficial microorganisms (Bifidobacterium animalis and Lactobacillus casei). The higher susceptibility of pathogenic microorganisms supports the use of Monarda EO’s as antimicrobial agents with a favourable profile of selectivity. Applications are envisaged in food preservation, plant protection and human health. © 2017 Har Krishan Bhalla & Sons.

To tackle the epidemiological role of the latency phase of Pseudomonas syringae pv. actinidiae (Psa) in susceptible asymptomatic host plants, the pathogen survival and colonization were studied in seven-year-old plants of Actinidia chinensis ‘Hort16A’. The plants were inoculated with a virulent Psa gfp-expressing/rifampicin-resistant strain (Psagfp-Rifres) and the endophytic presence of Psa was determined by analysis of the whole plants to re-isolate Psagfp-Rifres and by PCR assays to confirm identity. Finally, the isolates were tested to verify their ability to induce disease symptoms and HR respectively in host and non-host plants. The Psagfp-Rifres presence inside the tissues of the experimentally contaminated plants was detected during the years following the inoculation. The data obtained showed that systemic colonization of host tissues by Psagfp-Rifres took place for a long period of time. The epidemiological significance of this finding raises questions about the effectiveness of the control measures to prevent bacterial canker solely based on antimicrobial treatment on plant surfaces.

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.

The purpose of this study was to investigate the effect of dietary vitamin E or EconomasE™ supplementation on the growth of several background/pathogenic bacteria on rabbit carcasses and hamburgers during refrigerated storage. For 51days, 270 New Zealand rabbits received either a basal diet, or experimental diets enriched with 100 or 200mg/kg of vitamin E or EconomasE™. The bacteria studied were Salmonella, Listeria monocytogenes, Pseudomonas, Enterobacteriaceae, Escherichia coli, coagulase-positive staphylococci, plus both mesophilic and psychrotrophic aerobes. The growth of Listeria monocytogenes on contaminated patties was evaluated through a challenge test. The potential protective or antimicrobial effect of vitamin E or EconomasE™ on Listeria monocytogenes or Pseudomonas aeruginosa was assessed in vitro. Diet did not influence the concentrations of bacteria found on rabbit carcasses and developing on hamburgers. Vitamin E (in vivo and in vitro) and EconomasE™ in vivo had a protective antioxidant role, while EconomasE™ in vitro had strong antibacterial activity against Listeria monocytogenes, but not against Pseudomonas aeruginosa.

During 1997, in occasion of the severe epidemic caused by Erwinia amylovora (Ea) on pear-tree in Emilia-Romagna, the first certified cases of fire blight were also reported in Veneto. Of the 14 ascertained foci, 11 were found in Rovigo province, ie. in areas facing the northern border of Emilia-Romagna and 2 were found in Padua and Verona provinces. In the following two years, the territorial distribution of the new foci in Verona, Padua and Venice provinces clearly pointed out that from Venetian areas close to the border with Emilia-Romagna the Ea was spreading mostly towards the north-west. In this study, the AFLP genomic analysis of 14 isolates from pear-tree showed that all Venetian isolates were referred to the same clone originally coming from Emilia-Romagna. After 1997 in Emilia-Romagna there has been a substantial increase in the official cases of fire blight on host plants other than pear-tree. From the clonal lineage of Ea, introduced in 1994, may originate secondary lines different for pathogenic traits selected by the new host plant or a new clonal lineage could have occurred. The AFLP of the Ea strains isolated from host plants other than pear-tree was carried out in order to study the genetic variability between the Emilian strains which were found to belong to the original clone. Moreover, to test the hypothesis of continuity of the original clonal lineage, the changes of the virulence grade among the strains was studied. The pathogenicity test on immature pear fruitlets was performed measuring the minimum/maximum axis of the lesion induced by each strain (14 Venetian and 47 Emilian strains). The strain OMP-BO1077/7, associated to the primary foci in Emilia-Romagna on pear during 1994, was used as a reference strain. For each strain the virulence index was calculated and statistical analysis was used to determine the significant difference with the reference strain

Eight strains of the mycoparasite Coniothyrium minitans were analysed for intraspecific variation by three different approaches: analysis of morpho-physiological characteristics; random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analysis of the genomic DNA. A high variability in colony colour, gross morphology and conidial size was found depending on strain and culture conditions. All individual strains could be distinguished using the three type of analyses. However, a close correspondence between RAPD, AFLP patterns and morpho-physiological characteristics of C. minitans strains was not observed. Our results suggest AFLP analysis constituted a more efficient and reliable tool than RAPD analysis or morpho-physiological studies to detect intraspecific variability in C. minitans and to follow its fate after application in the field.

Infiltration of protein-lipopolysaccharide complexes (prLPS) (250 Cg/ml) of Pseudomonas syringae pv. aptata into tobacco leaves protected against subsequent elicitation of the hypersensitive response (HR) by Erwinia amylovora. The effect of prLPS on the expression of E. amylovora hrp (hypersensitive response and pathogenicity) genes was tested in protected tobacco leaves and in a defined medium in which hrp genes were highly expressed. Two E. amylovora hrp loci transcriptionally fused with the Escherichia coli β-glucuronidase (Gus) coding sequence were used as chromosomal reporter genes. The prLPS treatment did not affect hrp gene expression of Hrp − mutants both in planta and in vitro, so the effect of prLPS treatment on hrp gene expression of two E. amylovora Hrp + transformants was assayed during HR development in unprotected tissue and during the same time in prLPS protected tissue. A plasmid containing the same Gus fusions previously located in the chromosome was introduced into a wild-type strain of E. amylovora. Gus activity was significantly lower in protected tissue. We suggest that HR inhibition by prLPS treatment requires the entire hrp gene cluster in order for the bacteria to send a signal to the plant, which, in turn, inhibits the expression of hrp genes

Autumn infections of pear scions through wounds in nursery might be responsible of latent infections and the infected plants could not be recognized by the nurserymen during the uprooting and selection by late autumn. Consequently, symptomless pear scions harbouring cells of Erwinia amylovora sold at the end of the winter can introduce the pathogen in the farm where the new plantation is set up. In this research work the E. amylovora survival in symptomless scions was investigated to evaluate if it were compatible with the role of primary source of inoculum in new plantations. 274 pear scions were contaminated in autumn through a pruning cut in the main stem with a virulent strain resistant to rifampicin. The young pear plants grown singularly in pots were inspected for two consecutive vegetative seasons and after one and two years, pieces of stem and shoot (10 cm in length) were cut in slices (2-3 mm in thickness) and analyzed to detect the presence of the antibiotic resistant strain. The pear plants were symptomless in both vegetative seasons. The rifampicin resistant strain was re-isolated after one year (8 out of 51), but not after two years (0 out of 146). Several pear plants used as a control excluded the endophytic presence of local strains, and the absence of an environmental inoculum. The results of this and of an analogous experiment indicate that latent infections of scions may cause visible infections during the months following the plant out, but not in the following year

The survival of Erwinia amylovora during cold storage or in the open air might be a relevant factor in the spread of fire blight. Pear fruits, the wooden or plastic container surfaces and the packaging paper may be vehicles for the spread over short, medium and long distances associated with the marketing of fruit, the movement of bins and trays, and the disposal of packaging paper after storage. Survival in cold storage (-0.5°C) and/or in the open air of a rifampicin-resistant strain was studied on pears, bins, trays and packaging paper. The surfaces of highly standardised samples were contaminated with a bacterial suspension. The washing liquid was centrifuged and the final concentrate was quantitatively streaked on selective medium. Although it was no longer possible to isolate the bacteria from the pear skin after 24 hours, survival in the calycine cavity reached at least 101 days. Survival was (considerable) on oak (20–30 days) and poplar wood (40–60 days); maximum survival was estimated at 60–70 days and 70–80 days respectively. After 24 hours the bacteria could no longer be isolated from polyvinyl chloride. Survival on packaging paper was 14-24 days. The grey paper used to line the sides was less conducive to survival than that used for the bottom. The survival curves were calculated for each type of material. An estimation of phytosanitary risk periods for the transmission of Ea based on the critical time of survival is hypothesised.

Periplasmic oligosaccharides, extracted from Pseudomonas syringae pv. aptata and purified by column chromatography, modify the homologous interaction between tobacco leaves and P. syringae pv. tabaci. The osmoregulating oligosaccharides can act as signal molecules to plant cells, but the molecular basis of this function is not known. Genetical analysis of pathogenicity determinants in P. s. pv. aptata strain NCPPB2664 allowed the identification of hrp genes which are essential in governing the ability of the bacteria to cause disease on host plants (such as sugar beet, lettuce), and the ability to induce the hypersensitive response on non-host plants (such as tobacco). The current study addresses whether a correlation between the pathogenicity and the production of oligosaccharides exists. To test the hypothesis that in the early stages of plant-bacteria interactions the hrp genes might be involved in the production of the periplasmic oligosaccharides, the latter has been studied in P. syringae pv. aptata Hrp- mutants.

The hypothesis of superoxide anion generation as a direct bactericidal mechanism exercised by hypersensitive tobacco leaf tissue against an incompatible phytopathogenic pseudomonad was tested. The detection of O2− was made directly by reducing Cytochrome c and by producing nitrite from hydroxylamine chloride and indirectly by measuring the death of endophytic bacteria in the presence of superoxide dismutase. At 4 h and 6 h the low net specific reduction of Cytochrome c in the hypersensitive tissue was not confirmed by the specific production of nitrite. At the same time active superoxide dismutase did not give significant protection to the incompatible bacteria as compared to inactive superoxide dismutase. The protection provided by active superoxide dismutase as compared with the controls (bovine serum albumine treated tissue and non-treated tissue) at 4 h and at 6 h was not correlated to its superoxide anion scavenger activity. The results indicated that the death of the incompatible pseudomonad during the hypersensitive reaction was not directly linked to O2− generation.