Explore the vast range of titles available.

In autumn 2013, the presence of Xylella fastidiosa , a xylem-limited Gram-negative bacterium, was detected in olive stands of an area of the Ionian coast of the Salento peninsula (Apulia, southern Italy), that were severely affected by a disease denoted olive quick decline syndrome (OQDS). Studies were carried out for determining the involvement of this bacterium in the genesis of OQDS and of the leaf scorching shown by a number of naturally infected plants other than olive. Isolation in axenic culture was attempted and assays were carried out for determining its pathogenicity to olive, oleander and myrtle-leaf milkwort. The bacterium was readily detected by quantitative polymerase chain reaction (qPCR) in all diseased olive trees sampled in different and geographically separated infection foci, and culturing of 51 isolates, each from a distinct OQDS focus, was accomplished. Needle-inoculation experiments under different environmental conditions proved that the Salentinian isolate De Donno belonging to the subspecies pauca is able to multiply and systemically invade artificially inoculated hosts, reproducing symptoms observed in the field. Bacterial colonization occurred in prick-inoculated olives of all tested cultivars. However, the severity of and timing of symptoms appearance differed with the cultivar, confirming their differential reaction.

After the first confirmed outbreak of Xylella fastidiosa in the European Union (EU), associated with an olive disease denoted olive quick decline syndrome, mandatory surveys are now carried out in the member States and inspections increased at EU entry points such as ports. Such activities led to the interception of X. fastidiosa -infected coffee plants in consignments originating from Central America. Similarly, the geographic expansion of the olive decline epidemic area of the Apulia region (southern Italy) prompted investigations to identify new host plants. Here we report the interception of three novel bacterial sequence types in Italy, based on multi-locus sequence typing, that cluster with different X. fastidiosa subspecies, illustrating the risk of the introduction of additional pathogen genetic diversity into Europe. In the epidemic area of Apulia, new foci as well as host plant species positive with X. fastidiosa , including cherry, myrtleleaf and rosemary, were found to be all infected with the same sequence type of this bacterium (ST53, or CoDiRO strain). This work highlights the limited knowledge of X. fastidiosa phylogenetic and phenotypic diversity, the risk of novel X. fastidiosa introductions via contaminated plant material, and corroborates other studies indicating that the Apulia epidemic emerged from a single introduction of this pathogen into the region.

The isolation in pure culture of the Xylella fastidiosa strain associated with the quick decline syndrome of olive, recently observed in Apulia (Salento peninsula, southern Italy) was attempted from symptomatic, naturally infected olive and oleander plants, and a periwinkle seedling that had been exposed to, and was infected by Xylella-positive spittlebugs. Prior to isolation, the presence of Xylella was ascertained in all donor hosts by PCR, indirect immunofluorescence and electron microscopy. Isolations from olive failed because of the heavy contamination by bacteria other than Xylella. By contrast, pure bacterial cultures were obtained from oleander and periwinkle extracts plated in periwinkle wilt gelrite (PWG) and buffered cysteine-yeast extract (BCYE) media. In both media, colonies were slow-growing, small-sized (less than 1 mm 25 days from plating), non pigmented, opalescent and exhibited the same morphology, except for the margin that was entire in BCYE and somewhat irregular in PWG. Bacterial cells were rod-shaped with rounded ends, had a thick and rippled cell wall, an average width of 0.35 μm, and a maximum length of ca. 5 μm. They gave a positive reaction in immunofluorence assays and were clearly decorated by colloidal gold in immunogold labelling tests. Sequenced PCR products amplified from periwinkle and oleander colonies shared 97-99% sequence identity with known X. fastidiosa strains from database and were 100% identical to one another and to comparable sequences obtained from infected olive trees. These sequences grouped in a distinct cluster of a branch comprising X. fastidiosa isolates belonging to the subspecies pauca.

Xylella fastidiosa (Xf) is a xylem-inhabiting, vector-transmitted bacterium and the agent of a variety of diseases in a broad range of plant hosts. A destructive Xf outbreak has recently emerged in some olive groves of the province of Lecce (south-eastern Italy), caused by a strain (called CoDiRO from the Italian for Rapid desiccation complex of olive) of Xf subsp. pauca, a pathogen known to infect coffee and citrus in South America. Taking into account that this is the first confirmed record of the presence of Xf in the European Union (EU) territory and fairing its possible spread from the affected site, the EU has issued the Commission Implementing Decision L 45/29-31 (February 24, 2014), which prohibits the transfer from the province of Lecce of all plant genera and species not listed in the Annex I of the cited Decision. A survey was therefore initiated to verify the health status of a number of ornamental and forestry plants growing mostly in nurseries, but also in public and private gardens, which are or can be naturally exposed to high inoculum pressure. Samples collected from 207 conifers, 105 members of the family Arecaceae, Musaceae and Cycadaceae, and 208 succulent plants (totalling 520) were comparatively examined by serological (DAS-ELISA) and molecular (PCR) methods. None of the tested plant species proved to be infected, providing a strong indication that they may not be susceptible to field infection by the Xf subsp. pauca strain currently present in the province of Lecce.

A survey on viruses infecting olive trees was performed in Croatian Istria. Major olive viruses were detected using molecular (RT-PCR), biological (bioassay on indicator plants) and serological (DAS-ELISA) diagnostic techniques. Specifically, fifteen olive varieties from a total of 62 olive trees have been tested for the presence of eight viruses. Cherry leaf roll virus (CLRV), Strawberry latent ring spot virus (SLRSV) and Olive latent virus-1 (OLV-1) were detected by one-step RT-PCR in ten autochthonous and five introduced olive varieties. The most frequent was CLRV, detected in 11.3% of tested trees both in autochthonous and in introduced varieties, while OLV-1 and SLRSV were detected only in autochthonous varieties and in lower incidence. In order to confirm the results obtained by one-step RT-PCR, inocula prepared from the flowers were used for inoculation of herbaceous indicator plants. Symptoms on herbaceous hosts were monitored and CLRV and SLRSV were detected by DAS-ELISA, confirming the results of molecular test. The most successful mechanical transmission was on Chenopodium quinoa, while the most evident symptoms were observed on Cucumis sativus L. cv. Sunčani potok plants.

A dot-blot hybridization protocol using digoxigenin-labelled riboprobes was finalized for the detection of Citrus psorosis virus (CPsV) and Citrus variegation virus (CW). Both viruses were readily identified in different organs of screenhouse-grown and, throughout a 9-month period, in-field-grown citrus plants. With CPsV, strong hybridization signals were obtained by dot blot hybridization from flowers (ovaries) and young leaves and, with CVV, from young leaves and shoots. In tissues prints, CPsV was satisfactorily detected from ovaries and CVV from petioles, ovaries, young leaves and tender shoots. The sensitivity threshold of the assay was determined to be 15 pg of in vitro transcripts for both CPsV and CVV, allowing detection of both viruses even when their titres decreased in plant tissues and ELISA tests failed.

Xylella fastidiosa has recently been identified in the Apulian province of Lecce (south-eastern Italy) in olive trees affected by a devastating disease denoted Olive Quick Decline Syndrome (OQDS), that appeared suddenly in 2010. Symptoms of OQDS consist of withering and desiccation of scattered terminal shoots, which rapidly expands to the rest of the canopy, and results in the collapse and death of the tree. The identification of X. fastidiosa in OQDS-affected trees represents the first confirmed detection of this bacterium in the European Union (EU), but its exact role in the aetiology of this disease is yet to be determined. Since X. fastidiosa is a regulated quarantine pathogen in the EU, upon request of the Apulian Plant Protection Service, surveys were initiated in order to delineate the contaminated area. To this effect, diagnostic protocols based on ELISA and conventional PCR for X. fastidiosa detection in olive samples were compared and validated via an interlaboratory ring-test in which three accredited laboratories, all located in Italy, participated. Both procedures proved to be equally effective but, due to the simplicity of sample preparation, ELISA was chosen for the large-scale X. fastidiosa monitoring programme now in progress.

In 2005 a survey was conducted in the main citrusgrowing areas of Epirus. Commercial groves and nurseries were inspected for symptoms of virus and viruslike diseases and a total of 123 samples were collected. Molecular hybridisation was used to test for Citrus tristeza virus (CTV), Citrus psorosis virus (CPsV), Citrus infectious variegation virus (CW), Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd). Although the results are from a low number of samples, they give a significant insight into the sanitary conditions of the Greek citrus industry, disclosing the presence of CTV mild strains in the North-western area.

Polyclonal sera raised to Escherichia coli-expressed movement proteins encoded by ORF 3 (p8K) and ORF 4 (p6K) of olive latent virus 1, were used for their immunodetection in infected Nicotiana benthamiana plants. In subfractionated locally infected tissues 4 days post inoculation (d.p.i.) that were analysed by Western blot, p8K was found in the fast-sedimenting fractions P1 and P30 containing membranous material and/or cell organelles and, likely, the fibrous structures mentioned below, but not in the soluble protein-containing supernatant. No p6K could be detected in these extracts. In locally inoculated leaves p8K began to accumulate from 2 d.p.i onwards reaching its peak at 4 d.p.i. Intracellular immunogold labelling of cells from locally and systemically infected tissues localized p8K primarily in fibrous inclusions made up of thin filaments with a helical structure present in the cytoplasm of locally and systemically infected cells. In systemic infections a light and scattered labelling was observed in the cytoplasm and near the cell wall. The specific serum to p6K did not label the fibrous structures and failed to recognize its antigen in systemically and locally infected tissues except at 4 d.p.i., when scattered labelling was observed in the cytoplasm and near plasmodesmata.