Following the introduction and establishment of the plant pathogenic bacterium Xylella fastidiosa (Xf) in the Apulia Region (southern Italy), olive turned to be the main host of the Salentinian bacterial strain and the majorly devastated crop. The mechanism of pathogenicity of Xf is still not completely understood and no means to cure the bacterium in the infected plants are available yet. Nevertheless, the alteration of microbial communities and effects in the expression of symptoms of Xf-infected plants is poorly studied. We are investigating the microbiome of Xf-infected olives by a shotgun metagenomic DNA sequencing approach that avoids the limitations of amplicon sequencing. Data obtained (28,333,924 and 29,096,610 reads from Xf-infected and healthy plants) were analyzed by MetaPhlAn, a metagenomic abundance estimation tool which maps reads to a set of selected marker sequences. Libraries from xylem tissues revealed a complex community in which small symbiotic bacteria of insects, i.e. Candidatus Zinderia insecticola and Candidatus Carsonella ruddii represented the 31% and 22% of the total population. Xf reaches in infected plants the 12% of the total microbial community. Studies are ongoing to characterize the microbial communities in the xylem sap of tolerant and susceptible olive cultivars, to envisage a control strategy based on the manipulation of these resident communities and to identify endosymbiont(s) which may be used to reduce the severity of symptoms. To this end, the evaluation of an endosymbiont bacterium for its potential to colonize Xf-infected olive tissues is underway.

The detection of the four grapevine viruses (GLRaV-1, GLRaV-3, GFLV and ArMV) regulated in European Union plant material certification, requires sensitive and specific diagnostic tools. A strategy of simultaneous detection in a real-time single tube amplification was developed, based on the EvaGreen binding dye. The melting curve analysis (MCA) of the amplicons allows a qualitative detection of the four different virus targets in multiplex analysis. A plasmid dilution assay calculated an analytical sensitivity with an amplification threshold up to 100 copies of the target sequences. A small cohort of field grapevine samples, with a known status of infection by mixtures of the target viruses or free of them, respectively, was successfully tested for the evaluation of the amplicons Tm.

The recent Xylella fastidiosa subsp. pauca (Xfp) outbreak in olive (Olea europaea) groves in southern Italy is causing a destructive disease denoted Olive Quick Decline Syndrome (OQDS). Field observations disclosed that Xfp-infected plants of cv. Leccino show milder symptoms, than the more widely grown and highly susceptible cv. Ogliarola salentina. To ascertain whether these field observations underlie a tolerant condition of cv. Leccino, which could be exploited for lessening the economic impact of the disease on the local olive industry, transcriptional changes occurring in plants of the two cultivars affected by Xfp were investigated. A global transcriptome profiling comparing susceptible (Ogliarola salentina) and tolerant (Leccino) olive cultivars, infected or not by Xfp, was done on messenger RNA (mRNAs) extracted from xylem tissues. The study revealed that 659 and 447 genes were differentially regulated in cvs Leccino and Ogliarola upon Xfp infection, respectively, whereas 512 genes were altered when the transcriptome of both infected cultivars was compared. Analysis of these differentially expressed genes (DEGs) shows that the presence of Xfp is perceived by the plants of both cultivars, in which it triggers a differential response strongly involving the cell wall. Up-regulation of genes encoding receptor-like kinases (RLK) and receptor-like proteins (RLP) is the predominant response of cv. Leccino, which is missing in cv. Ogliarola salentina. Moreover, both cultivars react with a strong re-modelling of cell wall proteins. These data suggest that Xfp elicits a different transcriptome response in the two cultivars, which determines a lower pathogen concentration in cv. Leccino and indicates that this cultivar may harbor genetic constituents and/or regulatory elements which counteract Xfp infection. These findings suggest that cv. Leccino is endowed with an intrinsic tolerance to Xfp, which makes it eligible for further studies aiming at investigating molecular basis and pathways modulating its different defense response.

The quarantine bacterium Xylella fastidiosa (Xf) is responsible for diseases of a wide range of cultivated and wild plants. Few ef- forts have been made to investigate the potential use of endophytic symbionts on the disease phenotype of Xf-infected plants. The aim of our study was to evaluate if Paraburkholderia phytofirmans PsJN strain, a plant growth-promoting rhizobacterium, whose beneficial effects in the reduction of symptom severity caused by Xf in grape- vine affected by Pierce's Disease have recently been proven, may play a role as biocontrol agent against Xf CoDiRO strain, the agent of a severe disease of olives in Apulia (southern Italy). Greenhouse trials are being conducted to test the ability of P. phytofirmans to colonise xylem vessels of olive, Nicotiana benthamiana and oleander, following inoculation of bacterial suspensions by needle puncture and root dipping. A conventional PCR assay for detection of P. phytofirmans movement in plants has been developed to be used in combination with plate isolation and a qPCR specific assay. Preliminary results showed that needle-inoculated bacterial cells were detectable in the leaf petioles of the three hosts, away from the inoculation site. Root dipping proved successful in infecting in vitro-cultured olive plantlets. Double-infection assays, currently underway, will prove if P. phytofirmans PsJN shows a beneficial interaction with Xf CoDiRO.

The recent findings of the plant pathogenic bacterium Xylella fastidiosa (Xf), infecting several plant species in Italy and France, raised major concerns for its potential impact on the EU and Mediterranean agriculture. In the current EU outbreaks, olive is the predominantly affected crop, in which the bacterium has been consistently associated with a new severe syndrome, denoted Olive Quick Decline". So far, no effective treatments are available to cure infected plants. However, several approaches have been explored, mainly in grapevine and citrus, to reduce bacterial movement and multiplication or directly targeting Xf-cells for lysis.Current knowledge shows that the virulence of the pathogen relies on a fine balance between more motile bacterial forms, able to move and proliferate within xylem vessels, and sticky cells forming a biofilm, which are responsible for vessels blockage and insect acquisition. This different behavior is regulated in a cell density-dependent manner by a diffusible signaling factor (DSF), produced by rpfF-gene, that initiates a transduction cascade resulting in up- or down-regulation of several genes.The aim of our investigation is to explore "pathogen confusion" strategy, by altering DSF level in planta, for reducing the impact of Xf-infections in olives. To this end, a plant viral-based vector, harboring the rpfF-gene, has been engineered to induce transient DSF production. Experiments will verify if, upon DSF accumulation, the bacterium will be less motile and more adhesive to the surface of xylem vessels, thus showing a decreased virulence in infected plants.

A quince tree showing severe symptoms of a previously undescribed viral disease occurring in northern Apulia (Italy) was analysed using high-throughput sequencing of small RNA libraries, leading to the identification of a new strain of apple green crinkle associated virus (isolate AGCaV-CYD) showing peculiar traits. RT-PCR with specific primers detected AGCaV-CYD in consistent association with symptoms in the surveyed orchards. Molecular characterization of the reconstructed genome, together with phylogenetic analysis, showed it to be closely related to an AGCaV strain causing green crinkle disease in apple (AGCaV-AUR) and divergent from the type strain of apple stem pitting virus (ASPV-PA66).

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.

Plant pathogens cause significant losses to agricultural yields and increasingly threaten food security, ecosystem integrity and societies in general. Xylella fastidiosa is one of the most dangerous plant bacteria worldwide, causing several diseases with profound impacts on agriculture and the environment. Primarily occurring in the Americas, its recent discovery in Asia and Europe demonstrates that X. fastidiosa's geographic range has broadened considerably, positioning it as a reemerging global threat that has caused socioeconomic and cultural damage. X. fastidiosa can infect more than 350 plant species worldwide, and early detection is critical for its eradication. In this article, we show that changes in plant functional traits retrieved from airborne imaging spectroscopy and thermography can reveal X. fastidiosa infection in olive trees before symptoms are visible. We obtained accuracies of disease detection, confirmed by quantitative polymerase chain reaction, exceeding 80% when high-resolution fluorescence quantified by three-dimensional simulations and thermal stress indicators were coupled with photosynthetic traits sensitive to rapid pigment dynamics and degradation. Moreover, we found that the visually asymptomatic trees originally scored as affected by spectral plant-trait alterations, developed X. fastidiosa symptoms at almost double the rate of the asymptomatic trees classified as not affected by remote sensing. We demonstrate that spectral plant-trait alterations caused by X. fastidiosa infection are detectable previsually at the landscape scale, a critical requirement to help eradicate some of the most devastating plant diseases worldwide.

Gli istituti di ricerca pugliesi riportano i risultati ottenuti dall'impiego di attivatori delle difese della pianta e dell'N-acetilcisteina per il contenimentodei sintomi del complesso del disseccamento rapido dell'olivo.

Mulberry is a deciduous tree belonging to the Moraceae family. Although its economical importance and spreading all over the world, due mainly to the domesticated silkworm (Bombyx mori L.) breeding, to date, only few information are available about viral and virus-like diseases affecting this plant. In 2012 a small fragment of a Badnavirus DNA genome was sequenced after a DOP-PCR assay conducted on a mulberry plant, originated from Lebanon, showing symptoms of leaf mottling and vein yellowing. The virus, whose particles were observed with electron microscopy from a partially purified preparation and in tissue thin sections, was provisionally named Mulberry badnavirus-1 (MBV-1). Since different attempts of completing the full-length genome sequence through a conventional approach failed, a small RNA library was constructed for deep sequencing and run according to Illumina protocol ssRNAs analysis allowed the design of a set of primers used in PCR for the achievement of the full length sequence. The complete genome contains all the sequence features and the characteristic functional domains of the genus Badnavirus (highest nucleotide similarity shared with FBV-1 at 54%). By contrast to the badnaviruses, with genomes encoding for 3-4 ORFs, MBV-1 resembles genome organization of Petunia vein clearing virus (PVCV), which bears a single ORF. The study of the distribution of sRNA on the MBV-1 complete genome showed a tidy prevalence of 21- and 22-nt reads, differently from other viruses in the Caulimoviridae family, featuring a nuclear replication and typically supporting an accumulation of the 24-nt sRNAs involved in methylation processes.

Xylella fastidiosa (Xf) is a xylem-limited bacterium, regulated as a quarantine pest, that is causing a devastating disease on olive crops in the southern area of Apulia (Italy) and whose potential spread in the Mediterranean area poses a severe threat to EU agri- culture and landscape environment. Xf virulence is related to the expression of a cluster of rpf (regulation of pathogenicity factors) genes responsible for a signalling system based on small fatty acid molecules called DSF (Diffusible Signalling Factor). Since DSF regulation is involved in pathogenicity traits of Xf and biofilm for- mation, a "pathogen-confusion" strategy, based on the alteration of DSF levels in planta, has been proposed to contrast bacterial infection. In grapevine, the strategy is based on the transgenic ex- pression of the rpfF gene, which encodes the DSF-synthase. We are attempting to express the rpfF gene of the olive-infecting Xf strain CoDiRO in the heterologous Escherichia coli system. The gene product has been successfully detected by Western blot analysis in cell protein extracts. Chemical characterisation by Gas Chromatog- raphy-Mass Spectrometry analysis of the DSF molecules produced by this expression system, in addition to those naturally produced by Xf CoDiRO, are underway. Concurrently, a TMV-based vector has been engineered to harbour the same rpfF gene and induce its transient expression in planta. Biologically active transcripts of the vector have been inoculated to Nicotiana tabacum and N. ben- thamiana plants, to establish a model system on herbaceous hosts. RpfF expression was successfully proved by Western blot analysis, whereas movement and systemic colonisation of plant tissues were evaluated by RT-PCR assays. The same viral vector harbouring GFP in replacement of rpfF is used as a control. Following inoculation with Xf CoDiRO bacterial cells the system is now being tested to monitor the persistence of DSF expression and its efficacy to lower disease susceptibility or movement of bacterial cells behind the point of inoculation.

The CoDiRO strain of Xylella fastidiosa subsp. pauca (Xfp) is ravaging olive (Olea europaea) groves in southern Italy, causing a destructive disease denoted Olive Quick Decline Syndrome (OQDS). Field observations show that the Xfp-infected plants of the cv. Ogliarola salentina develop more severe symptoms than that of cv. Leccino. A global transcriptome profiling comparing the two olive cultivars, infected or not by Xfp, was performed to ascertain whether a tolerant condition of cv. Leccino exists, which could be exploited for lessening the economic impact of the disease on the local olive industry. The study revealed that 659 and 447 genes were differentially regulated upon Xfp infection, in cvs Leccino and Ogliarola salentina, respectively, whereas 512 genes resulted altered between the two infected cultivars. The analysis showed that plants of both cultivars perceive the presence of Xfp, mainly involving cell wall-associated proteins. The predominant response of cv. Leccino, which is missing in cv. Ogliarola salentina, consists on the up-regulation of genes encoding receptor-like kinases and receptor-like proteins. This different transcriptome response determines a lower pathogen concentration in the cv. Leccino, suggesting that it may harbor genetic constituents and/or regulatory elements which counteract Xfp infection. These findings suggest that cv. Leccino is endowed with an intrinsic tolerance to Xfp, which makes it eligible for further studies aimed at investigating molecular pathways underlying its different defense response.

The recent Xylella fastidiosa subsp. pauca (Xfp) outbreak in olive (Olea europaea) groves in southern Italy is causing a destructive disease denoted Olive Quick Decline Syndrome (OQDS). Field observations disclosed that Xfp-infected plants of cv. Leccino show much milder symptoms, than the more widely grown and highly susceptible cv. Ogliarola salentina. To determine whether these field observations underlie a tolerant condition of cv. Leccino, which could be exploited for lessening the economic impact of the disease on the local olive industry, transcriptional changes occurring in plants of the two cultivars affected by Xfp were investigated. Results: A global quantitative transcriptome profiling comparing susceptible (Ogliarola salentina) and tolerant (Leccino) olive cultivars, infected or not by Xfp, was done on messenger RNA (mRNAs) extracted from xylem tissues. The study revealed that 659 and 447 genes were differentially regulated in cvs Leccino and Ogliarola upon Xfp infection, respectively, whereas 512 genes were altered when the transcriptome of both infected cultivars was compared. Analysis of these differentially expressed genes (DEGs) shows that the presence of Xfp is perceived by the plants of both cultivars, in which it triggers a differential response strongly involving the cell wall. Up-regulation of genes encoding receptor-like kinases (RLK) and receptor-like proteins (RLP) is the predominant response of cv. Leccino, which is missing in cv. Ogliarola salentina. Moreover, both cultivars react with a strong re-modelling of cell wall proteins. These data suggest that Xfp elicits a different transcriptome response in the two cultivars, which determines a lower pathogen concentration in cv. Leccino and indicates that this cultivar may harbor genetic constituents and/or regulatory elements which counteract Xfp infection. Conclusions: Collectively these findings suggest that cv. Leccino is endowed with an intrinsic tolerance to Xfp, which makes it eligible for further studies aiming at investigating molecular basis and pathways modulating its different defense response.

Grapevine Pinot gris virus (GPGV) is a new virus reported in Europe and several other grape-growing countries. In an attempt to identify a vector for GPGV, samples of the eriophyid mite Colomerus vitis collected from buds and erinea in GPGV-infected vines were analysed by RT-PCR, using specific primers. Molecular analysis revealed the presence of GPGV in C. vitis. Transmission trials were conducted using C. vitis collected from GPGV-infected vines. Mites were able to transmit GPGV to healthy grapevines, suggesting that C. vitis is a potential vector of this virus.

Mulberry badnavirus 1 (MBV1) has been characterized as the aetiological agent of a disease observed on a mulberry tree in Lebanon (accession L34). A small RNA next-generation sequencing library was prepared and analysed from L34 extract, and these data together with genome walking experiments have been used to obtain the full-length virus sequence. Uniquely among badnaviruses, the MBV1 sequence encodes a single ORF containing all the conserved pararetrovirus motifs. Two genome sizes (6 kb and 7 kb) were found to be encapsidated in infected plants, the shortest of which shares 98.95% sequence identity with the full L34 genome. In the less-than-full-length deleted genome, the translational frame for the replication domains was conserved, but the particle morphology, observed under electron microscopy, was somehow altered. Southern blot hybridization confirmed the coexistence of the two genomic forms in the original L34 accession, as well as the absence of cointegration in the plant genome. Both long and deleted genomes were cloned and proved to be infectious in mulberry. Differently from other similar nuclear-replicating viruses or viroids, the characterization of the MBV1-derived small RNAs showed a reduced amount of the 24-mer class size.