The draft genome sequence of Xylella fastidiosa CO33 isolate, retrieved from symptomatic leaves of coffee plant intercepted in northern Italy, is reported. The CO33 genome size is 2,681,926 bp with a GC content of 51.7%.

We determined the draft genome sequence of the Xylella fastidiosa CoDiRO strain, which has been isolated from olive plants in southern Italy (Apulia). It is associated with olive quick decline syndrome (OQDS) and characterized by extensive scorching and desiccation of leaves and twigs.

Molecular features and genomic organization were determined for Citrus yellow vein clearing virus (CYVCV), the putative viral causal agent of yellow vein clearing (YVC) disease of lemon trees, reported in Pakistan, India and more recently in Turkey and China. CYVCV isolate Y1 from Adana, Turkey, was used for deep sequencing analysis of the virus induced small RNA fractions and for mechanical and graft inoculation of herbaceous and citrus indicator plants. A polyclonal antiserum was developed from CYVCV-Y1 purified from Phaseolus vulgaris and used in western blot assays to characterize the coat protein of CYVCV-Y1 and determine its serological relationship with related viruses. Contigs assembled from the Illumina sequenced short reads was used to construct the whole genome of CYVCV, consisting in a positive-sense RNA of 7,529 nucleotides and containing six predicted open reading frames. The CYVCV genome organization and size resembled that of flexiviruses, and search for sequence homologies revealed that Indian citrus ringspot virus (ICRSV) (Mandarivirus, Alphaflexiviridae) is the most closely related virus. However, CYVCV had an overall nucleotide sequence identity of ~74% with ICRSV. Although the two viruses were similar with regard to genome organization, viral particles and herbaceous host range, CYVCV caused different symptoms in citrus and was serologically distinct from ICRSV. Primer pairs were designed and used to detect the virus by conventional and quantitative RT-PCR on YVC symptomatic field trees as well as graft- and mechanically inoculated host plants. Collectively, these data suggest that CYVCV is the causal agent of YVC disease and represents a new species in the genus Mandarivirus.

The advent of next generation sequencing (NGS) technologies dramatically advanced our ability to comprehensively investigate diseases of unknown etiology and expedited the entire process of virus discovery, identification, viral genome sequencing and, subsequently, the development of routine assays for new viral pathogens. Unlike traditional techniques, these novel approaches require no preliminary knowledge of the suspected virus(es). Currently, the RNA-Seq approach has been widely used to identify new viruses in infected plants, by analyzing virus-derived small interfering RNA populations, single- and double-stranded RNA (dsRNA) molecules extracted from infected plants. The method generates sequence in an unbiased fashion, likely allowing to detect all viruses that are present in a sample. We applied the Illumina NGS, coupled with metagenomic analysis, to generate large sequence dataset in different woody crops affected by diseases of unknown origin or infected with uncharacterized viruses or new strains. This approach allowed the identification of five novel viral species and, in addition, the sequencing of the whole genome of several viruses and viroids infecting Citrus spp., Prunus spp., grapes, fig, hazelnut, olive, persimmon and mulberry. Combined analysis of the datasets generated by using either siRNA fractions and dsRNA templates, enhanced the characterization of the whole virus-derived sequences in the infected tissues. Furthermore, profiling small RNAs from virus-infected plants led to a better understanding of host-plant response to virus and viroid infections in perennial plants. A general bioinformatic pipeline and an experimental validation strategy were developed and its application illustrated.

Citrus tristeza virus (CTV) is a filamentous virion (genus Closterovirus, family Closteroviridae) that contains a single-stranded, positive-sense RNA genome of 19.3 kb consisting of 12 open reading frames (ORFs). CTV is the responsible of the current devastation of cultivated citrus trees especially of the widely used rootstock sour orange (Citrus aurantium), which appears to be one of the most susceptible species causing severe economic losses. Moreover, the relative species of trifoliate orange (Poncirus trifoliata) appears to be resistant to the disease while, the Citrange carrizo, derived from the cross between C. sinensis L. x P. trifoliate, appears to be tolerant. The resistance to the virus is due to the presence of a locus, called Ctv locus, which is available an accurate genetic map. Eight retrotransposons, 61 Simple Sequence Repeats (SSRs), more than 400 Miniature Inverted-repeats transposable Elements (mites) and several genes, with a high homology sequence to known function genes of other species are present into the Ctv locus. Among these the Ctv.20 gene showed homology with a plant virus movement-like protein. This membrane protein is involved, through the formation of channels in plasmodesma, in the protein transfer and viral RNA movement from cell to cell. The aim of the present work was to carry out different siRNA 21:24 nucleotides libraries by Illumina sequencing, from susceptible plants of sour orange and tolerant of Citrange carrizo ones, both healthy and infected with different strains of CTV virus. The presence of a homologous locus in susceptible species such as Citrus aurantium suggests that the mechanisms of resistance and therefore of regulating the expression of the genes present in the locus cannot be fully explained by DNA sequence alone. On this basis we moved to verify the role of siRNA through an epigenetic regulation of the methylation status of Ctv locus. The difference between methylated and unmethylated condition in tolerant and susceptible plants was performed by PCR analysis on DNA digested by sensitive and unsensitive enzymes to cytosine methylation