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Molecular features and genomic organization were determined for Citrus yellow vein clearing virus (CYVCV), the putative viral causal agent of yellow vein clearing 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 were used to construct the whole genome of Citrus yellow vein clearing virus (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, Alpha-flexiviridae) 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 reverse transcription-polymerase chain reaction on yellow vein clearing symptomatic field trees as well as graft- and mechanically inoculated host plants. Collectively, these data suggest that CYVCV is the causal agent of yellow vein clearing disease and represents a new species in the genus Mandarivirus. [ABSTRACT FROM AUTHOR]

Sanitary selection and certification of olive cultivars require sensitive diagnostic methods and effective sanitation protocols. Although much attention has been paid in the past few years to the development of diagnostic tools for reliable virus identification, the need to define a common and standardized diagnostic protocol led to the implementation of a ring test among nine Italian diagnostic laboratories. A one-step RT-PCR protocol and different primer sets, targeting the most common olive viruses covered by phytosanitary rules, were tested in each laboratory, using the same batch of positive and healthy controls as well as the same amplification conditions and reaction components. The one-step RT-PCR, performed using several specific primer sets, was able efficiently to detect the target viruses in all laboratories. Furthermore, a one-step RT-PCR protocol was used successfully for the first time for detection of Tobacco necrosis virus (TNV) and Olive mild mosaic virus (OMMV). Results showed that all target viruses were not uniformly distributed in the canopy, and that at least two subsets of samples must be collected from each plant. This standardized protocol is now being used to produce nuclear stocks for 70 different Italian olive cultivars, in the framework of the national project OLVIVA, which involves 25 national research institutions. [ABSTRACT FROM AUTHOR]

The advent of next generation sequencing (NGS) technologies has dramatically advanced our ability to comprehensively investigate diseases of unknown aetiology 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 is widely used to identify new viruses in infected plants, by analyzing virus-derived small interfering RNA populations, or single- and double-stranded RNA (dsRNA) molecules extracted from infected plants. The method generates sequence in an unbiased fashion, thus allowing the detection of 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 virus strains. This approach has allowed the identification of five novel viral species and the sequencing of the whole genome of several viruses and viroids infecting Citrus spp., Prunus spp., grapevines, fig, hazelnut, olive, persimmon and mulberry. Combined analysis of the datasets generated by using either siRNA fractions or dsRNA templates, favoured the characterization of the whole virus-derived sequences in 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. [ABSTRACT FROM AUTHOR]

A dramatic outbreak of Xylella fastidiosa decimating olive was discovered in 2013 in Apulia, Southern Italy. This pathogen is a quarantine bacterium in the European Union (EU) and created unprecedented turmoil for the local economy and posed critical challenges for its management. With the new emerging threat to susceptible crops in the EU, efforts were devoted to gain basic knowledge on the pathogen biology, host, and environmental interactions (e.g„ bacterial strain(s) and pathogenicity, hosts, vector(s), and fundamental drivers of its epidemics) in order to find means to control or mitigate the impacts of the infections. Field surveys, greenhouse tests, and laboratory analyses proved that a single bacterial introduction occurred in the area, with a single genotype, belonging to the subspecies pauca, associated with the epidemic. Infections caused by isolates of this genotype turned to be extremely aggressive on the local olive cultivars, causing a new disease termed olive quick decline syndrome. Due to the initial extension of the foci and the rapid spread of the infections, eradication measures (i.e.. pathogen elimination from the area) were soon replaced by containment measures including intense border surveys of the contaminated area, removal of infected trees, and mandatory vector control. However, implementation of containment measures encountered serious difficulties, including public reluctance to accept control measures, poor stakeholder cooperation, misinformation from some media outlets, and lack of robust responses by some governmental authorities. This scenario delayed and limited containment efforts and allowed the bacterium to continue its rapid dissemination over more areas in the region, as shown by the continuous expansion of the official borders of the infected area. At the research level, the European Commission and regional authorities are now supporting several programs aimed to find effective methods to mitigate and contain the impact of X. fastidiosa on olives, the predominant host affected in this epidemic. Preliminary evidence of the presence of resistance in some olive cultivars represents a promising approach currently under investigation for long-term management strategies. The present review describes the current status of the epidemic and major research achievements since 2013. [ABSTRACT FROM AUTHOR]