Small interfering RNAs (siRNAs), play a vital role in epigenetics of plant virus-host plant interactions. It has been extensively studied at both the transcriptional and post-transcriptional levels. In plants, siRNAs initiate and manage gene silencing by directing DNA methylation and/or histone methylation. In Arabidopsis, the ~24 nt siRNAs directs DNA methylation (RNA-directed DNA methylation, RdDM) and chromatin remodeling at their target loci. Recent advances in highthroughput sequencing techniques has enabled thorough exploration of small RNAs populations and allow rapid analysis of massive datasets to assemble complete full-length genome sequence for different plant species. This large database of sequence information also allows identification of genome regions specifically matched by siRNAs that likely differ among tolerant, resistant or susceptible hosts and advance epigenetic studies on diseased plants. Resistance to Citrus tristeza virus (CTV), the most severe virus affecting Citrus spp., associated with a single dominant gene locus Ctv occurring in Poncirus trifoliata while all Citrus spp. are considered susceptible. This locus contains 22 putative genes, but their regulation and mechanism for resistance remains unknown. In our study, CTV was graft-inoculated on Carrizo citrange (Poncirus trifoliata x C. sinensis (I think) ) and C. aurantium (sour orange) seedlings, and the population of siRNA characterized by high-throughput sequencing using an ILLUMINA platform. The Ctv-derived siRNA (~2% of the total short reads) were dominated in both hosts by the 24-nt. However, CTV infection caused an increase in accumulation of 24-nt siRNA sequences homologous to the Ctv gene in Carrizo but it decreased in sour orange. Distribution of the 24nt along the Ctv gene locus (282Kb) had a clearly different distribution between the two host. The predominant hot spot of siRNA in Carrizo mapped in the putative gene Ctv-20, whereas in sour orange it associated to the intergenic region between the putative genes Ctv-11 and Ctv-12, where a Copia-like retrotransposon C is located. This distribution profile was conserved for each species between CTV-infected and uninfected plants but, as previously mentioned, the frequency of the 24nt siRNAs was altered by the presence of the virus. We supposed that the different profile of 24nt between the two host in the locus ctv is due to RdDM mechanisms. To demonstrate the methylation status of the resistance locus we performed a bisulfite treatment of DNA. in which unmethylated cytosine was converted to uracile, while methylated cytosine did not react. A methylcytosines mapping was carried out on Ctv-11 and Ctv-12 sequences. By specific software were found 5 different CpG islands in the Copia-likeretrotransposon sequence and 42 primer pair were designed. The PCR analyses have been carried out using MSP and BSP primers followed by combined bisulfite restriction analysis (COBRA).

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.

Huanglongbing (HLB) is a serious disease of citrus worldwide. Three different ‘Candidatus Liberibacter’ species are associated with HLB: ‘Ca. Liberibacter asiaticus’, ‘Ca. L. africanus’, and ‘Ca. L. americanus’ (1). ‘Ca. L. africanus’ and its vector, Trioza erytreae, are both heat sensitive, and when present, occur in citrus when temperatures remain below 30 to 32°C. In Africa, ‘Ca. L. africanus’ and T. erytreae have been reported in South Africa, Zimbabwe, Malawi, Burundi, Kenya, Somalia, Ethiopia, Cameroon, and Madagascar (1). Inspection of citrus trees in orchards and budwood sources in nurseries located in the warmer citrus-growing areas of Tigray and North Wollo in northern Ethiopia revealed nearly 100 trees with symptoms of leaf yellowing with a blotchy mottle pattern, dead branches, and decreased fruit quality and yield. Two symptomatic sweet orange budwood trees and three symptomatic orchard plants were sampled in April 2009, along with three healthy-looking sweet orange plants. DNA was extracted from 200 mg of desiccated leaf midribs using the CTAB method (4) and subjected to conventional PCR using the primer pairs A2/J5 (2) and OI2/23S1 (3) that amplify the ribosomal protein gene in the rplKAJL-rpoBC operon and the 16S/23S ribosomal intergenic regions, respectively, of ‘Ca. L. africanus’ and ‘Ca. L. asiaticus’. Positive PCR reactions were obtained for all five symptomatic samples with both primer pairs. PCR amplicons of 703 bp (A2/J5) and 892 bp (OI2/23S) recovered from two of these samples were purified, cloned, and sequenced. BLAST analysis revealed that the nucleotide sequences we obtained for the ribosomal protein (GenBank Accessions Nos. GQ890155 and GQ890156) shared 100% identity with each other and 99% identity with sequences of ‘Ca. L. asiaticus’ from Brazil (DQ471904), Indonesia (AB480161), China (DQ157277), and Florida (CP001677). Similarly, the 16S/23S ribosomal intergenic sequences (GU296538 and GU296539) shared 100% identity with each other and 99% identity with homologous ‘Ca. L. asiaticus’ sequences from Brazil (DQ471903), Indonesia (AB480102), China (DQ778016), and Florida (CP001677) and contained two tRNA genes as occurs in ‘Ca. L. asiaticus’ but not in ‘Ca. L. africanus’ (3). To our knowledge, this is the first report of ‘Ca. L. asiaticus’ in Africa. The presence of ‘Ca. L. asiaticus’ is a threat for warmer citrus-growing areas of Africa that are less favorable for ‘Ca. L. africanus’ and T. erytreae. In areas where ‘Ca. L. asiaticus’ was confirmed, symptomatic trees must be promptly eradicated and surveys to determine spread of the disease and its vectors are necessary.

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.

In the attempt to identify the causal agent of Citrus chlorotic dwarf disease (CCDD), a virus-like disorder of citrus, the small RNA fraction and total DNA from symptomatic citrus plants were subjected to highthroughput sequencing. DNA fragments deriving from an apparently new geminivirus-like agent were found and assembled by NGS to re-construct the entire viral genome. The newly identified virus has a circular single-stranded DNA genome comprising five open reading frames (ORFs) with sequence homologies with those encoded by geminiviruses. PCR and qPCR assays were successfully used for determining its presence in the CCDD-affected plants obtained by graft propagation. The larger genome size (3.64 vs. 2.5–3.0 kb) and a number of differences in its structural organization, identified this virus as a highly divergent member of the family Geminiviridae, to which the provisional name of Citrus chlorotic dwarf-associated virus (CCDaV) is assigned.

Discovery of Xylella fastidiosa from olive trees with “Olive quick decline syndrome” in October 2013 on the west coast of the Salento Peninsula prompted an immediate search for insect vectors of the bacterium. The dominant xylem-ßuid feeding hemipteran collected in olive orchards during a 3-mo survey was the meadow spittlebug, Philaenus spumarius (L.) (Hemiptera: Aphrophori- dae). Adult P. spumarius, collected in November 2013 from ground vegetation in X. fastidiosa-infected olive orchards, were 67% (40 out of 60) positive for X. fastidiosa by polymerase chain reaction (PCR) assays. Euscelis lineolatus Brulle ́ were also collected but tested negative for the pathogen. Transmission tests with P. spumarius collected from the Salento area were, therefore, conducted. After a 96-h inoculation access period with 8 to 10 insects per plant and a 30-d incubation period, PCR results showed P. spumarius transmitted X. fastidiosa to two of Þve periwinkle plants but not to the seven olive plants. Sequences of PCR products from infected periwinkle were identical with those from X. fastidiosa-infected Þeld trees. These data showed P. spumarius as a vector of X. fastidiosa strain infecting olives trees in the Salento Peninsula, Italy.

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.

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. solations 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.

Citrus tristeza virus (CTV) outbreaks have been reported in the main citrus-growing regions of Italy in the past 10 years. In some areas where eradication efforts failed to suppress spread, high CTV incidence is now observed. Recently, potentially severe CTV strains were detected in Calabria (southern Italy), one of the major citrusgrowing area. As a result, investigations of the virulence and molecular features of CTV populations spreading in this region were undertaken. Virus was detected by enzyme-linked immunosorbent assay (ELISA) using a broad spectrum polyclonal antiserum, and was differentiated into potential virulent categories with the severe-strain discriminating monoclonal antibody MCA13. Isolate genotyping was conducted using reverse-transcription polymerase chain reaction (RT-PCR) with multiple molecular markers (MMM), single-strand conformation polymorphism (SSCP) analysis of the amplicons from the genes coding for the coat protein (CP) p25 and the non-structural p20 protein as well as sequence analysis. Based on the serological reactivity, the isolates were differentiated in two distinct serogroups: MCA13-reactive and MCA13 non-reactive. Similarly, based on the molecular profile, the isolates were grouped in two genetically distinct phylogenetic clusters, and associated either with a T30-like or with a T3-like genotype. These data were related to the results of biological indexing on standard indicator plants, which distinguished isolates causing mild or severe seedling yellow reactions. The study has demonstrated the presence of MCA13-reactive isolates associated with a T3-like genotype and causing severe seedling yellows in sour orange, grapefruit and lemon seedlings, and stem pitting in Mexican lime.

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 is one of the agent of devastating cultivated citrus trees especially if grafted on sour orange (Citrus aurantium), which appears to be the most susceptible species. The trifoliate orange (Poncirus trifoliata) appears to be resistant to the disease compared to the sour orange, the sweet orange and the grapefruit, while, the Citrange carrizo, derived from cross to C. sinensis L. x P. trifoliate, appears to be tolerant. In Citrange carrizo, the virus replicates and spreads throughout the tolerant plant without showing any symptoms. On the contrary in the resistant plants of Poncirus, the virus replication is not blocked, but it seems likely that it acts by preventing cell to cell and/or long distance moviment (Karasev et al., 2010). The resistance to the virus is due to the presence of a locus, called locus CTV, which is available an accurate genetic map (Yang et al., 2001; Rai, 2006). Within this region are present 22 genes seven of which (Ctv.4, Ctv.7, Ctv.8, Ctv.11, Ctv.17, Ctv.18 and Ctv.21), are denoted R(1-7) genes, since they show a high homology with Arabidopsis resistance genes encoding to CC-NBS-LRR proteins (coil-coil-nucleotide binding site-leucine rich repeat) (Deng et al., 2000; Yang et al., 2003). In addition, six other genes, with a high homology sequence to known function genes of other species are present. In particolar the Ctv.20 showed homology with a plant virus movement-like protein (Karasev and Hilf 2010). This membrane protein, through the formation of channels in plasmodesma, is involved in the transfer of proteins and viral RNA from cell to cell. Ctv.20 was predicted to contain three open reading frames (ORFs) by GenScan Web server (Burge and Karlin, 1997), but BLAST searches indicated that both the first and the third ORFs were highly homologous with Petunia vein-clearing virus (PVCV) ORF1 (Yang et al. 2003). Yang and colleagues (2003), performed the northern hybridization analyses using DNA fragments from the first and the third ORFs and both these fragment hybridized with the same band of about 9 kb, that presented a size compatible with the GenScan prediction (data not shown). Northern hybridization indicated that Ctv.20 and its orthologous are highly expressed in P. trifoliata and sweet orange leaves and in P. trifoliata bark tissues, but are relatively lowly expressed in the phloem of sweet orange (data not shown). The ortholog of Ctv.20 in sweet orange is about 8.5 kb, which is slightly smaller than Ctv.20 (9 kb) in P. trifoliata. CTV tends to accumulate in phloem tissue of infected plants, which suggests that Ctv.20 could also be considered as a candidate gene for Ctv resistance (Yang et al., 2003). The aim of the present research 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 with particular reference to Ctv.20 gene. 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.

The recent introduction of Xylella fastidiosa in Europe and its involvement in the Olive Quick Decline Syndrome (OQDS) in Apulia (Salento, Lecce district, South Italy) led us to investigate the biology and transmission ability of the meadow spittlebug, Philaenus spumarius, whichwas recently demonstrated to transmit X. fastidiosa to periwinkle plants. Four xylem-sap-feeding insect species were found within and bordering olive orchards across Salento during a survey carried out from October 2013 to December 2014: P. spumarius was the most abundant species on non-olive vegetation in olive orchards as well as on olive foliage and was the only species that consistently tested positive for the presence of X. fastidiosa using real-time PCR. P. spumarius, whose nymphs developwithin spittle on weeds during the spring, are likely to move from weeds beneath olive trees to olive canopy during the dry period (May to October 2014). The first X. fastidiosainfective P. spumarius were collected in May from olive canopy: all the individuals previously collected on weeds tested negative for the bacterium. Experiments demonstrated that P. spumarius transmitted X. fastidiosa from infected to uninfected olive plants. Moreover, P. spumarius acquired X. fastidiosa from several host plant species in the field, with the highest acquisition rate from olive, polygala and acacia. Scanning electron microscopy (SEM) revealed bacterial cells resembling X. fastidiosa in the foreguts of adult P. spumarius. The data presented here are essential to plan an effective IPM strategy and limit further spread of the fastidious bacterium.

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

A number of important citrus pathogens are spread by graft propagation, arthropod vector transmission and inadvertent import and dissemination of infected plants. For these reasons, citrus disease management and clean stock programs require pathogen detection systems which are economical and sensitive to maintain a healthy industry. To this end, multiplex quantitative real-time PCR (qPCR) assays were developed allowing high-throughput and simultaneous detection of some major invasive citrus pathogens. Automated high-throughput extraction comparing several bead-based commercial extraction kits were tested and compared with tissue print and manual extraction to obtain nucleic acids from healthy and pathogen-infected citrus trees from greenhouse in planta collections and field. Total nucleic acids were used as templates for pathogen detection. Multiplex reverse transcription-qPCR (RT-qPCR) assays were developed for simultaneous detection of six targets including a virus, two viroids, a bacterium associated with huanglongbing and a citrus RNA internal control. Specifically, two one-step TaqMan-based multiplex RT-qPCR assays were developed and tested with target templates to determine sensitivity and detection efficiency. The first assay included primers and probes for 'Candidatus Liberibacter asiaticus' (CLas) and Citrus tristeza virus (CTV) broad spectrum detection and genotype differentiation (VT- and T3-like genotypes). The second assay contained primers and probes for Hop stunt viroid (HSVd), Citrus exocortis viroid (CEVd) and the mitochondrial NADH dehydrogenase (nad5) mRNA as an internal citrus host control. Primers and TaqMan probes for the viroids were designed in this work; whereas those for the other pathogens were from reports of others. Based on quantitation cycle values, automated high-throughput extraction of samples proved to be as suitable as manual extraction. The multiplex RT-qPCR assays detected both RNA and DNA pathogens in the same dilution series as singleplex assays and yielded similar quantitation cycle values. Taken together, high throughput extraction and multiplex RT-qPCR assays reported in this study provided a rapid and standardized method for routine and simultaneous diagnosis of different RNA and DNA citrus pathogens.