Citrus tristeza virus (CTV) is the causal agent of the most important virus disease of citrus. CTV isolates differing in biological and molecular characteristics have been reported worldwide. Recently, CTV was detected in Syria in citrus groves from two Governorates (Lattakia and Tartous) and several CTV outbreaks have been reported in Apulia (southern Italy) since 2003. To molecularly characterize the CTV populations spreading in Syria and Italy, a number of isolates from each region was selected and examined by different molecular approaches including: Multiple Molecular Markers analysis (MMM), real time RT-(q)PCR, single strand conformation polymorphism (SSCP) of the major coat protein (CP) gene (P25), and sequence analysis of the CP (P25), P18, P20 and RdRp genes. SSCP analysis of CP25 yielded two distinct simple patterns among the Syrian isolates and three different patterns in the Italian isolates. Based on MMM analysis, all Syrian CTV isolates were categorized as VT-like genotype, whereas the Italian isolates reacted only with the markers specific for the T30 genotype. These findings were also confirmed by RT-qPCR and by sequencing analysis of four genomic regions. The Italian isolates had nucleotide identities which varied: from 99.5 to 99.8 for the CP gene; from 97.4% to 98.3% for the P18 gene; from 98.6% to 99.8% for the P20 and from 97.8% to 99.1% for the partial RdRp sequenced. High sequence identity was found for all genomic regions analyzed between the Syrian isolates (from 98.9% to 99.6%). These results show that the CTV populations spreading in Apulia and Syria are associated with different genotypes, indicating different potential impacts on the citrus trees in the field. Since in both areas the introduction of the virus is relatively recent, infected plants resulted to contain a single and common genotype, suggesting that CTV is spreading from the first outbreaks by aphids or local movement of autochthonous infected plant material.

The complete nucleotide sequence and the genome organization were determined of a putative newmember of the family Tymoviridae, tentatively named Olive latent virus 3 (OLV-3), recovered in southernItaly from a symptomless olive tree. The sequenced ssRNA genome comprises 7148 nucleotides excludingthe poly(A) tail and contains four open reading frames (ORFs). ORF1 encodes a polyprotein of 221.6 kDain size, containing the conserved signatures of the methyltransferase (MTR), papain-like protease (PRO),helicase (HEL) and RNA-dependent RNA polymerase (RdRp) domains of the replication-associated proteinsof positive-strand RNA viruses. ORF2 overlaps completely ORF1 and encodes a putative protein of43.33 kDa showing limited sequence similarity with the putative movement protein of Maize rayado finovirus (MRFV). ORF3 codes for a protein with predicted molecular mass of 28.46 kDa, identified as thecoat protein (CP), whereas ORF4 overlaps ORF3 and encodes a putative protein of 16 kDa with sequencesimilarity to the p16 and p31 proteins of Citrus sudden death-associated virus (CSDaV) and Grapevinefleck virus (GFkV), respectively. Within the family Tymoviridae, OLV-3 genome has the closest identitylevel (49-52%) with members of the genus Marafivirus, from which, however, it differs because of thediverse genome organization and the presence of a single type of CP subunits.

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.

Citrus, one of the most economicallyimportant crops, is susceptible to a number ofarthropod- and graft-transmissible pathogens. Rapidand reliable methods for detecting multiple pathogensare important for routine diagnosis by reducing time,labour and costs. To this end, primers and TaqManprobes for Citrus psorosis virus (CPsV) and Citrusvariegation virus (CVV) detection by singleplex realtime(q) reverse transcription (RT)- PCR were initiallydesigned. Further optimizations included the developmentof a multiplex (m) RT-qPCR assay to detectsimultaneously CPsV, CVV, and Citrus tristeza virus(CTV) in a single reaction. When 10-fold serialdilutions prepared using total RNAs from CPsV- andCVV-infected plants were tested, RT-qPCR assaysproved to be 100 and 1000 times more sensitive thanconventional RT-PCR, respectively. The target viruseswere effectively identified by mRT-qPCR in fieldinfectedclementine and sweet orange trees. Theoptimized multiplex assay proved to be as sensitiveas the singleplex tests, thus providing a valuablealternative tool for detection of these citrus viruses.

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%. Isolate CO33, cultured from a coffee plant intercepted in northern Italy, represents a novel multilocus sequence typing profile, ST72 (G. Loconsole, personal communication). Isolates genetically related to CO33 were found in several coffee plants imported in October 2014 from Costa Rica through the Netherlands (European Food Safety Authority [EFSA] 2015).

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.

The Italian olive germplasm comprises one of, if not the highest number of authoctonous cultivars in the world. The recent finding of severe infections of Xylella fastidiosa in Olea europaea, a relatively new host for this harmful pathogen, prompted a survey for a preliminary evaluation of the susceptibility of different of its cultivars

In 2013, an outbreak of Xylella fastidiosa was first identified in Apulia (southeastern Italy) in olive trees severely affected by a novel disease denoted Olive Quick Decline Syndrome (Complesso del Disseccamento Rapido dell'Olivo, CoDiRO in Italian). CoDiRO was first widely observed in 2010 and is characterized by extensive scorching and desiccation of leaves and twigs. At a preliminary examination, many of such trees appeared to be impacted by the presence of three different agents: (i) the leopard moth (Zeuzera pyrina), which drills galleries in the branches and trunks of olives; (ii) a set of xylem-inhabiting fungi of different genera (Phaeoacremonium and Phemoniella, in particular) (Nigro et al. 2014), which invade the sapwood taking also advantage of the moth galleries; and (iii) the bacterium X. fastidiosa. As time passed and a better insight into the disease was gained with field and laboratory observations, it became evident that the role of the leopard moth is minor, whereas the fungi could play the role of aggravators. Although the etiological role of X. fastidiosa is yet to be determined, the finding of this quarantine bacterium in the European Union prompted urgent investigations to address the many open questions and to enforce actions for its containment to avoid further spread in the neighboring areas and in other countries.

Molecular features and genomic organization were determined forCitrus yellow vein clearing virus (CYVCV), the putative viral causalagent 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), consistingin a positive-sense RNA of 7,529 nucleotides and containing sixpredicted 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 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.

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 high-throughput 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.

Xylella fastidiosa is an important pathogen of commercial crops, landscape trees and ornamentals in North and South America. In Europe, symptoms resembling those caused by X. fastidiosa have occasionally been observed, but the presence of this EPPO quarantinable pathogen has never been confirmed. Recently, a rapidly spreading decline of aged olive trees has taken place in a large area of the Salento peninsula (Apulia, southern Italy). PCR assays on extracts from leaf veins and petioles of diseased trees gave positive reactions using X. fastidiosa gene-specific primers. In particular, PCR amplicons were generated by primers targeting the conserved hypothetical HL protein (Francis et al., 2006), the RNA polymerase sigma-70 factor, and the 16S rDNA genes (Rodrigues et al., 2003). Furthermore, molecular tests extended to almond and oleander trees with leaf scorching symptoms, growing next to diseased olive orchards, were also positive for X. fastidiosa. PCR products amplified from diseased olive trees were sequenced in duplicate and the sequences (EMBL-EBI provisional accession Nos HX2000034932- HX2000035003) showed 95 to 99% identity with the homologous genomic regions of X. fastidiosa. Tests for ascertaining the presence of X. fastidiosa by DAS-ELISA using two commercial kits (Agadia, USA and Bio-Rad, USA) were also positive, thus confirming molecular tests. Studies aimed at isolating the bacterium, determining the strain, evaluating its pathogenicity, and identifying the putative local vector(s) are currently in progress. X. fastidiosa has an extensive natural host range, including olive, from which the bacterial genotype A, pathogenic to oleander and almond, but not to grapevine, has been isolated in California (Krugner et al., 2010).

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-fluid feeding hemipteran collected in olive orchards during a 3-mo survey was the meadow spittlebug, Philaenus spumarius (L.) (Hemiptera: Aphrophoridae). 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 Brullé 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 five periwinkle plants but not to the seven olive plants. Sequences of PCR products from infected periwinkle were identical with those from X. fastidiosa-infected field trees. These data showed P. spumarius as a vector of X. fastidiosa strain infecting olives trees in the Salento Peninsula, Italy. © 2014 Entomological Society of America.

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.

Sanitary selection and certification of olive cultivars require sensitive diagnostic methods and effectivesanitation protocols. Although much attention has been paid in the past few years to the developmentof diagnostic tools for reliable virus identification, the need to define a common andstandardized diagnostic protocol led to the implementation of a ring test among nine Italian diagnosticlaboratories. A one-step RT-PCR protocol and different primer sets, targeting the most commonolive viruses covered by phytosanitary rules, were tested in each laboratory, using the same batch ofpositive 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 detectthe target viruses in all laboratories. Furthermore, a one-step RT-PCR protocol was used successfullyfor 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, andthat at least two subsets of samples must be collected from each plant. This standardized protocol isnow being used to produce nuclear stocks for 70 different Italian olive cultivars, in the framework ofthe national project OLVIVA, which involves 25 national research institutions.

Citrus ringspot is a graft-transmissible disease and at least two taxonomically distinct viral species are associated with this syndrome: Citrus psorosis virus (CPsV) and Indian citrus ringspot virus (ICRSV). Neither of these two viruses was detected, however, by serological or molecular assays, in symptomatic tissues from citrus trees in southern Iran, where the ringspot syndrome is widespread. By contrast, electron microscopy and molecular assays revealed the presence of a rhabdovirus-like virus, which was graft-transmitted to several citrus species and mechanically to herbaceous hosts. Virus particles were bacilliform and resembled rhabdovirus nucleocapsids deprived of the lipoprotein envelope. Partial sequences of the viral nucleoprotein and RNA polymerase genes showed a distant genetic relatedness with cytorhabdoviruses. This virus appears to be a novel species for which the name Iranian citrus ringspot associated virus (IrCRSaV) is suggested.

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 mu m, and a maximum length of ca. 5 mu m. 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.

Il virus della tristezza (CTV) causa una delle più gravi malattie in grado di colpire diverse specie di agrumi in tutto il mondo, favorita dall’uso dell’arancio amaro come portinnesto. Il virus viene trasmesso sia attraverso alcune specie di afidi vettori sia mediante l’uso di materiale di propagazione infetto, spesso importato fuori dalle norme di quarantena. Tra i quadri sintomatologici con cui la malattia si manifesta, i più rilevanti sono la morte fulminante della pianta, la butteratura del legno e il giallume dei semenzali. La variabilità dei sintomi dipende da fattori quali le condizioni ambientali, il vettore, la virulenza del ceppo, la combinazione d’innesto. La malattia da tempo è stata segnalata in Italia, tuttavia i vasti focolai rinvenuti negli ultimi anni in Sicilia, Puglia e Calabria, ripropongono l’urgenza di intensificare gli interventi per un piano incisivo di prevenzione e controllo che metta al riparo l’'agrumicoltura dai rischi di gravi epidemie nel nostro paese.

L'introduzione di nuovi metodi di sequenziamento stà rivoluzionando le potenzialità diagnostiche e di ricerca in campo vegetale. Queste tecnologie di sequenziamento di nuova generazione (NGS), non necessitano di alcuna conoscenza preliminare sul contenuto in acidi nucleici, virali e dell'ospite, dei tessuti sottoposti ad analisi. Il loro utilizzo permette di condurre un'analisi metagenomica sul campione in esame, che può essere costituito da una singola pianta o provenire da più piante. Da un punto di vista fitopatologico, queste nuove tecnologie rivelano la complessità del "viroma" (l'insieme di virus e viroidi) del campione e delle sue interazioni con il genoma dell'ospite, laddove questo è noto. Dal punto di vista diagnostico il metodo velocizza la scoperta e l'identificazione di nuovi o già noti virus, la sequenza del loro genoma e lo sviluppo di nuovi e specifici metodi di diagnosi.

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 citrus-growing 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.

Taxonomically, Xylella fastidiosa (Xf) constitutes a single species with four recognized subspecies (Xf. fastidiosa, multiplex, pauca and sandyi). For identifying subspecies and determining the taxonomic allocation of novel bacterial isolates, a widely used approach is multilocus sequence typing (MLST) analysis that encompasses seven different loci. This approach was used for the identifcation of the Xf strain isolated from olive trees affected by the quick decline syndrome (OQDS) in the the Salento peninsula (Apulia, south-eastern Italy).

In the course of surveys carried out in June 2014 in the Salento (Apulia, southern Italy) area affected by an epidemic of a strain of Xylella fastidiosa subsp. pauca (Cariddi et al., 2014) denoted CoDiRO (abbreviation from the Italian name "Complesso del Disseccamento Rapido dell'Olivo"), the following symptomatic plants were observed: (i) cherry (Prunus avium), 13 trees showing scanty vegetation and bud failure, but no leaf scorching; (ii) myrtle-leaf milkwort (Polygala myrtifolia), three shrubs showing extensive desiccation of twigs and scorched leaves; (iii) coastal rosemary (Westringia fruticosa), one shrub with extensive chlorosis and desiccation of the leaves. Samples collected from all these hosts (except for two of the 13 cherry plants) were ELISA- and PCR-positive upon testing with the protocols described by Loconsole et al. (2014). Sequencing of the amplified products from five housekeeping genes (gyrB, 16S rRNA, dnaK, tonB, RNA polymerase sigma factor) and of the PCR products obtained using the X. fastidiosa strain-specific primers 272-1int/272- 2int, showed that all these amplicons, regardless of the host of origin, had 100% sequence identity with the homologous products amplified from diseased olive trees (Cariddi et al., 2014). These results provide evidence that all the analyzed positive samples contain the same X. fastidiosa strain infecting olives in the same area. With the exception of cherry, for which there is a recorded infection by X. fastidiosa subsp. fastidiosa in California (Hernandez-Martinez et al., 2007), to the best of our knowledge P. myrtifolia and W. fruticosa are hitherto unreported hosts of this bacterium.

To investigate the role of herbaceous plants as hosts of Xylella fastidiosa, monthly sampling of the native flora of two heavily infected olive groves and of the sides of the adjacent dirt roads was conducted from January 2014 onwards. One of the orchards had not been subjected to weeding, whereas periodic tillage had been carried out in the other. Overall, more than 100 species of 40 monocotyledonous and dicotyledonous families were collected.

To investigate the role of herbaceous plants as hosts of Xylella fastidiosa, monthly sampling of the native flora of two heavily infected olive groves and of the side of the adjacent dirt roads was conductedfrom January 2014 onwards. One of the orchards had not been subjected to weeding, whereas periodic tillage had been carried out in the other. Overall, more than 100 species of 40 monocotyledonous anddicotyledonous families were collected, photographed and identified, their phenological stage was recorded using the Keller-Baggiolini scale, their period of presence and the type of distribution prevailing in the field (e.g. whether the species scattered or concentrated under olive trees or along the edges of the dry stone walls) was assessed, as well as the abundance-dominance indices according to the Braun-Blanquet method. At all sites monitored, Philenus spumarius, the main and most important vector of Xylella fastidiosa so far found in the area of the outbreak, was present with abundant populations on herbaceous species in the spring and on olive trees from the middle of May throughout the summer. All weed samples collected, in pools of 4-5 plants, were tested by DAS-ELISA and the uncertain results were verified by PCR. So far, none of the samples analyzed, in excess of 600, proved to host Xylella fastidiosa, confirming the preliminary observations that, by and large, weeds may not have a major role in the epidemiology of X. fastidiosa in the considered area. However, since sampling of the summer/autumn flora is yet to be done, a better insight into the epidemiological role of these plants will become available in the coming months, in conjunction with the migration of the vectors from the olive trees onto the native flora with the consequent possible inoculation of the bacterium.

The RNA genome of pathogenic and non-pathogenic variants of citrus Hop stunt viroid (HSVd) differ by five to six nucleotides located within the variable (V) domain referred to as the "cachexia expression motif". Sensitive hosts such as mandarin and its hybrids are seriously affected by cachexia disease. Current methods to differentiate HSVd variants rely on lengthy greenhouse biological indexing on Parson's Special mandarin and/or direct nucleotide sequence analysis of amplicons from RT-PCR of HSV-dinfected plants. Two independent high throughput assays to segregate HSVd variants by real-time RTPCR and High-Resolution Melting Temperature (HRM) analysis were developed: one based on EVAGreen dye; the other based on TaqMan probes. Primers for both assays targeted three differentiating nucleotides in the V domain which separated HSVd variants into three clusters by distinct melting temperatures with a confidence level higher than 98%. The accuracy of the HRM assays were validated by nucleotide sequencing of representative samples within each HRM cluster and by testing 45 HSVd-infected field trees from California, Italy, Spain, Syria and Turkey. To our knowledge, this is the first report of a rapid and sensitive approach to detect and differentiate HSVd variants associated with different biological behaviors. Although, HSVd is found in several crops including citrus, cachexia variants are restricted to some citrus-growing areas, particularly the Mediterranean Region. Rapid diagnosis for cachexia and non-cachexia variants is, thus, important for the management of HSVd in citrus and reduces the need for bioindexing and sequencing analysis. (C) 2013 Elsevier Ltd. All rights reserved.

A multiplex Taqman-based real-time reverse transcription (RT)polymerase chain reaction (PCR) assay was developed to identifypotential severe strains of Citrus tristeza virus (CTV) and separategenotypes that react with the monoclonal antibody MCA13. Three strainspecificprobes were developed using intergene sequences between themajor and minor coat protein genes (CPi) in a multiplex reaction. ProbeCPi-VT3 was designed for VT and T3 genotypes; probe CPi-T36 for T36genotypes; and probe CPi-T36-NS to identify isolates in an outgroupclade of T36-like genotypes mild in California. Total nucleic acidsextracted by chromatography on silica particles, sodium dodecyl sulfatepotassiumacetate, and CTV virion immunocapture all yielded highquality templates for real-time PCR detection of CTV. These assays successfullydifferentiated CTV isolates from California, Florida, and a largepanel of CTV isolates from an international collection maintained inBeltsville, MD. The utility of the assay was validated using field isolatescollected in California and Florida.

Xylella fastidiosa (Xf) is a xylem-inhabiting, vectortransmitted 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.

Xylella fastidiosa (Xf) was identified in September 2013 in olive trees affected by the Olive quick decline syndrome (OQDS) in the Salento peninsula (southern Italy) and denoted Xf strain CoDiRO. Xf is comprised of a group of genetically diverse bacteria in the class Gammaproteobacteria that causes severe plant diseases in many crops and ornamentals. The bacterium is acquired and transmitted by xylem-sap feeding hemipterans such as sharpshooter leafhoppers (Cicadellidae, Cicadellinae), froghoppers and spittlebugs (Aphrophoridae and Cercopidae) and, possibly, cicadas (Cicadidae and Tibicinidae.)

Oleander (Nerium oleander) is a host of different genotypes of Xylella fastidiosa (Xf) including the CoDiRO strain, which is associated with the quick decline syndrome of olive (OQDS) in the Salento peninsula, and was shown by MLST analysis to belong to the subspecies pauca, The CoDiRO strain, however, is molecularly distinct from the Xf pauca isolate reported from coffee and citrus, of which oleander is not a host.

The olive quick decline syndrome (OQDS) is a disease that appeared suddenly a few years ago in the province of Lecce (Salento peninsula, southeastern Italy). Among the factors that may be involved in its aetiology, the most relevant is Xylella fastidiosa, a quarantine pathogen of American origin, whose presence in Italy represents its first confirmed record in the European Union. X. fastidiosa is a Gram-negative bacterium that invades the xylem of a wide range of hosts, from which it is acquired by xylem-feeding insect vectors and transferred to other plants. The bacterium multiplies within the plant vessels and occludes them, thus impairing water uptake. Besides olive, the Salentian strain of X. fastidiosa infects in nature a number of woody (almond, cherry) and shrubby (oleander, broom, Acacia saligna, Polygala myrtifolia, Westringia fruticosa, Rosmarinus officinalis, Rhamnus elaternus, Myrtus communis) hosts, with no evidence for grapevines or citrus being hosts. The bacterium was isolated in culture and identified as a genotype of X. fastidiosa subsp. pauca, molecularly identical to an isolate from Costa Rica. Philaenus spumarius (meadow spittlebug), a froghopper quite common in the Salento area where it thrives on olive, was identified as the main vector. Disease eradication and sanitation of infected olives are unfeasible. However, strategies are being enacted for restraining the spread of pathogen and vector(s) within the boundaries of the currently infected zone.

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. Published by Elsevier B.V.

The discovery of Xylella fastidiosa (Xf) in olive trees affected by the quick decline syndrome (OQDS) in the Salento peninsula of Apulia (southern Italy) had prompted a series of studies which, among other aspects, investigated the presence of the bacterium in the xylem vessels of infected olives. These observations have now been extended to hosts other than olive that showed leaf scorching and desiccations of the canopy. In these hosts (Acacia saligna, Nerium oleander, Polygala myrtifolia, Prunus amygdalus) the presence of X. fastidiosa had previously been ascertained by PCR and ELISA.