The need of accurate and reliable methods for DNA isolation and plant species identification in foodstuffs is of great importance, especially in the protection of high added value products. Fresh foods, which are not subjected to any modifications, are suitable for many kind of analysis; for processed products, such as musts, wines, olive oils, and pasta, the situation may be more complicated due to DNA fragmentation and, in the worst case, by its degradation. This work aimed to establish an exhaustive and reproducible analytical procedure for table grape DNA tracing in industrial musts. Three different DNA extraction methods were initially compared and DNA was tested in PCR for its suitability for the amplification reaction of microsatellite markers or simple sequence repeats (SSRs). An optimized DNA extraction method for microsatellite amplification was developed and adapted for industrial musts. Two SSR-based molecular methods, High Resolution Melting and capillary electrophoresis, were tested and the markers VrZAG62 and VrZAG79 were found to be the most informative. High Resolution Melting analysis, here applied for the first time on musts, proved to be the method of choice for a preliminary screening using four cultivars chosen as references and different DNA mixtures prepared in laboratory. Capillary electrophoresis, providing allele size, allowed a fine genotyping of musts in comparison with reference cultivars. The LOD6 of a single grape cultivar in mixture with other varieties was also determined at 2.5 ng. Merging the information of the two molecular analyses applied to real samples, we demonstrated that is possible to discover case of musts adulterated with table grapes, and we propose our procedure in controlling musts quality and origin certification.

Virus-free accessions of peach, apricot, cherry and plum were graft-inoculated in 2004 with buds from a peach tree cv. Missour infected by Apricot latent virus (ApLV), then analysed by RT-PCR using a specific primer set. The expected DNA fragment of 200 bp in size was amplified from 33 different Prunus sources, thus extending the woody host range of ApLV to new cultivars of Prunus persica, P. avium, P. armeniaca and, for the first time, to European (P. domestica) and Japanese (P. salicina) plum cultivars. Several peach cultivars and apricot cv. Tirynthos were symptomatic. To determine the best source material for ApLV detection, RTPCR assays were carried out during the growing season. ApLV was homogeneously distributed in flowers, leaves, petioles, barks and fruits of peach and apricot but was detected only in the leaves of cherry and plum. The genetic variability of the ApLV coat protein gene from graft-inoculated hosts and comparable sequences from databases ranged between 63.5 and 100%.

Summary. 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 new member of the family Tymoviridae, tentatively named Olive latent virus 3 (OLV-3), recovered in southern Italy from a symptomless olive tree. The sequenced ssRNA genome comprises 7148 nucleotides excluding the poly(A) tail and contains four open reading frames (ORFs). ORF1 encodes a polyprotein of 221.6 kDa in 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 proteins of positive-strand RNA viruses. ORF2 overlaps completely ORF1 and encodes a putative protein of 43.33 kDa showing limited sequence similarity with the putative movement protein of Maize rayado fino virus (MRFV). ORF3 codes for a protein with predicted molecular mass of 28.46 kDa, identified as the coat protein (CP), whereas ORF4 overlaps ORF3 and encodes a putative protein of 16 kDa with sequence similarity to the p16 and p31 proteins of Citrus sudden death-associated virus (CSDaV) and Grapevine fleck virus (GFkV), respectively. Within the family Tymoviridae, OLV-3 genome has the closest identity level (49–52%) with members of the genus Marafivirus, from which, however, it differs because of the diverse genome organization and the presence of a single type of CP subunits.

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.

Sharka is a severe apricot viral disease caused by the plum pox virus (PPV) and is responsible for large crop losses in many countries. Among the known PPV strains, both PPV-D (Dideron) and PPV-M (Marcus) are virulent in apricot, the latter being the most threatening. An F1 apricot progeny derived from Lito, described in the literature as resistant, crossed to the susceptible selection BO81604311 (San Castrese 9 Reale di Imola) was used to study the genetic control of resistance to PPV. A population of 118 individuals was phenotyped by inoculating both PPV-D and PPV-M strains in replicated seedlings and scored for 3 years. An additional set of 231 seedlings from the same cross was also phenotyped for 2 years. SSRbased linkage maps were used for quantitative trait locus (QTL) analysis. A major QTL of resistance to both PPV-M and PPV-D strains was found in the top half of the Lito linkage group 1, where a QTL was previously described in Stark Earli-Orange, the donor of Lito resistance. The LOD score was considerably enhanced when the recovery of plants from infection was taken into account. The results obtained in Lito were compared with those observed in a second apricot cross progeny (Harcot 9 Reale di Imola) in which QTL of resistance to sharka were also mapped in the same linkage group 1 for both PPV strains. Several models of resistance to sharka disease are discussed considering the segregation frequencies, the QTL alignment in the two maps and the information

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 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.

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.