The necrotic disorder of freesia (Freesia refracta hyb., Family Iridaceae) was first described in The Netherlands before 1970. In following years, the disorder was widely reported in other European countries and more recently also in the United States and in New Zealand. The presence of the Freesia sneak virus (FreSV, Ophiovirus genus) has been widely associated with the necrotic disease, but some uncertainty remains. Freesia leaves showing necrotic disease were subjected to Ophiovirus purification and the product obtained was analyzed. Several differently shaped virus particles were visualized by TEM and a new virus-like agent, with a ca. 30 kDa coat protein was detected by western blotting. Previously uncharacterized viruses, possibly transmitted by the same vector, might have a role in the disease, at least in mixed infection, and are now thickening the plot about the necrotic disease causal agent.

Bright mosaic, consisting of white and yellow rectangular or oval to linear patches and stripes, often confluent, was observed on Agapanthus leaves, flower stalks and buds in Italy. To identify and characterize the causal agent, deep-sequencing was carried out and the resulting sequences were assembled. De novo assembly of contigs, along with RACE experiments, resulted in the complete RNA molecule of 13,100 nt sharing 85.6% identical nucleotides with the genome of Eggplant mottled dwarf virus (EMDV). Sequence analysis and comparisons of the Agapanthus isolate of EMDV showed five conserved motifs in the L gene, which are localized in the N-terminal region (amino acid no. 531 to 770).

A survey to identify the presence of Eggplant mottled dwarf virus (EMDV) in cultivated and wild plants in Latium region, Italy, was conducted during September 2012. In the same area, potted plants of Agapanthus sp., with symptoms suggestive of virus infection (bright mosaic consisting of white and yellow rectangular/oval to linear patches and stripes, often confluent, on leaves, flower stalks and buds) were observed in a private garden of Minturno (Fig. 1). Symptoms included ‘colour-breaking’ and flower distortion. Electron microscopic examination of leaf-dip preparations, by applying negative staining with 2% uranyl acetate, and ultrathin sections of symptom-bearing leaves showed only the presence of bacilliform enveloped rhabdovirus-like particles in the cytoplasm of Agapanthus cells. Symptomatic samples were further tested for the presence of EMDV using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), reverse transcription-polymerase chain reaction (RT-PCR) and mechanical inoculation of herbaceous test plants assays. When tested by DAS-ELISA using a commercial polyclonal antiserum, extracts of symptomatic leaves showed positive reaction for EMDV (genus Nucleorhabdovirus, family Rhabdoviridae). The virus was mechanically transmissible to Chenopodium quinoa, whereas repeated attempts to transmit the virus by sap inoculation to different test plants (Nicotiana benthamiana, N. glutinosa, N. rustica, N. tabacum cvs. Samsun and White Burley) failed. Total RNA was extracted from 100 mg of fresh Agapanthus plant tissue using the RNeasy Plant Kit (Qiagen GmbH, Germany). Amplification of a portion of the viral genome by RT-PCR was done with EMDV polymerase (P) gene specific primers (Rhab for: 5'-GGAGTGGGTAGTATTGGATCAGAA-3', Rhab rev: 5'-CTTGGAAAAGACAGGATGGC-3'). A distinct DNA fragment of about 1,028 base pairs (bp) in size was amplified. The PCR amplicon was directly sequenced in both directions. Sequence homology using BLAST analysis showed high nucleotide sequence identity with the P gene of EMDV (GenBank Accession No. AM922322). The RNA genome of the virus was partially characterised. Through primer-walking, a total of 6499 nucleotides (nt) of the 3' half of the viral genome was obtained as cDNA, cloned and sequenced. The assembled 6499 nt cDNA sequence (KF410949) had an open reading frame (from 518 to 6358) potentially coding for a 1946 amino acid (aa) protein, and the aa sequence of this putative RNA-directed RNA polymerase (RdRp) shared 97% identity with that of EMDV (Pappi et al., 2), confirming the identity of the virus isolated from diseased Agapanthus as EMDV. To our knowledge, this is the first report of natural infection of Agapanthus by EMDV. EMDV continues to be an economically important viral pathogen of several crops in Europe (Miglino et al., 1; Parrella et al., 3) and this report of EMDV infection of an ornamental plant suggests its increasing host range and that infected Agapanthus might act as a potential reservoir for EMDV.

Pepino mosaic virus (PepMV), a member of the genus Potexvirus, was first described in 1980 on pepino (Solanum muricatum) and was later isolated from different wild species of the genus Solanum (formerly Lycopersicon) (Soler et al., 2002). PepMV has been reported in several European countries and in North America, causing disease in tomato. In July 2008, plants of basil (Ocimum basilicum) showing interveinal chlorosis on young leaves were observed in three greenhouses in Sicily, in the area where tomato plants were found to be infected by PepMV in 2005 (Davino et al., 2006). The disease was transmitted mechanically to Nicotiana benthamiana, producing chlorotic spots and leaf deformation. Electron microscopy of negatively-stained preparations from basil and N. benthamiana leaves, both with symptoms, revealed the presence of flexuous, filamentous virus particles, typical of potexviruses. Leaves from 100 plants with or without symptoms were analysed by double-antibody sandwich (DAS) ELISA using a polyclonal antibody to PepMV (Loewe Biochemica). The presence of the symptoms was correlated with positive ELISA results. Total RNA was extracted from the same samples using an RNeasy Plant Mini Kit (Qiagen) and analysed by RT-PCR using the PepMV-specific primers PepMV-TGB-F and PepMV-UTR-R (Mumford & Metcalfe, 2001). The 840 bp RT-PCR product was sequenced in both directions and the sequence deposited in GenBank (Accession No. EU888637). The sequence had 99% sequence identity with an isolate of PepMV found in Sicily in 2005 (DQ517884). This is the first report of PepMV infecting basil. Although the disease does not appear too severe on basil, the species is frequently cultivated next to tomatoes and PepMV is transmitted mechanically easily; therefore basil may act as a virus reservoir.

During the 2007 growing season, melon (Cucumis melo) samples from the state of Guerrero in Mexico showing mosaic and other virus-like symptoms were collected for analysis. Electron microscopic examination of negatively stained leaf-dip extracts revealed the presence of abundant virus-like particles with features characteristic of the family Bunyaviridae. No other viral particles were observed in these preparations. However, enzyme-linked immunosorbent assays (ELISAs) specific for the most common Tospovirus spp. gave negative results. Antibodies raised against purified nucleocapsids reacted specifically with the infected leaf extracts in Western blots and double-antibody sandwich ELISA. The viral RNA was used as a template for a cDNA library, and nucleotide sequence analysis identified cloned cDNAs representing sequences corresponding to the three Tospovirus genome segments. Sequence comparisons showed that the new virus had the highest similarity to Chrysanthemum stem necrosis virus (CSNV). Phylogenetic analysis of two genome regions confirmed that this virus, provisionally named Melon severe mosaic virus (MeSMV), is a previously undescribed Tospovirus sp. belonging to the “new world” clade of Tospovirus spp. An initial survey of various cucurbit crops in various states of Mexico confirmed the widespread occurrence of this virus.

Ourmia melon virus (OuMV), Epirus cherry virus (EpCV) and Cassava virus C (CsVC) are three species placed in the genus Ourmiavirus . We cloned and sequenced their RNA genomes. The sizes of the three genomic RNAs of OuMV, the type member of the genus, were 2814, 1064 and 974 nt and each had one open reading frame. RNA1 potentially encoded a 97.5 kDa protein carrying the GDD motif typical of RNA-dependent RNA polymerases (RdRps). The putative RdRps of ourmiaviruses are distantly related to known viral RdRps, with the closest similarity and phylogenetic affinity observed with fungal viruses of the genus Narnaviridae . RNA2 encoded a 31.6 kDa protein which, expressed in bacteria as a His-tag fusion protein and in plants through agroinfiltration, reacted specifically with antibodies made against tubular structures found in the cytoplasm. The ORF2 product is significantly similar to movement proteins of the genus Tombusviridae , and phylogenetic analysis supported this evolutionary relationship. The product of OuMV ORF3 is a 23.8 kDa protein. This protein was also expressed in bacteria and plants, and reacted specifically with antisera against the OuMV coat protein. The sequence of the ORF3 protein showed limited but significant similarity to capsid proteins of several plant and animal viruses, although phylogenetic analysis failed to reveal its most likely origin. Taken together, these results indicate that ourmiaviruses comprise a unique group of plant viruses that might have evolved by reassortment of genomic segments of RNA viruses infecting hosts belonging to different eukaryotic kingdoms, in particular, fungi and plants.

During spring and summer of 2007 and 2008, a number of onion fields in Emilia Romagna region (northern Italy) showed various virus-like symptoms. DAS-ELISA carried Out in 2007 and early 2008 for Impatiens necrotic spot virus (INSV), Iris yellow, spot virus (IYSV) and Tomato spotted wilt virus (TSWV) showed the occasional presence of TSWV, whereas a number of samples also reacted weakly with IYSV antiserum a number of TSWV-negative samples, electron microscopy of leaf extracts revealed the presence Of tospovirus-like particles. Western blot analysis on the same set of samples gave positive results with antisera against Tomato fruit yellow ring virus (TFYRV) and TYSV, two serologically related Virus species. Sequence analysis of RT-PCR fragments confirmed that the virus In the onion samples was IYSV with approximately 98% identity at the amino acid level with reported Serbian and Spanish sequences of the same virus Phylogenetic analysis confirmed that the Italian IYSV isolates belong to a newly defined Southern European clade. Our data suggest that IYSV is made up of it heterogeneous and serologically distinct group of isolates.

Electron microscopy of extracts from diseased Polygonum convolvulus plants from Piedmont (Italy) revealed particles with the morphological features of a tospovirus. Sequencing of the full-length small (S) and medium (M) genome segments indicated that the virus is a member of a new Tospovirus species provisionally named Polygonum ringspot virus. A feature distinguishing it from members of other Tospovirus species was the presence of a very short intergenic region on the S segment lacking the potential for formation of the predicted hairpin structure involved in subgenomic expression. Antibodies made against purified nucleocapsids allowed serological comparison with other tospovirus isolates and revealed a relationship with tomato yellow ring virus, and to a lesser extent, to iris yellow spot virus. Serological tests detected the virus in various locations in northern and central Italy. The experimental host range was wide, although in nature the virus appeared restricted to two Polygonum species.

We were able to mechanically transmit a small isometric virus from field tomato samples showing severe necrotic symptoms, collected in the Culiacan area of Sinaloa state (Mexico). After gradient purification and three rounds of single-lesion passage on Chenopodium quinoa, the virus was back-inoculated to tomato plants and reproduced the original apical necrosis symptoms. The virus could be transmitted to a wide range of experimental hosts, including a number of solanaceous plants. Purified virus was used to produce specific polyclonal rabbit antibodies and serological tests such as enzyme-linked immunosorbent assay, Western blot analysis, and an immunochromatographic lateral flow assay. Such assays confirmed the wide distribution of this virus in symptomatic field plants in the area of the epidemic. Purified particles contained two genomic RNA molecules of ca. 7 kb (RNA1) and 5 kb (RNA2) estimated length. Analysis of clones from a cDNA library provided 6.5 and 3.0 kb of sequence for RNA1 and RNA2, respectively. Sequence analysis of the encoded replicase showed greatest similarity with members of the Sequiviridae family, and indicated that the virus we isolated is a new virus species, provisionally named Tomato apex necrosis virus.

We isolated a potyvirus from Tradescantia fluminensis that was causing leaf distortion and mild mosaic. We cloned and sequenced a 1500 bp cDNA obtained by RT-PCR corresponding to the 3' proximal region of the genome. We determined the host range and tested a series of potyviral antisera against our tradescantia virus isolate by immuno-enzymatic methods. Based on our results, we suggest that our viral isolate could be considered a new potyvirus species named Tradescantia mild mosaic potyvirus. Phylogenetic analysis confirmed that Tradescantia mild mosaic virus belongs to the genus Potyvirus within the family Potyviridae, but the virus could not be assigned to any of the potyvirus groupings recently defined.

While Mexico is the main cactus pear-producing country, Italy is the most important producer in the Mediterranean basin. No phytoplasma disease of cactus pear has been reported, despite previous detection of phytoplasmas in related species such as Opuntia tuna (Casper et al., 1970) and Opuntia linguiformis showing witches’ broom symptoms (Cai et al., 2002). In 2003, three cactus pear plants showing abnormal growth were observed in the DISTEF collection. The plants showed severe proliferation of cladodes with lack of flowers, fruits and spine production. Viral particles were not observed by transmission electron microscopy in sap from any of the affected plants. Total DNA was extracted from the affected plants and from two symptomless cactus pears as described by Cai et al. (2002). This was used as template for phytoplasma-specific 16S rDNA PCR amplification using one of three universal primer pairs: P1/P7, R16f2/r2 (Lee et al., 2000) or fU5/rU3 (Lorenz et al., 1995). DNA preparations from phytoplasma reference strains maintained in periwinkle were used as positive controls. To produce enough amplicon for further characterization by RFLP analysis, nested primer pair R16f2/r2 was used to reamplify P1/P7-primed rDNA products. RFLP analysis was performed with restriction enzymes AluI, HhaI, HpaII, MseI and TaqI. Phytoplasma-specific PCR products were amplified from all three plants showing symptoms with P1/P7 and fU5/rU3 primers by direct PCR, and with R16f2/r2 primers by nested PCR, but symptomless plants were always negative in all three PCR assays. The RFLP patterns obtained from analysis of rDNA amplicons from the former samples were identical to the pattern obtained for reference strain faba bean phyllody phytoplasma, a member of the 16S rDNA RFLP subgroup 16SrII-C. This is the first report of a phytoplasma infecting O. ficus-indica.

The genus Ophiovirus comprises five official species, and two of them, Tulip mild mottle mosaic virus (TMMMV) and Ranunculus white mottle virus (RWMV) naturally infect ornamentals. For some years a severe disease in freesia has been noted in the area round Sanremo, Northern Italy, similar to that earlier reported elsewhere in Europe, with symptoms of chlorotic interveinal lesions on the leaves, later coalescing and becoming sunken and necrotic. Examination by electron microscopy of negatively stained sap extracts of the diseased freesia material showed the presence of particles resembling those of ophioviruses. Partial characterization of the putative new ophiovirus is presented.

Highbush blueberry ( Vaccinium corymbosum ) has been planted in various areas of northern Italy for the past 30 years, in an effort actively to maintain cropped hillside and mountainside areas with acidic soil. The crop has also gained some economic importance for the fresh fruit market. During the summer of 2004, a number of plants from a blueberry crop field in southern Piedmont (Costigliole Saluzzo, Cuneo Province) showed symptoms generally associated with blueberry scorch disease (Martin & Bristow, 1988; Bristow et al. , 2000). Towards the end of the season, 23 leaf samples were collected from various plants showing symptoms of different cultivars: Blueray, Berkeley and Bluecrop. Leaf extracts were examined by electron microscopy using negatively stained preparations. A filamentous virus with longitudinal ribbing typical of the carlaviruses was found in some samples. Specific ELISA testing was then carried out according to the manufacturer’s instructions (Agdia) for a number of viruses often found in blueberry crops. Thirteen of the samples collected tested positive for Blueberry scorch virus (BlScV), whereas none tested positive for Blueberry shock virus (BlShV) or Blueberry leaf mottle virus (BLMoV). Four samples containing carlavirus particles were mechanically inoculated onto a range of herbaceous test plants. These did not show any symptoms, as expected from previous experience with BlScV, which was shown to be not mechanically transmissible to a range of herbaceous test plants (Martin & Bristow, 1988). The virus was then partially purified according to previous protocols (Martin & Bristow, 1988), using leaves taken from BlScV ELISA-positive plants. RNA was extracted from the purified virus and RT-PCR was carried out (Invitrogen) using random hexamers for reverse transcription, and the oligonucleotides 5 ′ -GAAAGAAGCACCGGCTCAATC-3 ′ and 5 ′ -GGAGATCTTGGCCATTTGCTC-3 ′ for PCR. The resulting amplification product of ≈ 380 bp was cloned using pGEM-T vector (Promega) and sequenced. The resulting nucleotide sequence of the insert was deposited in GenBank (Accession No. AY823507). A pairwise amino acid sequence comparison, using the amino-terminal region of the coat protein of BlScV isolates so far sequenced, showed that the Italian isolate has the highest similarity to the NJ1 strain of BlScV (Cavileer et al ., 1994). To our knowledge this is the first report of this potentially damaging virus outside North America.

The known natural hosts of tospoviruses, in particular Tomato spotted wilt virus (TSWV), are steadily increasing worldwide. The Liguria region of north-west Italy is important for the cultivation of vegetable and ornamental crops, both in open fields and under protection. TSWV and Impatiens necrotic spot virus (INSV) were first reported in Liguria in 1989, when many infected crops were found to be severely affected (Vaira et al ., 1993). The virus vector, Frankliniella occidentalis , is present in Liguria on protected crops during the winter and on both protected and field-grown crops during the summer. In 2000, approximately 20% of a batch of around 10 000 glasshouse-grown pot plants of Euphorbia eritrea showed chlorotic and necrotic spots in the stem, starting from the attachment point of true leaves. TSWV, but not INSV, was detected by both ELISA and immunochromatographic lateral flow assay in tissues with symptoms but not in symptomless tissues of the same plants. Only tospovirus particles were detected in tissues with symptoms by electron microscopy. The virus was transmitted mechanically to common herbaceous test plant species, but not to healthy E. eritrea plants. Thus it seems possible that viruliferous thrips fed on, and infected, true leaves and then moved only partly into the stem. Such infected plants were unmarketable. In 2002, a few potted plants of Asclepias curassavica in a glasshouse showed malformation and some necrosis of apical leaves. Again TSWV, but not INSV, was detected by ELISA and immunochromatographic lateral flow assay, and was transmitted to several herbaceous hosts of TSWV. Electron microscopy of negatively stained sap of affected plants revealed only the presence of tospovirus particles. After removal of necrotic tissues, some plants continued to develop malformation and sometimes necrosis on leaves and stems. The Asclepiadaceae and Euphorbiaceae include several host of TSWV (Campbell et al ., 2003; Peters, 2003), but this is the first report of TSWV infection in Euphorbia eritrea and Asclepias curassavica.