First Report of Plum pox virus Strain M Isolates in Apricot in Sicily, Italy.

Sharka or plum pox disease is one of the most economically important virus diseases of stone fruits. Plum pox virus (PPV), the causal agent, is a member of the genus Potyvirus of the family Potyviridae transmitted by aphids in a non-persistent manner and by grafting. To date, nine PPV strains have been described on the basis of their biological, serological, and molecular properties: M and D are the most widespread and economically important strains, PPV-Rec and PPV-C have been reported mainly in Europe, PPV-EA confined to Egypt, PPV-T to Turkey, PPV-W from Canada, Ukraine, Latvia, and Russia, PPV-CR detected in Russia, and finally a putative PPV strain infecting plum in Albania described as the ancestor of the M. PPV-M is responsible for major epidemics in many Italian regions and despite phytosanitary measures, the infection rate increases each year. The D and Rec isolates are sporadically reported while PPV-C, once signaled in Apulia, has been successfully eradicated. Except for a report from the 1980s, which is no longer traceable, Sicily was considered free from the virus (2). In 2012, two new foci of sharka in a coastal area of Catania in Sicily were first reported by the national plant protection service to the European Commission (DG-SANCO). In spring 2013, plants of different varieties of apricot (Prunus armeniaca) and peach (P. persica) showing typical symptoms of flower color break, yellowing and leaf deformation, chlorotic spots or rings, and malformation on fruits were tested positive to PPV by DAS-ELISA using polyclonal antibodies. In order to characterize two isolates from apricot varieties (Carmen Top and Ninfa), total RNAs, extracted using the RNeasy Plant Mini Kit (Qiagen) from ELISA-positive samples, were analyzed by RT-PCR with primers P1/P2, targeting the 3'-terminal region of the coat protein (CP) gene (5) followed by RFLP analysis after digestion with Rsa1. Subsequently total RNAs were analyzed with the type-specific primers P1/PM and P1/PD (3), P3M/P4b and P3D/P4b amplifying the N-terminal region of the CP gene (1) and, finally, with primers mD5/mD3, mM5/mM3, and mD5/mM3, amplifying the region 3'NIb-5'CP, including the recombination site of Rec isolates (4). Only primer pairs P1/P2, P1/PM, P3M/P4b, and mM5/mM3 produced amplicons of the expected size (243, 198, 466, and 459 bp, respectively). The RFLP assay confirmed both isolates belonging to the M strain. Moreover, no reaction was obtained with primer pair mD5/mM3, excluding isolates belonging to Rec-type. Isolate characterization was completed by direct sequencing in both directions of the of P1/P2 and P3M/P4b amplicons obtained from apricot samples L9-1 (Carmen Top isolate) and 9-335 (Ninfa isolate). The P1/P2 sequences (KJ994235, KJ994237) showed 98% similarity with PPV-M or PPV-Rec isolates. The P3M/P4b sequences (KJ994236, KJ994238) confirmed that Sicilian isolates belong to the PPV-M strain showing 99% similarity with those already present in GenBank, thus ruling out the possibility of an infection with a PPV-Rec isolate. This outbreak of the Marcus strain of PPV in Sicily represents a high risk for the expanding production of stone fruit in southern Italy. An eradication plan was quickly activated by the regional phytosanitary service. References: (1) T. Candresse et al. Phytopathology 101:611, 2011. (2) EPPO. PQR-EPPO database on quarantine pests (available online). http://www.eppo.int , 2014. (3) A. Olmos et al. J. Virol. Methods 68:127, 1997. (4) Z. Subr et al. Acta Virol. 48:173, 2004. (5) T. Wetzel et al. J. Virol. Methods 33:355, 1991.

Characterization of Bois noir isolates by restriction fragment length polymorphism of a Stolbur-specific putative membrane protein gene.

Bois noir phytoplasma (BNp), widespread in wine-producing areas of Europe and endemic in France and Italy, is classified in the 16SrXII-A subgroup, whose members are referred to as Stolbur phytoplasmas. The 16S rDNA gene of Stolbur phytoplasma shows low variability, and few non-ribosomal genes are available as markers to assess variation among isolates. We used the Stolbur-specific stol-1H10 gene, encoding a putative membrane-exposed protein, to investigate genetic diversity of French and Italian BNp isolates from plants and insects. Amplification of stol-1H10 from infected grapevines, weeds, and Hyalesthes obsoletus produced fragments of three sizes, and restriction fragment length polymorphism analysis divided these amplicons further into 12 profiles (V1 to V12). French BNp isolates were more variable than Italian ones, and different profiles were present in infected grapevines from France and Italy. Isolate V3, most abundant among Italian affected grapes but present among French ones, was found in one Urtica dioica sample and in all H. obsoletus collected on this species. Four Italian-specific profiles were represented among infected Convolvulus arvensis, the most frequent of which (V12) was also detected in H. obsoletus collected on this species. Most of the variability in the stol-1H10 sequence was associated with type II on the tuf gene.

First Report of Cucumber mosaic virus Infecting Solanum jasminoides in Italy.

Solanum jasminoides Paxton (potato vine or jasmine nightshade) is a vegetatively propagated ornamental species within the Solanaceae family. Recently, symptomless plants of this species were reported as natural hosts of the quarantine pest, Potato spindle tuber viroid (PSTVd) in Italy (1). In January 2008, approximately 1,000 potted, 2-year-old plants of S. jasminoides growing in an ornamental nursery in Sicily showed virus-like mosaic and malformation of leaves. Symptoms were observed on approximately 60% of the plants. Leaf tissue, collected from 30 symptomatic and 10 symptomless plants, was analyzed by double-antibody sandwich-ELISA with polyclonal antisera specific to Cucumber mosaic virus (CMV), Tomato spotted wilt virus, and Impatiens necrotic spot virus (Loewe Biochemica, Sauerlach, Germany). The same samples were also analyzed by tissue-printing hybridization with a PSTVd-specific digoxigenin-labelled riboprobe. All the symptomatic samples tested positive only with antisera against CMV, but negative in all other tests. The symptomless samples were negative in all the performed tests. To confirm the association of CMV with the diseased plants, total RNA was extracted from the same samples (RNeasy Plant Mini Kit; Qiagen, Hilden, Germany) and analyzed by reverse transcription (RT)-PCR using CMV-specific primers MP+5'-CATGGCTTTCCAAGGTACCAG-3' and MP-5'-CTAAAGACCGTTAACCACCTGC-3' that amplify a 844-bp fragment from the MP gene (2). The expected fragment was amplified only from samples of symptomatic tissue. CMV was also detected in mother plants grown in the same nursery and showing same mosaic symptoms. Definitive identification of the pathogen was obtained by cloning and sequencing the RT-PCR product. The obtained sequence (GenBank Accession No. EU828783) had 99 and 98% similarity with the subgroup I-A isolates CMV-LUN (GenBank Accession No. EU432183) and CMV-Fny (GenBank Accession No. DI0538), respectively. To our knowledge, this is the first report of CMV infecting S. jasminoides and it adds a new host to the more than 1,000 species (85 plant families) infected by this virus. The high incidence of the disease in the nursery could be due to propagation of cuttings from an infected source. References: (1) F. Di Serio. J. Plant Pathol. 89:297, 2007. (2) H. X. Lin et al. J. Virol. 78:6666, 2004.

Polygala myrtifolia as a New Natural Host of Cucumber mosaic virus.

Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV), and Impatiens necrotic spot virus (INSV) are among the most important viral pathogens of ornamental plants (1). Polygala myrtifolia L. (myrtle-leaf milkwort), originating from South Africa, and a member of the Polygalaceae, was recently introduced in Italy as a cultivated ornamental shrub for its fast and attractive free-flowering growth and drought-resistant characteristics. It can become an invasive plant and is now considered a serious problem in coastal areas of Australia where it was introduced as a garden plant. In Italy, P. myrtifolia is propagated by cuttings in commercial nurseries during the summer. In the winter of 2002, plants of P. myrtifolia growing in pots in an ornamental nursery in Sicily showed virus-like mosaic and malformation of leaves that appeared lanceolate with a lack of flowering. Leaf tissue was analyzed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with polyclonal antisera to CMV, TSWV (Lettuce type), and INSV. Positive ELISA results were obtained only with the CMV polyclonal antisera. Complete remission of symptoms was observed on new flushes after pruning and incubation of infected plants at warm temperatures (30 and 20°C, day and night, respectively). This evidence led to the hypothesis that the disease or virus was disseminated by transportation and propagation of plants without visible symptoms during the hot season. A survey was also performed in two historical gardens of Catania (Sicily) where a group of apparently healthy P. myrtifolia plants, from the previously mentioned ornamental nursery in Sicily, were introduced as a single specimen or to form a hedge. These plants showed the same leaf malformations and mosaic symptoms observed in the nursery. DAS-ELISA confirmed the presence of CMV but not TSWV and INSV. To our knowledge, this is the first report of CMV on P. myrtifolia and it adds a new host to over 1,000 species (85 plant families) infected by this virus. Reference: (1) M. L. Daughtrey et al. Plant Dis. 81:1220, 1997.

First Report of Mixed Infection of Hop stunt viroid and Peach latent mosaic viroid on Peach.

Hop stunt viroid (HSVd), belonging to the family Pospiviroidae, was first reported in hop, but infects several plant species, including herbaceous and woody hosts (e.g., grapevine, pear, peach, plum [2], apricot, almond, and pomegranate). In grapevine and apricot, the viroid appears to be latent. However, in other species, the viroid is associated with specific disorders: hop stunt, dapple fruit disease of plum and peach (2), and citrus cachexia. During spring 2000, stunted trees exhibiting delayed budbreak were observed in peach, cv. Redhaven, orchards in Sicily. In the summer of 2000, peach leaves were collected from symptomatic trees, triturated, and used to purify total nucleic acids by phenol extraction followed by CF-11 cellulose-chromatography. Viroid RNAs were detected by sequential polyacrylamide gel electrophoresis (sPAGE) and silver staining. Citrus exocortis viroid (371 nt) and Citrus viroid IIIb (294 nt) were used as standards. Nonradioactive hybridizations with digoxigenin (DIG)-labeled, Peach latent mosaic viroid (PLMVd) and HSVd RNA probes were used in viroid detection and identification. Predicted sizes of the viroid RNAs calculated in sPAGE and DIG probe hybridization demonstrated that 'Redhaven' peach trees were infected with PLMVd and HSVd. These results provide the first evidence of the contemporary presence of HSVd and PLMVd in peach trees. To our knowledge, this is the first report of HSVd in peach trees in Italy. Molecular characterization of this HSVd isolate is in progress to determine the nucleotide sequence of some of the variants in the population and to decide to which of the five proposed HSVd groups (1) this isolate should be assigned. Reference: (1) S. A. Kofalvi et al. J. Gen. Virol. 78:3177, 1997. (2) T. Sano et al. J. Gen. Virol. 70:1311, 1989.

Citrus psorosis virus Bark Scaling on Tarocco Sweet Orange.

Despite the wide distribution of vein flecking of citrus leaves in Italy, psorosis bark scaling has been reported only on Navelina sweet orange (Citrus sinensis L.), and Thompson and Washington navel oranges (1). Infection has not been found on any local sweet orange cultivars. Among these is Tarocco, a sweet orange cultivar that originated in Sicily, is very common because of the high fruit quality, has an attractive fruit appearance, and has blood-red pigmented flesh. In April 2001, classic psorosis bark scaling symptoms were observed on the main limbs of 10-year-old Tarocco trees grafted on sour orange rootstock, originally obtained by topworking Clementine (C. reticulata Blanco) trees with scions collected from 19-year-old Tarocco trees with no bark scaling at that time. The symptomatic trees first displayed one or two isolated circular patches of scales with gumming on the main or secondary limbs. As the disease progressed, the number of patches increased and coalesced to form bigger scales, resulting in bark flaking. Approximately 15% of trees in the field showed different stages of the disease. All of the affected trees showed vein flecking of young leaves. A leaf pattern was also present in a few plants without bark symptoms. Bark symptoms were correlated with the presence of Citrus psorosis virus (CPsV). Samples (110 representing 10% of the total number of trees in a field located in the area of Catania, Sicily) were collected during the spring flush using a W-pattern sampling method and tested by double-antibody sandwich enzyme-linked immunoassay (DAS-ELISA) (monoclonal antibody [MAb] PS29) (2). Of trees tested, 14% showed bark scaling, and 86% were symptomless. All symptomatic plants were tested and 70% of symptomless trees were positive based on DAS-ELISA. To confirm DAS-ELISA results, 10 field samples were also tested by bioassay on indicator plants (Navelina sweet orange ISA 315 and Pineapple sweet orange), triple-antibody sandwich enzyme-linked immunoassay (TAS-ELISA), and immunosorbent electron microscopy (ISEM) with a different antiserum (MAb 13C5). DAS-ELISA-positive samples produced vein flecking on indicator plants, were positive based on TAS-ELISA, and contained typical CPsV particles based on ISEM (R. G. Milne, IFA, CNR, Turin). To our knowledge, this is the first demonstration of psorosis bark scaling reaction of Tarocco sweet orange due to CPsV infection. References: (1) A. Catara et al. Proc. Int. Soc. Citri. 1:426, 1981. (2) M. Tessitori et al. Proc. 15th Conf. IOCV, IOCV, Riverside. In press.

[Diagnosis of total block in the bundle of His with rhythm of idioventricular substitution]. / Diagnosi di blocco totale intra-hissiano con ritmo di sostituzione idioventricolare.

His bundle (HB) recording does not allow the recognition of third degree intrahisian block in patients with complete atrio-ventricular block (AVB) associated with idioventricular rhythm, due to the absence of pacemaker activation in the distal HB region. We have observed fixed retrograde distal HB activation in the standard HB recording of a patient with complete AVB and ventricular rhythm at a rate of 28/min. Retrograde distal HB activation (h'r) did not disappear during apical right ventricular pacing, in association with the complete absence of retrograde nodal conduction: concealed retroconduction into the proximal HB did not allow the recording of anterograde hisian deflection when the interval between h'r deflection and the subsequent sinus atriogram was shorter than 200 msec. Distal HB bipolar pacing using low energy stimulus resulted in 1:1 ventricular response and normal QRS duration in the absence of nodal retroconduction, thus proving the localization of bidirectional block within the HB. The unmasking of retrograde V-h' conduction during idioventricular rhythm was likely related to phase 4 retrograde delay in the branch ipsilateral to the site of the emergency ventricular focus and to the subsequent trans-septal activation of the other side of His-Purkinje system. Referring to arrhythmic problems after DDD pace-marker implantation the localization of complete AV blocks and retrograde conduction patterns are discussed.