Italian Isolates Research Articles

MOLECULAR DIAGNOSTICS TO ASSESS THE PROGRESSION OF PHOMA TRACHEIPHILA IN CITRUS AURANTIUM SEEDLINGS AND ANALYSIS OF GENETIC DIVERSITY OF ISOLATES RECOVERED FROM DIFFERENT CITRUS SPECIES IN TUNISIA

SUMMARY This study was focused on the genetic diversity of a collection of Phoma tracheiphila isolates recovered from different orchards and host species in Tunisia. Knowledge of the sequences permitted molecular assessment of the pathogen’s downward progression from the point of inoculation on citrus leaves. Analysis of 58 isolates of P. tracheiphila including four Italian isolates assessed by ITS-RFLP molecular markers revealed that Tunisian isolates of P. tracheiphila are homogenous and genetically similar to the Italian isolates. Development of this pathogen is likely clonal under Mediterranean conditions and this should be taken into account when developing management strategies based on resistant varieties. The result made possible the molecular monitoring of the pathogen’s migration in sour orange vessels using only one representative and virulent isolate. PCR showed that within 10 days post inoculation (dpi) fungal DNA was detected 10 cm from the inoculation point and at 30 dpi it was present in the stem vessels at a distance of 50 cm and more. P. tracheiphila basipetal translocation is very quick and occurs prior to symptom expression. Consequently, diagnosis of mal secco based on typical symptom like shedding of leaves, wilting and the salmon pink discoloration of wood is not appropriate to estimate disease development and must be replaced by a molecular technique that allows earlier detection of the infection in still symptomless material.

Molecular Characterization and Phylogenetic Analysis of VP1 of Porcine Enteric Picornaviruses Isolates in Italy

Porcine enterovirus (PEV), Porcine Teschovirus and Porcine sapelovirus, belonging to the family Picornaviridae, are ubiquitous and mainly cause asymptomatic infections in pigs. In this study, a total of 40 Italian porcine picornavirus isolates were characterized by sequencing the capsid VP1-encoding gene. This procedure turned out to be a useful diagnostic tool for the molecular identification of porcine enterovirus, teschovirus and sapelovirus strains and for the study of molecular epidemiology and evolution of these viruses confirming the possibility of correlating virus genotype to serotype.

Metallo-β-Lactamase Expression Confers an Advantage toPseudomonas aeruginosaIsolates Compared with Other β-Lactam Resistance Mechanisms, Favoring the Prevalence of Metallo-β-Lactamase Producers in a Clinical Environment

The Pseudomonas aeruginosa isolate TS-832035 was responsible for an outbreak that occurred in an Italian hospital between 1999 and 2002. It exhibited a high-level resistance to carbapenems due to the contemporary presence of two independent mechanisms: the production of a carbapenemase, coded by a bla(VIM-1) determinant carried by the chromosomal class 1 integron In70.2 (containing also the aacA4, aphA15, and aadA1 genes in its cassette array), and the lack of the OprD porin. We compared TS-832035 with a strictly related isolate, TS-103, whose resistance to carbapenems was due to the lack of the OprD porin only, as it did not carry In70.2. We evaluated their growth kinetics, in both separate cultures and competition assays, under permissive conditions. These experiments highlighted a significant in vitro fitness cost associated with the integron. On the contrary, none of the resistance determinants other than the bla(VIM-1) seemed to confer a real selective advantage to its host. Comparison of these results with the in vivo behavior, showing that the In70.2-carrying isolates largely prevailed over the In70.2-lacking ones, besides the detection of similar integrons in other Italian clinical isolates, evidenced the need to investigate accurately the causes of their large distribution, as possible soft spots could exist in the ability of their hosts to adapt to the hospital settings.

Antimicrobial susceptibility to zinc bacitracin of Clostridium perfringens of rabbit origin.

Zinc bacitracin is widely used in Italian rabbit farms to control both Epizootic Rabbit Enteropathy (ERE) and clostridiosis, and field results demonstrate useful activity. Nevertheless, data regarding the in vitro efficacy of zinc bacitracin against clostridia of rabbit origin are not available. In this study, the minimal inhibitory concentrations (MICs) of zinc bacitracin were evaluated in 123 C. perfringens strains isolated from rabbits in Italian fattening units. The agar dilution method was performed in Brucella Agar supplemented with laked sheep blood, haemin and vitamin K1, as recommended in NCCLS document M11-A6. Most strains (94.3%) had low MIC values (£ 0.5 mg/ml), and a few strains (4%) were inhibited by a concentration of 1 mg/ml. Two isolates (1.6%) had a MIC value of 16mg/ml. The MIC values of ATCC reference strains showed a good fit between each batch. MIC required to inhibit the 90% of organisms was 0.5 mg/ml and the presence of only two strains with MIC=16 mg/ml revealed the susceptibility to zinc bacitracin of Italian isolates of C. perfringens from rabbit and the absence of acquired resistance.

First Record and Complete Nucleotide Sequence of Alfalfa mosaic virus from Lavandula stoechas in Italy.

During spring 2009, lavender plants (Lavandula stoechas L.) showing a bright yellow mosaic of calico type and light stunting were observed in a commercial nursery in Liguria Province in northern Italy. Of 300 plants inspected, ~2% were symptomatic. Preliminary observations of leaf sap with the transmission electron microscope revealed bacilliform virus-like particles in three symptomatic plants, whereas no virus-like particles were observed in asymptomatic plants. The same symptomatic plants were tested by double-antibody sandwich-ELISA with polyclonal antisera against Cucumber mosaic virus, Potato virus Y, Tobacco mosaic virus, and Alfalfa mosaic virus (AMV). All three plants reacted positively against AMV antibodies, but not the other antibodies. A crude sap extract obtained from a single symptomatic plant, hereafter referred to as the Lst isolate, was prepared by macerating 1 g of fresh leaves in 4 ml of sodium phosphate 0.03 M, containing 0.2% sodium diethyldithiocarbamate, 75 mg/ml of active charcoal, and traces of Carborundum (600 mesh). Sap extract was mechanically inoculated onto a set of herbaceous hosts. Inoculated plants were maintained in an insect-proof greenhouse with natural illumination and temperatures of 24 and 18°C day/night. Under these conditions, plants showed the following symptoms after 1 to 3 weeks: necrotic local lesions in bean (Phaseolus vulgaris L., cv. Borlotto) and cowpea (Vigna unguiculata L., cv. Black eye); necrotic local lesions followed by systemic necrosis in broad bean (Vicia faba L., cv. Super Simonia) and tomato (Solanum lycopersicum L., cv. San Marzano); and bright yellow mosaic (calico type) in basil (Ocimum basilicum L., cv. Gigante). To sequence the entire genome of the Lst isolate, total RNA was extracted from infected samples with the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and subjected to AMV-specific reverse transcription-PCR by using four primer pairs for each genomic RNA of overlapping oligonucleotides according to the complete genome sequence of AMV 425L isolate (GenBank No. L00163 for RNA1, X01572 for RNA2, and K03542 for RNA3). The 5'- and 3'-terminals regions of each RNA were amplified with the strategy described by Lozano et al. (1) and specific AMV oligonucleotides designed for the corresponding viral RNA. The complete genome of the AMV-Lst isolate comprised 3,643 nucleotides for RNA1 (No. FN667965), 2,593 nucleotides for RNA2 (No. FN667966), and 2,038 nucleotides for RNA3 (No. FN667967). Comparative sequence analyses revealed that the AMV-Lst isolate from Italy shared overall nucleotide sequence identities with the AMV isolate 425L of 97.1, 95.5, and 94.7% for RNA1, 2, and 3, respectively. P1 and P2 replicase genes and the movement protein and coat protein (CP) genes of AMV-Lst isolate showed, respectively, 97.2, 95.1, 96.2, and 97.8% identity with those from the 425L isolate. The AMV-Lst CP gene was shorter by nine nucleotides compared with the CP gene of 425L. A phylogenetic tree, obtained with neighbor-joining and maximum likelihood methods, showed that the Lst isolate grouped within subgroup I of AMV isolates confirmed that the differences between subgroups I and II correlate mainly with the geographic origin of isolates (2). Lst represents the first Italian isolate of AMV completely sequenced, and to our knowledge, this is the first report of this virus in L. stoechas.

First Report of Impatiens necrotic spot virus on Spiderlily in China.

Impatiens necrotic spot virus (INSV) (genus Tospovirus; family Bunyaviridae) is a devastating disease in the production of ornamental flowers (1). From 2007 to 2009, a survey of 10 major parks and recreation areas in Kunming, the capital of Yunnan Province, China, indicated that approximately 60 to 70% of Spiderlily (Hymenocallis littoralis Salisb.) plants from eight parks had symptoms of concentric ring spots and necrotic spots. Symptomatic plants were tested for INSV and Tomato spotted wilt virus (TSWV) with an immunostrip (Agdia Inc. Elkhart, IN). Results indicated that only the samples designated HDL were positive for INSV and all other samples were negative for both INSV and TSWV. Mechanically inoculated Emilia sonchifolia, Nicotiana glutinosa, Impatiens balsamina, and N. rustica showed chlorotic lesions, concentric rings, and severe necrosis, symptoms typical for INSV in these hosts. Electron microscope inspection found tospovirus-like spheroidal, enveloped particles that were 90 nm in diameter. Primer 5 software (Premier, Canada) was used to design 14 primers from GenBank Accession No. NC_003625 to amplify the L RNA, nine from NC_003616 to amplify the M RNA, and six from NC_003624 to amplify the S RNA. With total RNA extracted from infected plant tissue as templates in reverse transcription (RT)-PCR, these primers generated 29 target fragments of 250 to 900 bp. These fragments were cloned with the vector pMD19 simple-T vector (Takara Bio Inc., Dalian, China) and sequenced. The sequences of the clones were aligned with the software DNAman (version 2.5; Lynnon Biosoft, Quebec, Canada), showing that RNAs L, M, and S are 8,776 bp (GenBank Accession No. GU112505), 4,948 bp (GenBank Accession No. GU112503), and 2,875 bp (GenBank Accession No. GU112504), respectively. BLAST analysis of these Spiderlily INSV sequences against the NCBI sequence database indicated that the RdRp protein (L RNA) was 99.6% identical with the RdRp protein from an Italian isolate (No. DQ425094), the Nsm protein (M RNA) has 99.0% identity with the Nsm protein from an isolate from Italy (No. DQ425095) and one from the United States (No. NC_003616), the G1G2 polyprotein (M RNA) has 99.9% identity with the analogous protein from an Italian isolate (No. DQ425095), the N protein (S RNA) has 99.6% identity with the N protein from an Italian isolate (No. DQ425096), and the NSs protein (S RNA) has 98.7% identity with the NSs protein from an isolate from Japan (No. AB109100). To our knowledge, this is the first report of INSV on Spiderlily in China. Reference: (1) A. E. Whitfield et al. Annu. Rev. Phytopathol. 43:459, 2005.

Phylogenetic analysis of the VP2 gene of canine parvoviruses circulating in China

Sequences of the full-length gene encoding the main capsid protein VP2 of 22 strains of canine parvovirus (CPV), isolated from domestic dogs in China between 1983 and 2008, were determined and analyzed in comparison with the sequences of 30 other strains of CPV from China and reference CPV isolates retrieved from GenBank. Three types of CPV, including CPV-2, CPV-2a, and CPV-2b, were detected, and CPV-2a (with 297-Ala mutation) was predominant in China. The unique Ile-324 mutation in the VP2 of Chinese CPV isolates was detected, as compared with a Tyr-324 in the VP2 of the reference CPV strains. A phylogenetic tree constructed from the VP2 genes showed that most of the Chinese strains classified in a cluster consisting of Chinese and Korean field isolates, which were distinct from Thai, U.S., and Italian isolates.

Complete genome sequences of avian paramyxovirus serotype 6 prototype strain Hong Kong and a recent novel strain from Italy: Evidence for the existence of subgroups within the serotype

Complete genome sequences were determined for two strains of avian paramyxovirus serotype 6 (APMV-6): the prototype Hong Kong (HK) strain and a more recent isolate from Italy (IT4524-2). The genome length of strain HK is 16236 nucleotide (nt), which is the same as for the other two APMV-6 strains (FE and TW) that have been reported to date, whereas that of strain IT4524-2 is 16230 nt. The length difference in strain IT4524-2 is due to a 6-nt deletion in the downstream untranslated region of the F gene. All of these viruses follow the “rule of six”. Each genome consists of seven genes in the order of 3′N-P-M-F-SH-HN-L5′, which differs from other APMV serotypes in containing an additional gene encoding the small hydrophobic (SH) protein. Sequence comparisons revealed that strain IT4524-2 shares an unexpectedly low level of genome nt sequence identity (70%) and aggregate predicted amino acid (aa) sequence identity (79%) with other three strains, which in contrast are more closely related to each other with nt sequence 94–98% nt identity and 90–100% aggregate aa identity. Sequence analysis of the F-SH-HN genome region of two other recent Italian isolates showed that they fall in the HK/FE/TW group. The predicted signal peptide of IT4524-2 F protein lacks the N-terminal first 10 aa that are present in the other five strains. Also, the F protein cleavage site of strain IT4524-2, REP R↓L, has two dibasic aa (arginine, R) compared to the monobasic F protein cleavage site of PEP R↓L in the other strains. Reciprocal cross-hemagglutination inhibition (HI) assays using post-infection chicken sera indicated that strain IT4524-2 is antigenically related to the other APMV-6 strains, but with 4- to 8-fold lower HI tiers for the test sera between strain IT4524-2 and the other APMV-6 strains. Taken together, our results indicated that the APMV-6 strains represents a single serotype with two subgroups that differ substantially based on nt and aa sequences and can be distinguished by HI assay.

In Vitro Activity of Amphotericin B Against Candida lusitaniae Clinical Isolates

" In Vitro Activity of Amphotericin B Against Candida lusitaniae Clinical Isolates." Journal of Chemotherapy, 22(1), pp. 71–72

Identification of Merkel cell polyomavirus in the lower respiratory tract of Italian patients

Merkel cell polyomavirus (MCPyV) has been found to be integrated monoclonally in a rare skin cancer named Merkel cell carcinoma. More recently, MCPyV has been detected in the upper respiratory tract of pediatric and adult patients. However, the mode of transmission and pathogenic role of MCPyV in the respiratory system has not been determined. In this study, MCPyV was sought in the lower respiratory tract of adult patients admitted to the hospital. MCPyV DNA was detected in 15 (17.24%) out of 87 lower respiratory tract samples. Most of the patients with MCPyV were over 50 years old. Nucleotide sequence of the t-antigen of MCPyV identified in respiratory secretions showed a homology to those found in Merkel cell carcinoma. In addition, phylogenetic analysis undertaken on the t-antigen sequences of Italian isolates and other MCPyVs identified in healthy and cancer tissues showed that all these isolates belonged to the same clade. Selective pressure analysis for the t-antigen revealed the presence of five sites under positive selection (omega = 4.3), with a posterior probabilities above 0.99. The alpha parameter of the gamma distribution was 0.01, showing that this distribution has a characteristic L-shape and suggesting a strong nucleotide substitution rate heterogeneity across sites. This study shows that MCPyV can infect the lower respiratory tract, but further investigations are needed to define its pathogenic role in respiratory diseases. J. Med. Virol. 82:505-509, 2010. (c) 2010 Wiley-Liss, Inc.

Detection and molecular characterization of Italian Grapevine rupestris stem pitting‐associated virus isolates

The efficiencies of two published primer pairs (RSP13‐RSP14 and RSP2‐RSP21) were compared in reverse transcription‐polymerase chain reaction (RT‐PCR) to improve the diagnosis of Grapevine rupestris stem pitting‐associated virus (GRSPaV), which has consistently been found associated with rupestris stem pitting (RSP), one of the most common infectious diseases of grapevine. Fifty‐six grapevine accessions from different locations in Northern Italy were tested. Of these, 48 were positive with primers RSP13‐RSP14 and 37 with primers RSP2‐RSP21, suggesting that primer pair RSP13‐RSP14 has a broader detection spectrum than primers RSP2‐RSP21. The coat protein (CP) gene was also amplified by RT‐PCR and the amplicons analysed by single‐strand conformation polymorphism (SSCP) and sequencing. The results support the existence of wide variability within this virus, independent of cultivar or geographic origin. Sequence analysis of the 17 Italian GRSPaV isolates examined showed a molecular identity ranging from 78·8 to 99·7%, within which, four groups of sequence variants were recognized. Flexuous rod‐shaped particles, approximately 800 nm in length, were detected in plant extracts from infected grapevines and were positively decorated with the specific antiserum As7‐276 in immunosorbent electron microscopy (ISEM). This is the first report of GRSPaV particles in grapevines observed by transmission electron microscopy (TEM) in Italy.

Identification of recombination between subgenotypes IA and IB of hepatitis A virus

Co-circulation of subgenotypes IA and IB of hepatitis A virus (HAV) has been reported in South Africa, South America, Europe, and the United States. In this study, phylogenetic and recombination analyses were performed for the first time on 31 complete HAV genomes from infected humans and simians. Three potentially significant intra-genotypic recombination events (I-III) were identified by recombination detection analysis. Recombination events I and II occurred between the lineages represented, respectively, by the Japanese isolate AH2 (AB020565, subgenotype IA) and the North African isolate MBB (M20273, subgenotype IB), giving rise to the recombinant Uruguayan isolate HAV5 (EU131373). Recombination event III occurred between the lineages represented, respectively, by the North African isolate MBB (M20273, subgenotype IB) and the German isolate GBM (X75215, subgenotype IA), resulting in the Italian isolate FG (X83302). The findings demonstrate that humans can be co-infected with different HAV subgenotypes and provide valuable hints for future research on HAV diversity.

Field avian Metapneumovirus evolution avoiding vaccine induced immunity

Live avian metapneumovirus (AMPV) vaccines have largely brought turkey rhinotracheitis (TRT) under control in Europe but unexplained outbreaks still occur. Italian AMPV longitudinal farm studies showed that subtype B AMPVs were frequently detected in turkeys some considerable period after subtype B vaccination. Sequencing showed these to be unrelated to the previously applied vaccine. Sequencing of the entire genome of a typical later isolate showed numerous SH and G protein gene differences when compared to both a 1987 Italian field isolate and the vaccine in common use. Experimental challenge of vaccinated birds with recent virus showed that protection was inferior to that seen after challenge with the earlier 1987 isolate. Field virus had changed in key antigenic regions allowing replication and leading to disease in well vaccinated birds.

Characterization of Iris yellow spot virus isolates from onion crops in northern Italy.

SUMMARY 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. In 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 IYSV, 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 a heterogeneous and serologically distinct group of isolates.

An Amino Acid Deletion in Wheat streak mosaic virus Capsid Protein Distinguishes a Homogeneous Group of European Isolates and Facilitates Their Specific Detection.

The tritimovirus Wheat streak mosaic virus (WSMV) is widespread throughout the world and represents a severe threat to cereal crop production. To increase knowledge of genetic diversity of WSMV in Europe, until now scarce, capsid protein (CP) sequences of several Czech, French, Italian, Slovak, and Turkish isolates have been determined. A multiple alignment of CP nucleotide sequences using available WSMV sequences revealed only limited sequence variation among 3 previously sequenced European isolates and the 14 European isolates sequenced in this study. Moreover, these isolates were characterized by an identical 3-nucleotide deletion, resulting in the lack of the Gly2761 codon within the CP region of the polyprotein. The results indicate that this monophyletic group of isolates (designated as WSMV-ΔE) is common and widely dispersed throughout the European continent. The close relationship of WSMV-ΔE isolates implies a single common ancestor and, presumably, subsequent dispersal throughout Europe from a single focus. We developed two simple assays for specific and accurate detection of WSMV-ΔE isolates. First, a conserved ClaI restriction site in the core CP gene sequence unique to WSMV-ΔE isolates was used for restriction fragment length polymorphism analysis of amplified polymerase chain reaction (PCR) products. Second, the conserved and specific codon gap in WSMV-ΔE sequences was used as a target to design specific primers functional in one-step reverse-transcription PCR detection of WSMV-ΔE isolates.

Biological and Molecular Characterization of the Cucurbit aphid-borne yellows virus Affecting Cucurbits in Tunisia.

Surveys of yellowing viruses under nonheated and geothermal heated plastic tunnels and in open field crops of melon (Cucumis melo), cucumber (C. sativus), zucchini (Cucurbita pepo), squash (C. maxima), watermelon (Citrullus lanatus), and ware cucurbit (Ecballium elaterium) were carried out year-round during 2000-2001, 2003, and 2004 in the major cucurbit-growing areas in Tunisia. Severe yellowing symptoms on older leaves of cucurbits were observed in open fields and under plastic-tunnel production systems. These yellowing symptoms and large populations of aphids (Aphis gossypii) on a diversity of cucurbit crops in Tunisia support the hypothesis of a viral cause of the disease. Virus identification using double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), followed by reverse transcription-polymerase chain reaction (RT-PCR) and immunocapture (IC)-RT-PCR showed that Cucurbit aphid-borne yellows virus (CABYV) was largely distributed in melon, cucumber, zucchini, squash, and watermelon crops. Ware cucurbit (E. elaterium) and lettuce (Lactuca sativa) crops were identified as potential CABYV reservoirs. The RT-PCR-amplified partial coat protein (CP) and P4 genes were cloned and sequenced from nine Tunisian CABYV isolates. CP and P4 gene nucleotide and amino acid sequence comparisons as well as phylogenetic reconstructions showed that the Tunisian isolates clustered into two major subgroups. Comparisons with CABYV sequences retrieved from GenBank showed high nucleotide and CP amino acid identities, and close relationships of the Tunisian isolates with Italian and French isolates.

Undifferentiated nasopharyngeal carcinoma from a nonendemic area: Protective role of HLA allele products presenting conserved EBV epitopes

The role of genetic factors involved in the development of undifferentiated nasopharyngeal carcinoma (UNPC) in nonendemic areas has been poorly investigated. High-resolution human leukocyte antigen (HLA) class I genotyping carried out in 82 Italian UNPC patients and 286 bone marrow donors born in the same province showed that A*0201, B*1801, and B*3501, known to efficiently present Epstein-Barr virus (EBV)-derived epitopes, were significantly under-represented in UNPC patients. Moreover, the A*0201/B*1801 haplotype was significantly less frequent in UNPC cases, with a 90% reduced risk (odds ratio [OR] 0.1, 95% confidence interval [CI] = 0.0-0.5) to develop UNPC, suggesting an additive effect. Notably, all 5 BARF1 epitopes and 7 of the 8 LMP-2 epitopes known to bind A*0201 showed a fully conserved sequence in all the 31 Italian EBV isolates investigated. The 4 amino acid changes affecting the 436-447 LMP-2 epitope do not reduce, but rather increase in two cases, the predicted ability of "variant" epitopes to bind the HLA-A*0201 allele, as shown by immunoinformatic analysis. Moreover, a significantly increased risk for UNPC was associated with A*2601 (OR 2.4, 95% CI = 1.1-4.9) and B*4101 (OR 9.2, 95% CI = 2.5-34.3). These findings indicate that Italian UNPC patients have a distinct HLA-A and -B genotypic profile and suggest that the decreased risk for UNPC conferred by definite HLA class I molecules is probably related to their ability to efficiently present LMP-2 and BARF1 epitopes that are highly conserved in EBV isolates from this geographic region. These results have practical implications for the immunotherapy of UNPC.

First Report of Strawberry vein banding virus on Strawberry in Italy.

In the summers of 2007 and 2008, diseased strawberry plants (Fragaria x ananassa Duch.) were observed in production fields in Ferrara and Forlì-Cesena districts (Emilia-Romagna Region) in northern Italy. Plants exhibited poor growth, leaf chlorosis, decline, and reduced fruit production. Older leaves sometimes displayed a premature purplish discoloration, while the younger leaves appeared chlorotic and were reduced in size with a marked yellow edge. Symptom severity was dependent on the cultivar and growing conditions. Sixty-one leaf samples were collected from diseased plants from production fields and cultivar collections. Each sample was tested by grafting on Fragaria vesca (clone 'UC4 and UC5') and F. virginiana (clone 'UC10' and 'UC11'). Forty-five days after indexing different symptoms, characteristic of viral diseases, appeared on indicator plants. In particular, 24 samples showed Strawberry vein banding virus (SVBV)-like symptoms with chlorotic streaks along and on both sides of the main leaf veins of UC5, UC10, and UC11 indicator plants. Molecular methods were also used to better investigate the causal agent. Nucleic acids were extracted from young leaves of field and indicator plants by the cetyltrimethylammoniumbromide method (1). PCR analyses were performed with primer pair SVBVdeta/SVBVdetb as previously described to specifically amplify a product of 423 bp (2). SVBV was detected on all symptomatic indicator plants and corresponding field samples as well as on the positive control sample (supplied by J. D. Postman, National Clonal Germplasm Repository, Corvallis, OR and I. E. Tzanetakis, Oregon State University, Corvallis). No amplicons were detected from nucleic acids extracted from symptomless strawberry plants. PCR products, amplified from four Italian SVBV isolates, were cloned and sequenced and represent part of ORF IV of the SVBV genome that codes for the coat protein (CP). Italian SVBV isolates were more similar to the U.S. isolates than to the Chinese isolates (Genbank Accession Nos. AY862389, AY955374, X97304, AY605662, AY605663, and AY605664), showing 93 and 86% nt sequence identity, respectively. Strawberry vein banding disease has been reported previously in Italy in 1986 (3), but to our knowledge, this is the first finding of SVBV on strawberry field plants in Italy. SVBV was listed as a quarantine pest by the European and Mediterranean Plant Protection Organization (OEPP/EPPO) in 1978, but its spread has been increasing within European countries. Further studies should be done to ensure that strawberry propagating material is free of known viruses including SVBV.

Characterization of pABVA01, a Plasmid Encoding the OXA-24 Carbapenemase from Italian Isolates of Acinetobacter baumannii

Two epidemiologically unrelated carbapenem-resistant Acinetobacter baumannii isolates were investigated as representatives of the first Italian isolates producing the OXA-24 carbapenemase. Both isolates were of European clonal lineage II and carried an identical OXA-24-encoding plasmid, named pABVA01. Comparative analysis revealed that in pABVA01, bla(OXA-24) was part of a DNA module flanked by conserved inverted repeats homologous to XerC/XerD binding sites, which in other Acinetobacter plasmids flank different DNA modules, suggesting mobilization by a novel site-specific recombination mechanism.

‘Kwanzan Stunting’ syndrome: Detection and molecular characterization of an Italian isolate of Little cherry virus 1

Evident stunting was observed for the first time on Prunus serrulata ‘Kwanzan’ indicator trees in Southern Italy during the indexing of two sour cherry accessions from cultivars ‘Marasca di Verona’ and ‘Spanska’. Bud break and shooting were delayed and the developing leaves remained small. During the third year many Kwanzan plants died, regardless of the indexed cultivar. Electrophoretic analysis showed the presence of dsRNA pattern in extracts of stunted Kwanzan with a similar size to that of viruses of the family Closteroviridae. An identical pattern of more abundant dsRNA bands was obtained from GF305 seedlings grafted with the same sour cherry accessions. Observations by electron microscopy revealed the presence of long flexuous virus particles in both indicators (Kwanzan and GF305), characteristic of closteroviruses. Subsequent cloning work, starting from the dsRNA extracts of cultivar Marasca di Verona grafted on GF305 indicator, yielded 7 different clones, all showing high identity to the Little cherry virus 1 genome. Full sequencing of this virus isolate (ITMAR) was then done resulting in a complete genome composed of 16,936 nt. Primers designed on the obtained sequences for RT-PCR detection confirmed the presence of Little cherry virus 1 in Kwanzan and GF305 trees, inoculated with both sour cherry cultivars. Phylogenetic analysis of the minor coat protein grouped virus isolates into two clusters: one including Italian isolates of sweet cherry, Japanese plum, peach and almond, together with German sweet cherry UW1 isolate, and a second one containing the Italian isolates of sour cherry (ITMAR and ITSPA), that were found associated with strong symptoms of ‘Kwanzan Stunting’.