Italian Isolates Research Articles

First Report of Pitch Canker Caused by Fusarium circinatum on Pinus halepensis and P. pinea in Apulia (Southern Italy)

Since 2005, pitch canker symptoms have been observed in Apulia (southern Italy, 41°27'42.84″N, 15°33'0.36″E) on numerous trees of Pinus halepensis and P. pinea in urban parks and gardens. Trees showed crown decline as a consequence of dieback of twigs and branches and withering of needles. Bleeding cankers with abundant resin were visible on twigs and branches. The needles of affected twigs and branches wilted, faded, turned yellow, then red, and were discarded. Isolations from symptomatic needles, twigs, and branches were performed on water agar, potato dextrose agar (PDA), and pentachloronitrobenzene medium. A species of Fusarium was consistently isolated from all infected tissues, and pure cultures were obtained by single hyphal tip transfers on PDA and synthetic nutrient agar medium (2). Colonies were incubated at 22 ± 3°C for 7 to 10 days. They produced white aerial mycelia, violet pigment, typically 3-septate macroconidia with slightly curved walls, single-celled microconidia, and characteristic sterile hyphal coils. Microconidia were ovoid or allantoid and born in false heads on aerial polyphialides. The species was identified as Fusarium circinatum Nirenberg & O'Donnell (= F. subglutinans Wollenweb & Reinking) on the basis of morphological and cultural characteristics (3). The identification was confirmed by PCR with specific primers CIRC1A/CIRC4A. The specific primer pair amplified a 360-bp DNA fragment of the two nuclear ribosomal IGS region (4). The pathogenicity of three Italian isolates of F. circinatum from Pinus spp. (Fc1640, Fc1642, and Fc1643 stored in the collection of Dipartimento Scienze Agroambientali, Chimica and Difesa Vegetale, University of Foggia) was evaluated by artificial inoculations on 2-year-old potted seedlings of P. halepensis, P. pinea, P. nigra, P. sylvestris, P. domestica, P. pinaster, P. excelsa, P. radiate, and Pseudotsuga menziesii (10 seedlings for each species and fungal isolate). Small PDA plugs from actively growing colonies of F. circinatum were introduced into a U-shaped cut on the stem of the seedlings and wrapped with moist sterile cottonwool. An equal number of control plants of each Pinus spp. was inoculated with sterile agar. All plants were grown in a nursery at ambient temperature (20 to 28°C). Within 30 days after inoculation, resinous cankers appeared on the stem of the seedlings of P. halepensis, P. pinea, P. domestica, P. pinaster, and P. radiata. Basal needles began to wilt, turn yellow, then red, and were discarded. F. circinatum was reisolated from stems of symptomatic seedlings. No symptoms were observed on seedlings of Pseudotsuga menziesii, P. sylvestris, P. excelsa, and P. nigra or on control seedlings. In Europe, pitch canker caused by F. circinatum previously has been reported only in Spain on P. radiata and P. pinaster (1). There was an unconfirmed report of this disease in Italy ( http://www.eppo.org ), but to our knowledge, this is the first definite conclusive evidence of the presence of pitch canker of pine in Italy.

Pinnularia obscura Krasske (Bacillariophyceae, Bacillariophyta) from acidic environments: characterization and comparison with other acid-tolerant Pinnularia species

The genus Pinnularia Ehr. (Bacillariophyceae, Bacillariophyta) is very frequently found in algal communities of acidic environments. Several Pinnularia strains isolated from the thermal acidic area of Pisciarelli (Naples, Italy) have been investigated by using a combination of morphological, ecophysiological and molecular techniques and have been compared with 15 Pinnularia strains from acidic and neutral environments, obtained from Loras College Freshwater Algal Collection. Electron microscopy (EM) observations showed the presence of a single Pinnularia species in Italian isolates, Pinnularia obscura. The complete identity between the strains of P. obscura isolated from the thermo acidic hydrosystem of Pisciarelli and from freshwater environments revealed the ability of this species to disperse throughout the globe, colonizing both acidic and neutral habitats. Overall, the genus Pinnularia has been confirmed to show a very weak morphological variation between species; in fact, the main morphological traits helpful to discriminate among them have been identified only by EM. Low pH values were tolerated by the majority of Pinnularia strains and species, both from acidic and neutral environments. As shown by a 18S rDNA phylogenetic analysis, acid-tolerance within Pinnularia is homoplasious, as a reversion from intolerance to tolerance is documented at least in P. gentilis.

Expressed and Unexpressed tet (M) Genes and the erm (B)-Carrying Tn 1116 Transposon in Streptococcus pyogenes and Streptococcus pneumoniae

It was exciting to read, in an excellent countrywide survey of erythromycin-resistant Streptococcus pneumoniae in Spain just published by Calatayud et al. (3), that Tn1116—a novel erm(B)-carrying element that we have recently described in Italian isolates of Streptococcus pyogenes and published in a recent issue of this journal (2)—was detected in 16 out of their 125 test strains. However, more careful reading reveals that the element detected in the Spanish pneumococci is not Tn1116, but a new structure. Tn1116 is a composite element of ca. 50 kb (2) resulting from the insertion of an erm(B)-containing DNA fragment into a defective Tn5397, a Tn916-related transposon originally found in Clostridium difficile (6) where the conventional int (integrase) and xis (excisase) genes (4) are replaced by the tndX (resolvase) gene (7). Most interestingly, in Tn1116 the erm(B)-containing DNA is inserted into the coding sequence of the tet(M) gene of the defective Tn5397, at base 15,019 of its published sequence (accession no. {type:entrez-nucleotide,attrs:{text:AF333235,term_id:27818171,term_text:AF333235}}AF333235). Therefore, although detectable by PCR using suitable primers, the tet(M) gene in Tn1116 is incomplete and silent, due to the lack of the portion of the gene upstream of the insertion. [The nucleotide sequence of Tn1116 between erm(B) and tndX has been deposited under accession no. {type:entrez-nucleotide,attrs:{text:AM411377,term_id:134269469,term_text:AM411377}}AM411377.] Accordingly, a distinguishing feature of Tn1116-carrying strains, among S. pyogenes isolates with erm(B)-mediated erythromycin resistance, is the association between a tet(M) genotype and a tetracycline-susceptible phenotype. In contrast, all of the 16 Spanish pneumococci putatively carrying Tn1116 harbored the erm(B), tet(M), and tndX genes but were tetracycline resistant (3). This means that, unlike in Tn1116, tet(M) was complete and was regularly expressed. It should be noted that the tet(M)-specific primer pair (1) used by Calatayud et al. (3) would be unable to detect the incomplete tet(M) of Tn1116, because the forward primer targets the initial, lacking portion of the gene. If this pneumococcal element—sharing with Tn1116 an erm(B), tet(M), and tndX genotype but associated with an erythromycin-resistant and tetracycline-resistant phenotype instead of an erythromycin-resistant and tetracycline-susceptible phenotype—is not Tn1116, it is nevertheless an interesting new element warranting further investigation. The crucial issue is the location of the erm(B) gene: the insertion of erm(B)-containing DNA into a Tn5397-related element outside the coding sequence of tet(M) may be a working hypothesis, but an actual linkage of erm(B) with tet(M) and tndX is still to be proved. On the other hand, PCR data suggest that erm(B) is neither on Tn916-like elements with conventional int and xis genes (4, 5, 7) nor on Tn917 (8). A completely new organization cannot be ruled out. Finally, it seems likely (though not expressly stated by the authors) (3) that the 16 isolates in question also share the iMLSB phenotype of erythromycin resistance. This would entail that they represent a particular clone relatively widespread among erythromycin-resistant pneumococci in Spain but less prevalent in other countries, where iMLSB pneumococci are very uncommon.

MOLECULAR ANALYSIS OF A CHINESE CITRUS TRISTEZA VIRUS ISOLATE SHOWING ANOMALOUS SEROLOGICAL REACTIONS

A Chinese isolate of Citrus tristeza virus (CTV), denoted HB1, was not recognized by a polyclonal antiserum (PAb-I) raised to the Italian CTV isolate 0002 and ten monoclonal antibodies to the Chinese isolate S4 (MAbs-S4). However, it reacted with a polyclonal antiserum (PAb-L5) raised to the Chinese isolate L5 in both ELISA and Western blots. To understand this unusual serological behaviour, the CP gene sequence of HB1 and other CTV isolates was determined. The results showed that HB1 was closely related to L5 at the nucleotide and amino acid levels. However, three amino acid residues in the HB1 CP differed from those of L5, which were conserved in all other isolates sequenced, including CTV-0002 and S4. This suggested that the failure of PAb-I and MAbs-S4 to recognise HB1 could depend on these molecular changes. Prediction of Antigenic Indexes and Surface Probability in four isolates (HB1, S4, L5 and 0002) with the Peptidestructure and Plotstructure softwares, showed that the HB1 tracing contained two small peaks at aa 84 and 190 that did not appear in any of the other profiles, but no apparent difference in aa 98. However, HB1 and L5 showed a similar small peak at the 5' end which did not occur in the S4 and CTV-0002.

Sequence analysis within the RNA 3 of seven Spanish tomato isolates of Parietaria mottle virus (PMoV-T) reveals important structural differences with the parietaria isolates (PMoV)

The genomic regions encoding the putative movement protein (MP), coat protein (CP) and intergenic region (IGR) of seven Spanish isolates of the Parietaria mottle virus which infects tomato plants (PMoV-T) were sequenced. Values for the genetic diversity of the PMoV-T isolates were 0.056, 0.047 and 0.013 for the CP, MP genes and IGR, respectively. Nucleotide and amino acid sequence comparison of the seven PMoV-T isolates with those of PMoV revealed significant differences. All of them had a cytosine deletion at position 1366, also confirmed in an Italian tomato isolate, which involves a start codon for the CP gene different from that for the PMoV sequence, resulting in a CP 16 amino acids shorter than the PMoV CP. The certainty of a cytosine deletion only associated to the tomato isolates or the possibility of a mistake in the PMoV published sequence are the two hypotheses that could explain this difference. Structural motifs highly conserved in Ilarviruses were identified in PMoV-T MP and CP. A stable hairpin structure is proposed for IGR, by the initiation site for subgenomic RNA 4 synthesis. Phylogenetic analysis of CP and MP amino acid sequences showed that Spanish PMoV-T isolates form a separate group from PMoV and other members of the Ilarvirus genus. Comparative analysis with different PMoV isolates including tomato isolates from other regions and isolates from different hosts are necessary to confirm this differentiation.

Genetic Diversity of Cryphonectria hypovirus 1 in China, Japan and Italy

AbstractThe aim of this study was to examine the origin of Cryphonectria hypovirus 1 (CHV1) which infects chestnut blight fungus, Cryphonectria parasitica. The genetic diversity of 39 CHV1 isolates from China, Japan and Italy was assessed by combining sequencing and restriction fragment length polymorphic (RFLP) experiments. Based on their partial sequences, 26 CHV1 isolates within 26 haplotypes were grouped into three subtypes. The 17 viruses from China were distributed between subtypes I and III, while all four of the isolates from Japan were subtype II. Of the five isolates from Italy, four were subtype I, and one (IT 192) was subtype III. Our analysis of the geographic distribution of the isolates indicated that all but one of the Chinese subtype III CHV1s are present in northern China; meanwhile all but three of the Chinese subtype I CHV1s are present in South China. Unlike in Europe where there is one predominant CHV1 subtype, our distribution analysis indicates that there has been gene flow between the populations in Asia. Cluster analysis based on the RFLP banding patterns showed that the viral isolates could be separated into four clusters. Most of the viral isolates (29 of 39, 74.4%) were grouped into one large cluster. Greater genetic diversity was observed among the CHV1s from China than among those from Japan and Italy. The Italian isolates were genetically more similar to the Japanese and South Chinese isolates than to the North Chinese isolates, indicating that CHV1 in Italy may have originated from Japan and/or South China.

Convergent evolution of phytopathogenic pseudomonads onto hazelnut

Pseudomonas syringae pv. avellanae (synonym: P. avellanae, Pav) is the causal agent of hazelnut decline in Greece and Italy. The population structure and evolutionary relationships of 22 strains from these two countries were examined by multilocus sequence typing (MLST) of four housekeeping genes (gapA, gltA, gyrB and rpoD). Neighbour-joining and maximum-likelihood phylogenetic analysis revealed that Greek strains isolated from the original 1976 outbreak of hazelnut decline through 1990 were very similar to Italian strains isolated from 2002 through 2004. Other Italian strains that were isolated during the 1990s were very homogeneous and clustered in a clade that was quite distinct from the Greek isolates and Italian isolates from the 2000s. A split decomposition analysis found evidence for recombination between these two highly divergent clades in two of the four MLST housekeeping genes. Incorporating these data into a broad MLST analysis of the P. syringae species complex showed that the Pav Greek and Italian strains from the 2000s clustered with P. syringae phylogroup 1, which is predominantly composed of pathogens of tomato and Brassicaceae hosts, while the Pav Italian strains from the 1990s clustered in P. syringae phylogroup 2 and are most closely related to pea (Pisum sativum L.) pathogens. These results clearly indicate that the ability to infect hazelnuts has arisen twice. This evolutionary process may be due to de novo adaptation to hazelnut by local P. syringae strains (such as the colonizers of Leguminosae crops), or the result of genetic exchange from the original Greek Pav clonal group into a phylogroup 2 strain. The latter explanation is intriguing since there is no exchange of hazelnut propagative material between Italy and Greece, which would be a likely vector for the movement of these pathogens.

Anthrax in Red Deer (Cervus elaphus), Italy

To the Editor: Anthrax is hypoendemic in Italy; a few outbreaks occurred yearly involving unvaccinated herbivores on pastures in central and southern regions and the major islands. Multiple-locus variable-number tandem-repeat analysis (MLVA) with 8 variable-number tandem repeats (VNTRs) of Italian isolates of Bacillus anthracis has identified 9 genotypes belonging to cluster A1a (1). An isolate of cluster A3 has been identified recently in Sardinia, which suggests that such a strain could have been introduced into Italy from another country (1).

The Genetic Structure of Cypress Canker Fungus in Italy Using RAPD and Minisatellite Markers

Over the past half century a destructive blight of Cupressus spp., caused by Seiridium cardinale, has spread worldwide from North America, devastating forests, plantations, and ornamental cypresses. The epidemic has been particularly severe in the Mediterranean region, on C. sempervirens. Seventy-seven isolates of Seiridium cardinale have been collected for the genetic characterization of the North-Italian populations of the fungus. Five Seiridium spp. isolates from different countries and different hosts growing in different parts of the world were used for comparison. The structure of the population has been analysed by means of Random Amplified Polymorphic DNAs (RAPDs) and Direct Amplification of Minisatellite-Region DNA (DAMD) PCR marker techniques by using the M13 core sequence. The results indicated a very high level of homogeneity in the North-Italian population of the fungus, whereas a certain variability was recognized in isolates from other hosts and other species. The isolates belonged to the North-Italian population appear to be very similar from the molecular comparison with both type of markers. The isolate from Greece was included in the same group of the Italian isolates. Only the S. cardinale from Chile was clustered at significant distance from the other S. cardinale isolates from Italy and Greece. The genetic homogeneity of the fungus in Italy suggests that this population has gone through a recent genetic bottleneck, perhaps from the introduction in Europe of few genotypes of the fungus. This supports the hypothesis that the pathogen was introduced to Europe during World War II on infected wood material from the United States. The results are discussed in relation to the introduction and spread of the fungus in Europe.

Evaluation of Susceptibility of Carnation Cultivars to Fusarium Wilt and Determination of Fusarium oxysporum fsp. dianthi Races in Southwest Spain

Eighteen commercial cultivars of carnation were inoculated with eight isolates of F. oxysporum fsp. dianthi (Fod) from a collection of isolates obtained from diseased plants surveyed in the main growing area of Spain. Susceptible reactions were shown in most cultivars inoculated with six isolates characterized as race 2 when tested on differentials. However, cultivars Elsy and Westcristal were fully resistant to the six isolates, whereas ‘Scarlet King’ showed a variable response, suggesting genetic diversity within race 2 of Fod. In contrast, eight cultivars inoculated with three isolates of races 1 or 8 were usually resistant except for cultivars Nelson and Solar, which were fully susceptible, and ‘Elsy’, which showed resistance to only one of the isolates. Partial polygenic inheritance of resistance to race 2 determines the complexity in the host reactions. Nine cultivars used in the previous experiment were inoculated with 13 Italian isolates of a collection of races of Fod. Cultivar reactions to isolates of different races agreed with some reports but not with others, suggesting an effect of environmental conditions or inoculation method. Reactions under field conditions sometimes differed from reactions with artificial inoculation in the greenhouse and growth chamber, usually more prone to express susceptibility.

Genetic Variation among European Lophodermium piceae Populations - Preliminary Results

Lophodermium piceae is a common needle endophyte of Norway spruce (Picea abies). The aim of the present study was to examine the degree of differentiation within and among European populations separated by various distances and geographical obstacles. For this purpose, populations (including > 10 isolates/subpopulation) were collected along a north-south transect stretching from the northern timberline in Finnish Lapland to the southern border of the distribution area of Norway spruce in northern Italy. Differentiation between L. piceae populations was determined from DNA sequences of three genetic markers. One of the markers was the internal transcribed spacer (ITS) of the ribosomal DNA and the other two (LP1 and LP2) were based on sequence characterized amplified regions (SCAR) designed for L. piceae. Preliminary results including sequences of Finnish, Swiss and Italian isolates show low differentiation among populations. According to analysis of molecular variance the among population variation was 1%, 5% and 0% in ITS, LP1 and LP2 markers, respectively.

Genetic structure and evolution of natural populations of viruses causing the tomato yellow leaf curl disease in Spain

The population structure and genetic variation of two begomoviruses: tomato yellow leaf curl Sardinia virus (TYLCSV) and tomato yellow leaf curl virus (TYLCV) in tomato crops of Spain were studied from 1997 until 2001. Restriction digestion of a genomic region comprised of the CP coat protein gene (CPR) of 358 TYLC virus isolates enabled us to classify them into 14 haplotypes. Nucleotide sequences of two genomic regions: CPR, and the surrounding intergenic region (SIR) were determined for at least two isolates per haplotype. SIR was more variable than CPR and showed multiple recombination events whereas no recombination was detected within CPR. In all geographic regions except Murcia, the population was, or evolved to be composed of one predominant haplotype with a low genetic diversity (<0.0180). In Murcia, two successive changes of the predominant haplotype were observed in the best studied population. Phylogenetic analysis showed that the TYLCSV sequences determined clustered with sequences obtained from the GenBank of other TYLCSV Spanish isolates which were clearly separated from TYLCSV Italian isolates. Most of our TYLCV sequences were similar to those of isolates from Japan and Portugal, and the sequences obtained from TYLCV isolates from the Canary island of Lanzarote were similar to those of Caribbean TYLCV isolates.

First Detection of Grapevine leafroll associated virus-7 in California Vineyards.

Grapevine leafroll is one of the most important viral diseases in grapevine and occurs wherever grapevines are grown. At least nine different Grapevine leafroll associated viruses (GLRaVs) belonging to different genera within the Closteroviridae family have been reported. Typical leafroll symptoms include downward rolling and interveinal reddening or yellowing of leaves, reduced fruit size, sugar content, and yield. Our laboratory has detected the presence of GLRaV-7 in many grapevine cuttings collected in California vineyards. During the 2005-2006 fall/winter testing season, symptomatic and asymptomatic grapevine varieties collected from different vineyards in California were tested for the detection of economically important viruses and processed for reverse transcription (RT)-PCR and ELISA. The RT-PCR assays using primers specifically designed for the detection of GLRaV-7 (C. Turturo et al., 13th ICVG [Abstr.], 2000) amplified a single band of approximately 200 bp in samples from selections of Chardonnay, Merlot, Pinot Noir, and Sauvignon Blanc cultivars. The same samples showed low or no reactivity to GLRaV-7 specific antibodies (BIOREBA AG, Reinach, Switzerland). The variability of detection by ELISA can be due to the lower sensitivity of ELISA as compared with RT-PCR and the uneven distribution of GLRaVs in grapevines (1). To confirm that the amplified DNA was GLRaV-7 specific, different isolates were sequenced and compared with the nucleotide and derived amino acid sequences obtained by Turturo et al. (13th ICVG [Abstr.], 2000). The California isolates showed 81 to 98% similarity with the Italian isolates. To our knowledge, this is the first report of GLRaV-7 in California and the United States. Our initial testing data indicates that GLRaV-7 is wide spread in California vineyards. Further studies will allow us to determine the potential effect of this virus in grapevines grown in California. Reference: (1) J. Monis, and R. K. Bestwick. Am. J. Enol. Vitic. 47:199, 1996.

Molecular genetic variability of Italian binucleate Rhizoctonia spp. isolates from strawberry

Fifty-eight binucleate Rhizoctonia isolates were collected over six years from strawberry plants displaying symptoms of black root rot in Italy. Almost all isolates were able to produce necrosis on strawberry roots, most of them also showed this ability on faba bean and, with lower frequency, on a crucifer and a cereal crop used in rotation with strawberry in Italy. The sequence alignment of Internal Transcribed Spacer (ITS) regions of 51 binucleate Rhizoctonia were analyzed and compared with a set of eight sequences representative of Rhizoctonia isolate Anastomosis Groups (AG) already found to be pathogenic on strawberry (AG-A, AG-G, AG-I and AG-F). The neighbour-joining tree, based on ITS region sequences, divided Italian strawberry Rhizoctonia isolates into two main clusters corresponding to AG-A and AG-G. The results were confirmed by hyphal anastomosis tests. The clustering obtained with the phylogenetic tree was also confirmed using PCR-Restriction Fragment Length Polymorphism of 28S rDNA to compare some isolates, defined as AG-A and AG-G on the basis of ITS region sequence analysis, with representative AG isolates pathogenic on strawberry. The AG-A and AG-G Rhizoctonia spp. were widespread in Italian strawberry-growing areas, although with different relative frequencies: AG-G was most frequent in northern (latitude 44°N) and AG-A in southern (latitude 39–40°N) Italy. Analysis of MOlecular VAriance, based on geographic location, showed that Rhizoctonia molecular variations between northern and southern Italy accounted for 36.6% of the total, but most of the variations (61%) occurred within each of the four geographical regions from where the isolates originated.

Partial nucleotide sequence analysis of Turkish isolates of Beet necrotic yellow vein virus (BNYVV) RNA‐3

A survey was carried out to detect Beet necrotic yellow vein virus (BNYVV) in soil samples using RT‐PCR and bait plant techniques from the sugar beet production area of Tokat, Turkey in 2001. More than 80% of the soil samples analyzed were found to be contaminated with the virus. The partial nucleotide sequence of cDNA corresponding to RNA‐3 of BNYVV isolates were analyzed for six different regions of Tokat province. All isolates were assigned to type A strains based on RFLP analysis and DNA sequences. Sequence comparison revealed differences at amino acid positions 35, 68, 71 and 179 of the P25 coding region amongst Turkish isolates. Additionally, all Turkish isolates were compared with Japanese, French, Kazakh, Italian and Belgian isolates.

First Report of Cucumber mosaic virus in Viola hederacea in France and Italy.

Viola hederacea Labill. (Australian violet or trailing violet), family Violaceae, is native to eastern Australia and used for its ornamental quality in humid conditions, especially in terrariums. Mosaic and chlorotic ringspots associated with a mild crinkling on leaves of V. hederacea were observed in gardens in southeast France and La Mortola, Italy in 2001 and 2003, respectively. These symptoms were different from the small chlorotic spots reported in the same species in Queensland, Australia, from which a rhabdovirus was isolated (1). In samples collected from both locations, presence of Cucumber mosaic virus (CMV) was suspected on the basis of the symptoms exhibited by a range of inoculated plants (2) and the observation of isometric particles of approximately 30 nm with the electron microscope in sap preparations. This was confirmed by positive reactions with CMV polyclonal antiserum (3) in double-antibody sandwich-ELISA. Because of the viscosity of the V. hederacea extracts, 0.01% (w/v) of pectolyase in phosphate buffer was used to grind the samples and the homogenate was kept at room temperature for 2 h. In double-immunodiffusion analysis, each isolate was shown to belong to the group II of CMV. Tomato strains D and To were used as positive controls for CMV groups I and II, respectively. The virus was transmitted by aphids (Myzus persicae) in a nonpersistent manner from infected Nicotiana tabacum cv. Xanthi-nc plants to healthy Xanthi-nc plants. To determine if CMV was responsible for the symptoms observed, the French and Italian isolates were multiplied in Xanthi-nc tobacco plants after isolation from local lesions on Vigna unguiculata and mechanically inoculated to 10 plants of V. hederacea. A systemic vein clearing developed on leaves 10 days after inoculation, followed by mosaic and crinkling. CMV was detected in each plant 3 weeks after inoculation. To our knowledge, this is the first report of CMV in V. hederacea in France and Italy.

P876 Fluoroquinolone resistance in Clostridium difficile Italian clinical isolates

P876 Fluoroquinolone resistance in Clostridium difficile Italian clinical isolates

RNA INTERFERENCE FOR SHARKA DISEASE RESISTANCE

Sharka is the most important disease of stone fruits in terms of agronomic impact and economic importance. The disease is caused by Plum pox virus (PPV) a single-stranded RNA virus. In a recent work, we transformed Nicotiana benthamiana plants with four PPV sequences, covering the PI and HC-Pro genes, arranged to express self-complementary hairpin RNA. Two lines for each construct were challenged with PPV. All the transgenic plants were resistant to PPV infection, with the exception of a fraction containing the P1/HcPro construct. The P1/HcPro lines, in addition to the type of gene construct utilised, differ from the other lines in that they are multiple copy lines. In the current study, three additional P1/HcPro N. benthamiana lines were analysed. All thirty plants challenged with the Italian ISPaVe44 PPV isolate were fully resistant to the virus infection. Between one and three transgenic loci were present in the transformed lines suggesting that a single transgenic locus was not an absolute prerequisite for obtaining plants completely resistant to PPV infection. Additionally, in order to introduce PPV resistance in Prunus species, transformation techniques were developed for Prunus domestica (plum) 'Stanley'. Transgenic plums were obtained, as confirmed by PCR and histochemical GUS assays.

Molecular Characterization of a New Variant of Rotavirus P[8]G9 Predominant in a Sentinel-Based Survey in Central Italy

Rotavirus P[8]G9 was recognized as the most widespread genotype during a sentinel-based survey in Italy; phylogenetic analysis of the VP7 and VP4 genes showed that Italian isolates constituted a closely related genetic cluster distinct from the other G9 strains recently isolated in other European countries, America, and Asia.

Phylogenetic analysis of partial S1 and N gene sequences of infectious bronchitis virus isolates from Italy revealed genetic diversity and recombination

A total of ten infectious bronchitis virus (IBV) isolates collected from commercial chickens in Italy in 1999 were characterized by RT-PCR and sequencing of the S1 and N genes. Phylogenetic analysis based on partial S1 gene sequences showed that five field viruses clustered together with 793/B-type strains, having 91.3–98.5% nucleotide identity within the group, and one isolate had very close sequence relationship (94.6% identity) with 624/I strain. These two IBV types have been identified in Italy previously. The other three variant isolates formed novel genotype detected recently in many countries of Western Europe. For one of these variant viruses, Italy-02, which afterwards became the prototype strain, the entire S1 gene was sequenced to confirm its originality. In contrast, phylogenetic analysis of more conserved partial N gene sequences, comprising 1–300 nucleotides, revealed different clustering. Thus, three variant IBVs of novel Italy-02 genotype, which had 96.7–99.2% S1 gene nucleotide identity with each other, belonged to three separate subgroups based on N gene sequences. 624/I-type isolate Italy-06 together with Italy-03, which was undetectable using S1 gene primers, shared 97.7% and 99.3% identity, respectively, in N gene region with vaccine strain H120. Only one of the 793/B-type isolates, Italy-10, clustered with the 793/B strain sharing 99.3% partial N gene identity, whereas the other four isolates were genetically distant from them (only 87.7–89.7% identity) and formed separate homogenous subgroup. The results demonstrated that both mutations and recombination events could contribute to the genetic diversity of the Italian isolates.