SHORT COMMUNICATION FIRST REPORT OF CHRYSANTHEMUM …
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SUMMARY Chrysanthemum stunt viroid (CSVd) is a quarantine pathogen for chrysanthemum (Dendranthema spp.) in the European countries (Plant Health Directive 2000/29/EC), because this host is severely affected, thus comes down with a disease characterized by stunting, leaf chlorosis and floral disorders. CSVd spread in Eu- rope has efficiently been restrained so far, although sev- eral outbreaks were recorded in the past. Here we re- port the first occurrence of CSVd in Italy, as detected in several symptomless cultivars of Argyranthemum frutescens (marguerite daisy) by RT-PCR with specific primers and by Northern blot hybridization with a spe- cific digoxigenin-labeled riboprobe. Viroid identity was ultimately ascertained by cloning and sequencing cDNA amplicons. Molecular characterization of CSVd isolates from six different A. frutescens cultivars disclosed viroid RNA populations with a prevalent size of 354 nt and se- quences 98-100% identical to those of CSVd variants reported previously from D. grandiflora and A. frutescens. These results call for a prompt extension of surveys for assessing the presence of CSVd in symptom- less A. frutescens and other ornamentals, which could constitute hidden reservoirs of this pathogen. In view of this, a tissue-printing hybridization method for detect- ing CSVd in A. frutescens was tested and validated. Key words: viroids, detection, quarantine, RT-PCR, Northern blot hybridization, molecular characteriza- tion. Chrysanthemum stunt viroid (CSVd) (Diener and Lawson, 1973), a member of the genus Pospiviroid, family Pospiviroidae (Owens et al., 2012), included in the EPPO A2 list of quarantine pests (OEPP/EPPO 2011a), can cause serious damage to chrysanthemums (Dendranthema spp.) characterized by stunting, leaf yel- lowing and deformation of the flowers. Natural hosts of Corresponding author: F. Di Serio Fax: +39.080.5443071 E-mail: [email protected] CSVd (Bouwen and Zaayen, 2003) include also Argy- ranthemum frutescens, as reported from Germany (Menzel and Maiss, 2000) and, more recently, France (Marais et al., 2011). This work reports the identifica- tion of CSVd from several cultivars of A. frutescens in Italy. Symptomless A. frutescens plants of cvs Butterfly, Bright carmine, Sole mio, Meteor red, Reflection cream, Summersong white, Summersong yellow and Balzen roses were tested for the presence of CSVd. A silica-gel capture system (Foissac et al., 2001), modified accord- ing to Hajizadeh et al. (2012), was used to obtain total nucleic acid (TNA) from 100-200 mg of leaf tissues. TNA from a viroid-free chrysanthemum was used as negative control. Detection of CSVd by RT-PCR was done by synthe- sizing first strand cDNAs with random hexamers and the high-capacity cDNA reverse transcription kit (Ap- plied Biosystems, USA) following the manufacturer’s in- structions. PCR amplification reactions were carried out using GoTaq DNA polymerase and the specific primers CSVdRV (5’-CTCGCAGGAGTGGGGTCCTAAG-3’) and CSVdFW (5’-CTCCTGCGAGACAGGAG- TAATC-3’), complementary and identical, respectively, to positions 134-155 and 146-167 of the reference viroid variant (GenBank accession No. NC_002015), which are specific for amplification of CSVd full-length cDNA. The PCR amplification protocol (94°C for 30 sec, primer annealing for 30 sec at 57˚C and an exten- sion at 72˚C for 40 sec) encompassed 30 cycles after an initial denaturation at 94˚C for 3 min and a final elonga- tion step at 72˚C for 7 min. Amplification products were analyzed by electrophoresis on 1.2% agarose gels buffered in 1X TAE (0.04 M Tris-acetate, 1 mM EDTA, pH 7.2) and visualized under UV light after ethidium bromide staining. Amplicons of the expected size (354-356 bp) (Haseloff and Symons, 1981; Gross et al., 1982), were generated from all tested samples, but not from healthy controls (Fig. 1). CSVd infection was further confirmed by sub- mitting three of the RT-PCR-positive samples (cvs Mete- or red, Bright carmine and Sole mio) to Northern-blot hybridization. To this aim, TNA preparations were frac- tionated on denaturing 5% PAGE gels containing urea 8 Journal of Plant Pathology (2012), 94 (2), 451-454 Edizioni ETS Pisa, 2012 451 SHORT COMMUNICATION FIRST REPORT OF CHRYSANTHEMUM STUNT VIROID IN ARGYRANTHEMUM FRUTESCENS IN ITALY E.M. Torchetti 1 , B. Navarro 1 , V.N. Trisciuzzi 2 , L. Nuccitelli 3 , M.R. Silletti 2 and F. Di Serio 1 1 Istituto di Virologia Vegetale del CNR, UOS Bari, Via Amendola 165/A, 70126 Bari, Italy 2 Centro di Ricerca e Sperimentazione in Agricoltura Basile Caramia (CRSA), Via Cisternino 281, 70010 Locorotondo (BA), Italy 3 Servizio Fitosanitario Regionale del Lazio, Via Rosa Raimondi Garibaldi 7, 00145 Roma, Italy
Transcript of SHORT COMMUNICATION FIRST REPORT OF CHRYSANTHEMUM …
SUMMARY
Chrysanthemum stunt viroid (CSVd) is a quarantinepathogen for chrysanthemum (Dendranthema spp.) inthe European countries (Plant Health Directive2000/29/EC), because this host is severely affected, thuscomes down with a disease characterized by stunting,leaf chlorosis and floral disorders. CSVd spread in Eu-rope has efficiently been restrained so far, although sev-eral outbreaks were recorded in the past. Here we re-port the first occurrence of CSVd in Italy, as detected inseveral symptomless cultivars of Argyranthemumfrutescens (marguerite daisy) by RT-PCR with specificprimers and by Northern blot hybridization with a spe-cific digoxigenin-labeled riboprobe. Viroid identity wasultimately ascertained by cloning and sequencing cDNAamplicons. Molecular characterization of CSVd isolatesfrom six different A. frutescens cultivars disclosed viroidRNA populations with a prevalent size of 354 nt and se-quences 98-100% identical to those of CSVd variantsreported previously from D. grandiflora and A.frutescens. These results call for a prompt extension ofsurveys for assessing the presence of CSVd in symptom-less A. frutescens and other ornamentals, which couldconstitute hidden reservoirs of this pathogen. In view ofthis, a tissue-printing hybridization method for detect-ing CSVd in A. frutescens was tested and validated.
Key words: viroids, detection, quarantine, RT-PCR,Northern blot hybridization, molecular characteriza-tion.
Chrysanthemum stunt viroid (CSVd) (Diener andLawson, 1973), a member of the genus Pospiviroid,family Pospiviroidae (Owens et al., 2012), included inthe EPPO A2 list of quarantine pests (OEPP/EPPO2011a), can cause serious damage to chrysanthemums(Dendranthema spp.) characterized by stunting, leaf yel-lowing and deformation of the flowers. Natural hosts of
Corresponding author: F. Di SerioFax: +39.080.5443071E-mail: [email protected]
CSVd (Bouwen and Zaayen, 2003) include also Argy-ranthemum frutescens, as reported from Germany(Menzel and Maiss, 2000) and, more recently, France(Marais et al., 2011). This work reports the identifica-tion of CSVd from several cultivars of A. frutescens inItaly.
Symptomless A. frutescens plants of cvs Butterfly,Bright carmine, Sole mio, Meteor red, Reflection cream,Summersong white, Summersong yellow and Balzenroses were tested for the presence of CSVd. A silica-gelcapture system (Foissac et al., 2001), modified accord-ing to Hajizadeh et al. (2012), was used to obtain totalnucleic acid (TNA) from 100-200 mg of leaf tissues.TNA from a viroid-free chrysanthemum was used asnegative control.
Detection of CSVd by RT-PCR was done by synthe-sizing first strand cDNAs with random hexamers andthe high-capacity cDNA reverse transcription kit (Ap-plied Biosystems, USA) following the manufacturer’s in-structions. PCR amplification reactions were carried outusing GoTaq DNA polymerase and the specific primersCSVdRV (5’-CTCGCAGGAGTGGGGTCCTAAG-3’)and CSVdFW (5’-CTCCTGCGAGACAGGAG-TAATC-3’), complementary and identical, respectively,to positions 134-155 and 146-167 of the reference viroidvariant (GenBank accession No. NC_002015), whichare specific for amplification of CSVd full-length cDNA. The PCR amplification protocol (94°C for 30sec, primer annealing for 30 sec at 57˚C and an exten-sion at 72˚C for 40 sec) encompassed 30 cycles after aninitial denaturation at 94˚C for 3 min and a final elonga-tion step at 72˚C for 7 min. Amplification products wereanalyzed by electrophoresis on 1.2% agarose gelsbuffered in 1X TAE (0.04 M Tris-acetate, 1 mM EDTA,pH 7.2) and visualized under UV light after ethidiumbromide staining.
Amplicons of the expected size (354-356 bp) (Haseloffand Symons, 1981; Gross et al., 1982), were generatedfrom all tested samples, but not from healthy controls(Fig. 1). CSVd infection was further confirmed by sub-mitting three of the RT-PCR-positive samples (cvs Mete-or red, Bright carmine and Sole mio) to Northern-blothybridization. To this aim, TNA preparations were frac-tionated on denaturing 5% PAGE gels containing urea 8
Journal of Plant Pathology (2012), 94 (2), 451-454 Edizioni ETS Pisa, 2012 451
SHORT COMMUNICATION
FIRST REPORT OF CHRYSANTHEMUM STUNT VIROIDIN ARGYRANTHEMUM FRUTESCENS IN ITALY
E.M. Torchetti1, B. Navarro1, V.N. Trisciuzzi2, L. Nuccitelli3, M.R. Silletti2 and F. Di Serio1
1Istituto di Virologia Vegetale del CNR, UOS Bari, Via Amendola 165/A, 70126 Bari, Italy2Centro di Ricerca e Sperimentazione in Agricoltura Basile Caramia (CRSA), Via Cisternino 281, 70010 Locorotondo (BA), Italy
3Servizio Fitosanitario Regionale del Lazio, Via Rosa Raimondi Garibaldi 7, 00145 Roma, Italy
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452 CSVd in A. frutescens Journal of Plant Pathology (2012), 94 (2), 451-454
M and 1X TBE (90 mM Tris, 90 mM boric acid and 2mM EDTA) and electrotransferred in 0.5X TBE bufferto nylon membranes (Hybond-N, Amersham, UK).Membranes were hybridized with a digoxigenin (DIG)-labeled riboprobe specific for detecting the CSVd (+)strand; the probe was prepared by in vitro transcriptionwith the DIG RNA labeling kit SP6/T7 (Roche AppliedScience, Germany) using as template a plasmid contain-ing a full-length viroid cDNA insert kindly provided byR. Flores (Universidad Polytécnica de Valencia, Spain)Prehybridization, hybridization and washing of the mem-branes were done as previously reported (Hajizadeh etal., 2012). Hybridization signals were detected with anti-digoxigenin-AP Fab fragments and revealed with thechemi-luminescent substrate CDP-star (Roche AppliedScience) by exposure to an X-ray film. Hybridization sig-nals corresponding to bands co-migrating with CSVd cir-cular and linear forms were detected in the three testedsamples (Fig. 2).
Viroid identity was conclusively ascertained bycloning and sequencing the full-length cDNAs from sixcultivars (Sole mio, Reflection cream, Bright carmine,Meteor red, Summersong yellow and Butterfly). For thispurpose, cDNA was amplified using the same protocolreported above, but replacing the Taq DNA polymerasewith the Expand High Fidelity PCR system (Roche Ap-plied Science, Germany). Amplicons of the expectedsize were eluted from the gel, cloned in the pGEM-TEasy plasmid (Promega, USA) and automatically se-quenced (MWG-biotech, Ebersberg, Germany). Se-quencing of sixteen recombinant plasmids (two clonesfrom each isolate) showed cDNA inserts with the uni-form size of 354 nt (GenBank accession Nos fromJQ685734 to JQ685742). The sequenced inserts were98-100% identical to CSVd variants AF394452 andX16408 previously reported from chrysanthemums. In-terestingly, variants from these new CSVd isolates were98-99% identical to a 355 nt CSVd contig (AN:JF938538) recently assembled using sequences of three
different amplicons generated from A. frutescens inFrance (Marais et al., 2011). Sequence variants identi-fied in this study were compared with each other andwith those already deposited in databases by multiplealignments and generation of phylogenetic trees usingCLUSTAL W2 (Larkin et al., 2007) and the softwareMEGA version 5.05 (Tamura et al., 2011), respectively.Sequence variability was small, and no clustering withsignificant bootstrap values, that could highlight possi-ble epidemiological relationships between the Italianand other CSVd isolates previously reported, was iden-tified in the phylogenetic trees (data not shown).
Following CSVd identification in A. frutescens, thefirst in Italy to our knowledge, the Italian PhytosanitaryServices immediately traced the source of infection to a
Fig. 1. Agarose gel electrophoresis analysis of DNA products amplified from A. frutescens by RT-PCR.Lanes 1 and 3, viroid-free and CSVd-infected chrysanthemum used as negative and positive controls, re-spectively; lane 2, DNA molecular marker VI (Roche Applied Science, Germany) with migration positionsof the 298-bp and 394-bp DNA fragments indicated on the left; lanes 4, 6 and 11, cv. Meteor red; lanes 9,12, 16, 18, cv Sole mio; lane 7, cv. Reflection cream; lanes 13 and 14, cv. Bright carmine; lane 15, cv. Butter-fly; lanes 17, cv. Summersong white; lane 19, cv. Summersong yellow; lane 3, cv. Balzen rose.
Fig. 2. Detection of CSVd infections in A. frutescens by North-ern-blot analysis. Lanes 1-3, total nucleic acid preparations(TNA) from cvs Meteor red, Bright carmine and Sole mio, re-spectively; lane 4, TNA from a viroid-free chrysanthemum;lane 5, TNA from a positive control. Positions of circular andlinear viroid RNA forms are indicated on the left.
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Journal of Plant Pathology (2012), 94 (2), 451-454 Torchetti et al. 453
lot of plants imported from Brazil by a commercialnursery in Latium (central Italy), enforcing the destruc-tion of the infected lot and the disinfection of the green-houses (OEPP/EPPO, 2011b). Preliminary surveys inthe interested areas excluded spreading of the infectionto other crops. Although the presence of CSVd was as-certained in the same nursery in additional A. frutescensand chrysanthemum plants, none of these originatedfrom imports other than Brazil (F. Faggioli and L. Nuc-citelli, unpublished information).
Notwithstanding this encouraging finding, an exten-sion of field surveys appears desirable for assessing thesanitary status of A. frutescens cultivars and other symp-tomless ornamentals that could constitute hidden CSVdreservoirs. To this purpose, a tissue-printing hybridiza-tion (TPH) method for CSVd detection in A. frutescenswas tested and validated using membranes printed withfreshly-cut leaf petioles from plants already examinedfor the presence of the viroid. TPH has already beensuccessfully employed for CSVd detection in chrysan-themum (Hooftman et al., 1996; Tomassoli et al., 2004).We now confirm that this method, using the specificCSVd DIG-labelled probe reported above and the pro-tocol outlined by Di Serio (2007), is also effective fordetecting CSVd in A. frutescens (Fig. 3) and possibly inother ornamental species.
ACKNOWLEDGEMENTS
Our sincere thanks to Dr. C. Parodi for providing theplant material, the Italian Plant Protection Services for in-forming us about the undertaken measures, Prof. R. Flo-
res for kindly providing the transcription vector for theCSVd probe synthesis, and Dr. F. Faggioli for criticalreading the manuscript. This study was supported byfunds of the MIPAAF project OIGA-IN.OR.QUA (2009-2012) and PSTVd-free. EMT is recipient of a fellowshipfrom the Italian Society for Virology (SIV).
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Fig. 3. Detection of CSVd infection in A. frutescens by tissue-printing hybridization. The analysis was performed using tis-sues from the same samples tested by RT-PCR and Northern-blot (positions from A1 to C5); positive and negative controlswere spotted at positions D1 and D2, respectively; no sam-ples were applied to positions D3-D5.
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454 CSVd in A. frutescens Journal of Plant Pathology (2012), 94 (2), 451-454
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Received February 21, 2012Accepted March 19, 2012
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