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Journal of Plant Pathology (2009), 91 (3), 521-525
Edizioni ETS Pisa, 2009
521
DETECTION AND VARIABILITY OF OLIVE LATENT VIRUS 3 IN THE MEDITERRANEAN REGION A. Alabdullah1, T. Elbeaino2, A. Minafra3, M. Digiaro2 and G.P. Martelli1,3 1 Dipartimento
di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy 2 Istituto Agronomico Mediterraneo, Via Ceglie 9, 70010 Valenzano (BA), Italy 3 Istituto di Virologia Vegetale del CNR, Unità Organizzativa di Bari, c/o DPPMA, Via Amendola 165/A, 70126 Bari, Italy
SUMMARY
RT-PCR and dot blot hybridization assays were successfully used for the detection of Olive latent virus 3 (OLV-3), a newly discovered olive-infecting virus belonging to the family Tymoviridae. To assess the geographical distribution of OLV-3, a total of 224 olive samples were collected from eight Mediterranean countries and tested by RT-PCR and dot blot hybridization. According to RT-PCR assay, OLV-3 was detected in all surveyed countries with an overall average infection of 30.4%, ranging from 17% (Portugal) to 56% (Turkey). In 12 of 68 samples (c. 20%) shown to be infected by RT-PCR, dot blot hybridization failed to detect the virus, possibly because of the low concentration of target RNA. Single strand conformation polymorphism (SSCP) analysis of the viral RNA dependent RNA polymerase (RdRp) domain, conducted on the 68 PCR amplicons from infected trees of different geographical origin, yielded eleven heterogeneous patterns. Computerassisted analysis of the RdRp nucleotide sequences showed that the level of variability among the isolates ranged from 5% to 17%. In a tree constructed with RdRp nucleotide sequences, viral isolates grouped into three distinct clusters with no relation to their geographical origin. Even though preliminary, the results of the field survey indicate that OLV-3 ranks among the most widespread olive-infecting viruses in the Mediterranean basin. Key words: OLV-3, Olive, Mediterranean region, PCR, dot blot hybridization, SSCP, cloning.
INTRODUCTION
Since 1979, fifteen different viruses belonging to nine different genera have been recovered from olive (Fernandes Félix and Clara, 2006; Alabdullah et al., 2009),
Corresponding author: A. Minafra Fax: +39.080.5443608 E-mail:
[email protected]
mostly detected in symptomless olive trees, including Strawberry latent ringspot virus (SLRSV) and Olive leaf yellowing-associated virus (OLYaV) that are recognized agents of bumpy fruit and leaf yellowing diseases (Martelli, 1999). Olive latent virus 3 (OLV-3) is a newly described virus tentatively assigned to the family Tymoviridae, which was identified in an apparently healthy olive tree of cv. Cellina di Nardò from Apulia (southern Italy) (Alabdullah et al., 2009). OLV-3 has a single stranded positive-sense RNA genome most of which (6699 nucleotides) has been sequenced, disclosing that it comprises four open reading frames and an identity up to 54% at the amino acid level with members of the genus Marafivirus from which, however, it differs because of the diverse genome organization and the presence of a single type of CP subunits (Alabdullah et al., 2009). Since no information was available on the geographical distribution, epidemiology and genetic variability of this virus, these issues were addressed with the present study.
MATERIALS AND METHODS
Virus sources. Materials used for the development of molecular detection tools were from the following olive accessions: (i) cv. Cellina di Nardò (CN1) from a varietal collection at Palagiano (Apulia, southern Italy), from which the virus was first identified; (ii) cv. Cima di Bitonto (accession P4) growing in the Campus of the University of Bari; (iii) cv. Coratina (accession P5) growing on premises of the Mediterranean Agronomic Institute of Bari (IAM-B). Virus-free olive seedlings grown under screen at the Department of Plant Protection and Applied Microbiology of the University of Bari were used as negative controls. To gather information on the geographical distribution of OLV-3, olive samples of different cultivars were collected in May-July 2006 from eight Mediterranean countries. Samples, consisting of 1- to 2-year-old cuttings, came from varietal collections and commercial fields in Greece, Italy, Lebanon, Malta, Portugal, Syria, Tunisia and Turkey. Samples were stored at 4°C until
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use for testing by RT-PCR and dot blot hybridization. Nucleic acid extraction. Total nucleic acids (TNAs) were extracted from 0.2 g of cortical scrapings by adsorption on silica particles (Rott and Jelkmann, 2001). For molecular hybridization, TNA extracts were further concentrated by ethanol precipitation and the pellet dissolved in RNase-free water. Double stranded (ds) RNAs were extracted and purified from 20 g of cortical scrapings of young shoots by phenol/chloroform extraction and chromatography through cellulose CF-11 column in the presence of 17% ethanol according to Dodds (1993). RT-PCR. A pair of primers (OLV3f: 5’-CCCGTTGAGCAAGTTGTCTTCC-3’ and OLV3r: 5’GCAGTGGCTGGAGAGCATGGAG-3’) amplifying a fragment of 176 bp, potentially suitable for the detection of OLV-3 by RT-PCR, were designed on the sequence of the viral RNA dependent RNA polymerase (RdRp) domain. Reverse transcription was performed using 1 mg of random DNA hexanucleotide mixture (Roche, Germany) and 200 units of Moloney murine leukaemia virus (M-MLV) reverse transcriptase in a 50 ml reaction for 1 h at 42°C, following the manufacturer’s instructions (Invitrogen, USA). Two ml (of a total of 50 ml) of cDNA were mixed with 25 ml of the amplification mixture [1x Taq Promega buffer, 1.5 mM MgCl2, 0.5 mM of each dNTP, 0.2 mM of each primer and one unit of Taq DNA Polymerase (Promega, USA)]. After denaturation at 94°C for 2 min, cycling was as follows: denaturation for 30 sec at 94°C, annealing for 30 sec at 58°C, extension for 20 sec at 72°C for 35 cycles, and final extension for 5 min at 72°C. Amplified products were analyzed in 6% polyacrylamide gel electrophoresis and stained by silver nitrate. Molecular hybridization. A digoxigenin-labelled riboprobe (pOLV3) was synthesized from a recombinant plasmid containing a DNA fragment of OLV-3 RdRp domain (nt positions 5421-5684) using SP6/T7 DIG RNA Labeling kit (Roche, Germany). For dot blot hybridization, concentrated TNAs (20-30 mg), extracted from infected and healthy olive plants were NaOH-denatured (50 mM, 2.5 mM EDTA) and blotted on a Hybond N+ nylon membrane (Amersham, USA). For northern blots, TNAs were electrophoresed in a 1% agarose gel and transferred to nylon membranes by capillarity in 400 mM NaOH for 4 h. Hybridization was done according to Grieco et al. (2000). Blotted TNAs were fixed with UV cross-linking, spotted membranes were pre-hybridized in pre-warmed hybridizing solution (Dig Easy Hyb Granules, Roche, Germany) for 30 min at 56°C and hybridized for 12 h at 56°C after adding the denatured RNA probe. Hybridization signals were detected by Dig-chemiluminescent detection kit following the manufacturer’s instructions (Roche, Germany).
Journal of Plant Pathology (2009), 91 (3), 521-525
Search for possible vectors. Individuals of the olive psyllid Euphyllura olivina and of the black scale Saissetia oleae, that infested the OLV-3-infected accession P5 (cv. Coratina), were collected and analyzed for the presence of this virus. Total nucleic acids (TNAs) extracted by the Trizol method (Singh et al., 1995) from ten adults and instars of S. oleae and ten E. olivina instars were resuspended in 50 µl of RNase-free water, 5 µl of which were used for cDNA synthesis as described above. Two µl of synthesized cDNA were mixed with 25 µl of the PCR reaction mixture and cycled under the conditions mentioned above. PCR products were analyzed in 6% PAGE and silver stained. TNAs extracted from the same insect species collected from OLV-3-free olives were used as control. One-year-old olive seedlings, determined to be OLV-3free by RT-PCR using primers OLV3f/OLV3r, were used for transmission trials. Different instars of E. olivina, collected from accession P5, were individually transferred onto five olive seedlings (15-20 instars each) and allowed to feed for 15 days. Seedlings were then sprayed with an insecticide and placed in double net cages in a glasshouse. Controls consisted of olive seedlings artificially infested with insects collected from PCR-negative olives. All seedlings were PCR-analyzed for the presence of OLV-3 three months after the end of the experiment. Single strand conformational polymorphism (SSCP). The variability of the nucleotide sequence of OLV3 RdRp domain was investigated by SSCP analysis. One volume (3 µl) of the PCR product generated from TNA or DNA recombinant plasmids, was added to 3 vol. of a loading buffer (95% formamide, 20 mM EDTA, 10 mM NaOH and a few grains of bromophenol-blue), prior to denaturation at 96°C for 10 min and chilling for 3 min on ice (Martins-Lopes et al., 2001). Denatured ssDNAs of the amplified RdRp fragment were subjected to electrophoresis in a non-denaturing 10% polyacrylamide gel (Markoff et al., 1997). To improve band resolution, an electrophoretic pre-run was done for 1 h before loading the samples. Electrophoresis was made at 4°C, at a constant voltage of 100V for the first 30 min, then at 200V for 2 h. DNA conformation patterns were visualized by silver staining. Cloning, sequencing and computer-assisted analysis. PCR products showing different SSCP patterns were ligated to StrataCloneTM PCR Cloning vector pSC-A (Stratagene, USA), following the manufacturer’s instructions, and transformed into Escherichia coli SoloPACK cells. Plasmids were extracted from bacterial cells by boiling according to Sambrook and Russell (2001). Plasmids considered as representative of distinct SSCP profiles were subjected to automated sequencing (PRIMM, Italy). Nucleotide and protein sequences were analysed
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with the DNA Strider 1.1 program (Marck, 1988). Multiple alignments of nucleotide and amino acid sequences were obtained using the default options of CLUSTALX 1.8 (Pearson and Lipman, 1988). Search for homologies with proteins from the Protein Information Resources database (PIR, release 47.0) was done with BlastX and BlastN (Altschul et al., 1997). The phylogenetic tree was constructed using the NJPLOT package (Perrière and Gouy, 1996) with 1000 bootstrap replicates.
RESULTS AND DISCUSSION
Development of molecular tools for OLV-3 detection. RT-PCR. Successful amplification of a product of the expected size (176 bp) was first obtained from TNA templates (Fig. 1). However, when TNA extracts were compared with dsRNA extracts, the latter yielded better defined bands, likely because treatments with phenol/chloroform and nucleases had cleaned the recovered nucleic acid from plant contaminants interfering with enzymatic reactions (Rezaian and Krake, 1987). Nevertheless, TNA extraction using silica particles was used for routine detection of OLV-3, because dsRNA extraction is laborious, time consuming and requires larger amount of tissues. Molecular hybridization. Hybridization with the pOLV3 DIG-labelled riboprobe was successful on both dsRNA and TNA extracts. Positive signals were obtained only from OLV-3 infected plants (Fig. 2A). In Northern blot hybridization, the same probe recognized specifically OLV-3 genomic RNA (ca. 7 kb in size), which was present only in TNA extracts from virus-infected samples (Fig. 2B). Survey of OLV-3 in the Mediterranean region. A total of 224 samples collected from commercial fields and varietal collections in Italy, Syria, Malta, Tunisia, Portugal, Turkey, Lebanon and Greece (Table 1) were tested by RT-PCR. OLV-3 was detected by RT-PCR in all surveyed countries with an infection rate ranging between 17% (Portugal) and 55.6% (Turkey). Dot blot hybridization assays on TNA extracts using pOLV3 DIGlabeled riboprobe failed to detect the virus in 12 of 68 PCR-positive samples (ca. 20%), possibily because of the low concentration of target RNA. No positive hybridization signals were obtained from any of the plants found to be OLV-3-free by PCR. Although PCR was more sensitive than dot blot hybridization for virus detection, its use for large scale surveys, as mentioned above, may not be advisable because of the cost and the time consuming manipulations needed. Nevertheless, PCR assays are essential in the framework of sanitary improvement programs for checking the results of molecular hybridization tests whenever there is a negative response.
Alabdullah et al. 1
2
3
4
5
6
7
8
9
10 11
12
13
523
14 15
1000 500
200
176 bp
100
Fig. 1. Polyacrylamide gel electrophoresis of products (176 bp) amplified from TNA extracts of field-grown olives by RTPCR, using the virus-specific primers OLV3f and OLV3r. Lane 1, DNA marker 100 bp (Promega, USA); lanes 4, 7, 9, 12 and 13, infected samples; lanes 2, 3, 5, 6, 8, 10 and 11, OLV-3-free samples; lane 14, water control; lane 15, positive control (accession CN1 in which OLV-3 was first identified).
Fig. 2. A. Dot blot hybridization using pOLV3 DIG-labeled riboprobe of dsRNA and TNA extracts of OLV-3-infected samples (accessions CN1, P4 and P5). Row (-), negative controls, which are TNA extracts of OLV-3-free olive seedlings according to RT-PCR assay. Row (+), positive control, i.e. plasmid containing the DNA fragment complementary to the pOLV3 RNA probe. B. Northern blot hybridization of total RNA extracts from OLV-3-infected accession CN1 (+) and a healthy olive seedling (-). Arrow points at the viral genomic RNA.
Table 1. OLV-3 incidence in different Mediterranean olivegrowing countries, as determined by RT-PCR assays. Country Italy Syria Malta Tunisia Portugal Turkey Lebanon Greece Total
Tested/infected samples (No.) 60/18 35/8 35/10 25/10 24/4 18/10 15/3 12/5 224/68
Infection (%) 30 23 29 40 17 56 20 42 30
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IT17
IT17
IT15 IT15
Journal of Plant Pathology (2009), 91 (3), 521-525 TU20 TU20
GR2 GR2
TU11 TU11
CN1 CN1
P5 P5
P4 P4
TK13 TK13
MA30 MA30
Detection and variability of OLV-3
SY21 SY21
524
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P4
439
P5
Group I
CN1 MA30 459
IT15
581
Group II 470
Fig. 3. SSCP analysis of PCR amplicons from olive samples of different geographical origin: Syria (SY21), Morocco (MA30), Turkey (TK13), Portugal (P4 and P5), Tunisia (Tu11 and TU20), Greece (GR2), Italy (IT15 and IT17)
SY21 TUR13 GR2 936
410
TUN11
667
Group III TUN20
Search for possible vectors. PCR of extracts from E. olivina instars that had fed on infected accession P5, yielded a single amplified product of the expected size (176 bp). By contrast, there was no amplification from S. oleae extracts, nor from the negative controls (not shown). Following cloning and sequencing, E. olivina amplicons were identified as polymerase fragments of the OLV-3 genome. There was no PCR detection of OLV-3 in olive seedlings exposed to presumably viruliferous E. olivina, three months after inoculation. Whether this is due to a period of time too short for the build up of a detectable virus titre in olive tissues or to the lack of virus transmission remains to be ascertained. SSCP and phylogenetic analyses. All amplicons obtained by PCR from the 68 infected samples from Mediterranean countries were subjected to SSCP analysis. A total of eleven diverse patterns were observed
469
IT17
0.01
Fig. 4. Phylogenetic tree constructed with nucleotide sequences of the RdRp domains of OLV-3 isolates from different Mediterranean countries. Numbers are bootstrap values for 1000 replicates.
(Fig. 3), resulting from nucleotide sequence variability of viral isolates, in line with the expected quasi-species nature of OLV-3. Six different groups of SSCP profiles were given by Italian isolates, two groups by each Maltese, Syrian, Tunisian, and Turkish isolate and only one group by each Greek, Lebanese and Portuguese isolate. Some patterns were common to samples from more than one country. The 176 bp PCR product of a representative isolate
Table 2. Nucleotide (upper diagonal) and amino acid (lower diagonal) sequence identity of the RdRp domain of OLV3 isolates from different Mediterranean countries. Nt. A.a.
Group I P4
P4
Group II
Group III
P5
CN1
MA30
IT15
SY21
TK13
GR2
TU11
TU20
IT17
93
92
90
86
92
90
91
87
91
86
93
91
86
93
90
89
85
89
85
89
86
92
90
89
86
88
88
91
95
89
85
83
88
83
91
89
85
83
87
85
92
88
86
91
86
86
85
89
87
94
92
87
91
86
P5
98
CN1
98
100
MA30
98
100
100
IT15
96
98
98
98
SY21
96
98
98
98
96
TUR13
98
100
100
100
98
98
GR2
98
98
98
98
96
96
98
TUN11
91
93
93
93
91
91
93
91
TUN20
98
100
100
100
98
98
100
98
93
IT17
93
94
95
94
93
93
95
93
90
86 95
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of each SSCP pattern was cloned and sequenced, and the obtained nucleotide sequences were aligned by ClustalX program. These sequences showed an interisolate variability at the nucleotide level ranging from 5% to 17%, and a lower variability (0% to 10%) at the amino acid level (Table 2). A phylogenetic tree constructed with polymerase sequences showed that viral isolates clustered in three distinct groups (I, II and III) with no connection with their geographical origin (Fig. 4). Maximum nucleotide variability within isolates of the same cluster was 8%, 11% and 14% for groups I, II and III, respectively (Table 2). Considering that this study took into consideration only part of the viral polymerase, which is the most conserved region in the genome of all members of the family Tymoviridae (Sabanadzovic et al., 2000), higher levels of nucleotide and amino acid variability can be expected in other OLV-3 genomic regions. In fact, when specific primers designed on the coat protein (CP) gene were used with olive samples infected by the same 11 isolates of the phylotree, they failed to amplify some of them, in particular those of group III (data not shown). Although it is more likely that these isolates represent molecular variants of OLV-3 rather than different virus species, further sequencing, in particular of the viral CP gene, will be needed before a definitive answer to this question can be obtained.
ACKNOWLEDGEMENTS
The authors wish to thank Prof. K. Caglayan, M.K. University, Turkey; Prof. C. Vovlas, DPPMA, University of Bari; Dr. M. Saponari, CNR Institute of Plant Virology, Bari; Dr. M. Rosario Felix, University of Evora, Portugal, for supplying some of the olive samples examined in this study. REFERENCES Alabdullah A., Minafra A., Elbeaino T., Saponari M., Savino V., Martelli G.P., 2009. Nucleotide sequence and genome organization of Olive latent virus 3, a new putative member the family Tymoviridae. Virus Research (submitted) Altschul S.F., Madden T.L., Schäffer A.A., Zhang J., Zhang Z., Miller W., Lipman D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25: 3389-3402.
Received March 6, 2009 Accepted April 16, 2009
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