DETECTION OF FLMaV-1 AND FLMaV-2 IN THE ... - SIPaV

018_JPP485Toufic_425

25-06-2009

12:21

Pagina 425

Journal of Plant Pathology (2009), 91 (2), 425-431

Edizioni ETS Pisa, 2009

425

DETECTION OF FLMaV-1 AND FLMaV-2 IN THE MEDITERRANEAN REGION AND STUDY ON SEQUENCE VARIATION OF THE HSP70 GENE T. Elbeaino1, S. Nahdi1, M. Digiaro1, A. Alabdullah2 and G.P. Martelli2 1 Istituto

Agronomico Mediterraneo, Via Ceglie 9, 70010 Valenzano-Bari, Italy. di Protezione delle Piante e Microbiologia Applicata, Università degli Studi and Istituto di Virologia Vegetale del CNR, Unità Organizzativa di Bari, Via Amendola 165/A, 70126 Bari, Italy 2 Dipartimento

SUMMARY

The presence of Fig leaf mottle-associated virus 1 (FLMaV-1) and Fig leaf mottle-associated virus 2 (FLMaV-2) was investigated in fig orchards of six Mediterranean countries. A total of 415 samples coming from Albania, Algeria, Lebanon, Syria, Tunisia and Italy were collected and tested by PCR, which detected the presence of at least one of the tested viruses in all surveyed countries. Except for Algeria and Syria, where FLMaV-1 was absent, both viruses were found in all surveyed countries with mean infection rates of 34% and 18.5% for FLMaV-1 and FLMaV-2, respectively. More than 7% of the samples were infected with both viruses. As ascertained by dsRNA analysis, 25 out of 65 samples (ca. 38.5%) showed visible bands in polyacrylamide gel electrophoresis. Single strand conformation pattern (SSCP) analysis of a portion of the viral HSP70 gene from figs of different geographical origin showed heterogeneous patterns for both viruses. The HSP70 gene variability was confirmed by sequence analysis. The level of nucleotide variability between the isolates reached 18% for FLMaV-1 and 15% for FLMaV-2. While the deduced amino acid sequence variability maintained almost the same range of divergence in the case of FLMaV-1 (19%), this level decreased to 6% in the case of FLMaV2. The position of the isolates in phylogenetic trees constructed with HSP70 sequences did not correspond to their geographical origin, except for the Lebanese isolates which grouped together in a single cluster.

Spain, Tunisia, Greece and Syria (Anonymous, 2005). This crop is affected by a mosaic disease (FMD), characterized by a wide array of symptoms, i.e. discolorations of the leaves (chlorotic mottling and blotching, banding, clearing and feathering of the veins, chlorotic and necrotic ring spots and line patterns) and fruits, and malformations of the leaves. Over time, FMD has been the object of investigations especially intensive in the last few years (Martelli, 2009). A number of different viruses are associated with FMD (Martelli, 2009), among which two members of the family Closteroviridae, denoted Fig leaf mottle-associated virus 1 (FLMaV-1) and Fig leaf mottle-associated virus 2 (FLMaV), both occurring in symptomatic figs of Italian and Algerian origin (Elbeaino et al., 2006, 2007). The molecular information on these viruses is restricted to the nucleotide sequence of their HSP70 gene, a hallmark of the family Closteroviridae (Martelli et al., 2005), which has been used for designing virus-specific primers utlized for PCR detection of both viruses (Elbeaino et al., 2006, 2007). As reported in the present paper, the same molecular tools have now been used in a study aimed at investigating the geographical distribution and incidence of the two viruses in fig orchards of different Mediterranean countries and the variability of their HSP70 gene through the analysis of single strand conformation polymorphism (SSCP) and sequencing.

MATERIAL AND METHODS

Key words: Fig, clostroviruses, FLMaV-1, FLMaV-2, HSP70 gene, SSCP, sequence analysis.

INTRODUCTION

Fig (Ficus carica L.) is grown throughout the Mediterranean basin (Kuden, 1996), Turkey being the main producer, followed by Algeria, Morocco, Egypt, Corresponding author: T. Elbeaino Fax: +39.080.4606327 E-mail: [email protected]

Field survey. A field survey was carried out in autumn 2005 and 2006 for assessing the presence of FLMaV-1 and FLMaV-2 in six Mediterranean countries (Albania, Algeria, Italy, Lebanon, Syria and Tunisia). Plant material, consisting of 1- or 2-year-old cuttings, was collected from a total of 415 five- to ten-year-old symptomatic and symptomless fig trees representative of the main varieties and cultivated areas of the surveyed countries (Table 1). Samples were stored at 4°C or were rooted and maintained in a collection plot at the Mediterranean Agronomic Institute of Bari (Italy) before testing.


426

25-06-2009

12:21

Pagina 426

SSCP and HSP70 analysis of FLMaV-1 and FLMaV-2

dsRNA analysis. A number of samples proportional to that of the whole collection from each country (65 samples in total) was assayed for the presence of dsRNAs. Extraction was from 20 g of the main leaf veins by phenol and chromatography through cellulose CF-11 columns (Dodds, 1993). After treatment with RNase (60 µg/ml) and DNase (0.5 µg/ml) (Saldarelli et al., 1994), preparations were analyzed in 6% polyacrylamide gel electrophoresis at a constant voltage of 95 V for 5 h, and visualized by silver nitrate staining. Total nucleic acid extraction. Total nucleic acids (TNAs) were extracted from ca. 100 mg of leaf veins, homogenized in 1 ml of grinding buffer (4.0 M guanidine isothiocyanate, 0.2 M NaOAc pH 5.2, 25 mM EDTA, 1.0 M KOAc pH 5.0 and 2.5% PVP-40) and purified using silica particles as described by Foissac et al. (2001). RT-PCR amplification. First strand cDNA was synthesized using 1-2 µg of TNA extracts mixed with 0.5 µg of random hexamer primers (Boehringer, Germany) and 200 units of Moloney murine leukaemia virus (MMLV) reverse transcriptase, in accordance with the manufacturer’s instructions (Bethesda Research Laboratories, USA) in a final volume of 20 µl. Primers were the same designed on the sequence of the HSP70 gene of FLMaV-1 and FLMaV-2, which amplify segments of ca 350 bp and 360 bp, respectively (Elbeaino et al., 2006, 2007). A cDNA mixture (2.5 µl) was submitted to PCR amplification in a volume of 25 µl of a reaction mixture containing 1x Taq polymerase buffer (Promega, USA), 1 µM MgCl2, 0.5 mM dNTPs, 0.2 µl of Taq polymerase


(5U/µl) and 0.2 µM of each sense and anti-sense primers. Amplification was done in a Perkin Elmer Cetus Thermal Cycler apparatus with an initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 35 sec, annealing at 58°C for 35 sec, and extension at 72°C for 40 sec. A final extension was done at 72°C for 7 min. Amplicons were analysed by electrophoresis in 6% polyacrylamide gels (PAGE) and visualized by silver nitrate staining. Single strand conformation polymorphism analysis. SSCP analysis was performed directly on PCR amplicons of the positive FLMaV-1 and FLMaV-2 fig samples, or on PCR products obtained from amplification of transformed Escherichia coli colonies. One vol (5 µl) of PCR product was added to 2 vol of loading buffer (95% formamide, 20 mM EDTA, 10 mM NaOH and a few grains of bromophenol-blue), prior to denaturation at 95°C for 10 min and chilling for 3 min on ice (Martins-Lopes et al., 2001). Denatured ssDNAs of the amplified HSP70 fragment were subjected to electrophoresis in a non-denaturing 10% polyacrylamide gel using TBE as electrophoresis buffer (Markoff et al., 1997). To improve band resolution an electrophoretic pre-run was done for 1 h before loading the samples. Electrophoresis was at 4°C, at a constant voltage of 100 V for the first 30 min, then at 200 V for 4 h. DNA conformation patterns were visualized by silver staining. Further amplifications of the recombinant DNA of transformed bacterial colonies were made and the products were subjected to new SSCP analysis. Finally, representatives of each SSCP pattern were sequenced.

Table 1. Fig samples collected from different regions of Mediterranean countries. Country

No. of samples

Tunisia

Region (No. of samples)

Cultivar (No. of samples)

160

Mhamdia (63), Mednine (51), Baddar (20), Takelsa (20), Gabes (6)

Zidi (84), Bayoudhi (17), Bither (12), undetemined (47)

Lebanon

100

Al Bekaa (40), Mount Lebanon (25), North Lebanon (20), South Lebanon (15)

Bayadi ( 25), Shami (25), Houmairi (25), Asly (10), undetermined (15)

Italy

50

Apulia (30), Calabria (10), Sicily (10)

Dottato bianco (15), Brogiotto nero (10),Fiorone Di Ruvo (8), Barnachotte (7), San Giovanni (5), undetermined (5)

Syria

50

Idleb (20), Aleppo (15), Damascus (15)

Bayadi (15), Shami (13), Hamrini (10), Soltani (7), Bertati (5)

Albania

40

Tirana (15),Vlora (15), Shkodra (10)

Roshnik (10), Kraps (10), Patllixhan (10), undetermined (10)

Algeria

15

Biskra (15)

Bithara (12), undetermined (3)

Total

415

---

---


25-06-2009

12:22

Pagina 427


Elbeaino et al.

427

Fig. 1. Representative polyacrylamide electrophoresis gels of RT-PCR amplifications using FLMaV-1 and FLMaV-2 specific primers on fig samples from different Mediterranean countries. Left panel: infected samples are in lane 1-4; RT-PCR negative samples in lanes 5-7. Right panel: positive samples are in lane 1, 2, 4, 6 and 8. FLMaV-2-free samples are in lane 3, 5, 7 and 9. M: 100 bp DNA Marker (Promega, USA).

Cloning and sequence analysis of HSP70 gene. Three µl of PCR products were ligated to pGEM-T Easy Vector (50 ng/µl) following the manufacturer’s instructions (Promega, USA), and transformed into Escherichia coli DH5α cells. Plasmids were extracted from bacterial cells by boiling (Sambrook et al., 1989) and sequenced (PRIMM, Italy). Nucleotide and protein sequences were assembled using the Strider 1.1 Program (Marck, 1988), whereas CLUSTALX (Thompson et al., 1997) was used to generate multiple sequence alignments. Tentative phylogenetic trees were constructed according to Saitou and Nei (1987) and bootstrap analysis was made using NEIGHBOR, SEQBOOT, PROTDIST and CONSENSE programmes of the PHYLIP package (Felsenstein, 1989).

tion. dsRNA detection in different countries ranged from 25% of Algeria to 62.5% of Italy (Table 2). RT-PCR detection of FLMaV-1 and FLMaV-2. A fairly high presence of FLMaV-1 and FLMaV-2 was ascertained by PCR (Fig. 1), for the two viruses were detected in 188 fig trees (45.3% of the total), in single (38.1%) and mixed infections (7.2%). FLMaV-1 was the prevailing virus (34.0% in average), with a frequency of 60% in Italy, 45% in Lebanon, 36.3% in Tunisia and 20% in Albania, but was not detected in Algeria and Syria. FLMaV-2 occurred in all surveyed countries but with a lower incidence (18.6% in average). Its detection was particularly frequent in Syria (40%), followed by Lebanon (25%), Albania (20%) and Tunisia (13.2%). Less relevant was the incidence in Algeria (6.7%) and Italy (4%) (Table 2).

RESULTS

SSCP analysis and HSP70 gene variability. A total of 17 different SSCP patterns was observed in the analysed samples of FLMaV-1 (Fig. 2). In particular, five different groups of SSCP profiles were distinguished in Lebanese virus isolates, six groups in Albanian, five groups in Italian and three groups in Tunisian isolates. The sequenced

dsRNA analysis. Of the 65 fig samples examined for the presence of dsRNAs, 25 (38.5%) contained visible bands arranged in a variety of different electrophoretic profiles. Most of these patterns showed the heavy 16-18 kbp band commonly associated with closterovirus infec-

Table 2. Results of dsRNA isolation and RT-PCR assays on fig samples collected from different Mediterranean countries and infection level by FLMaV-1 and FLMaV-2.

Country Tunisia Lebanon Italy Syria Albania Algeria Total

dsRNA (No.) Tested Positive 25 9 15 6 8 5 7 2 6 2 4 1 65 25

RT-PCR No. of tested samples 160 100 50 50 40 15 415

FLMaV-1 No. 46 30 28 0 7 0 111

% 28.8 30 56 0 17.5 0 26.8

FLMaV-2 No. 9 10 0 20 7 1 47

% 5.7 10 0 40 17.5 6.7 11.3

Mixed infection No. % 12 7.5 15 15 2 4 0 0 1 2.5 0 0 30 7.2

Total infection No. 67 55 30 20 15 1 188

% 41.9 55 60 40 37.5 6.7 45.3


428

25-06-2009

12:22

Pagina 428



Fig. 2. Electrophoretic SSCP profiles of the cloned HSP70 fragment of FLMaV-1 (upper panel) and FLMaV-2 (lower panel) isolates coming from different Mediterranean countries. Abbreviated names of the clones below the gel refer to the isolate and country of origin (Albania, Alb; Algeria, Alg; Italy, It; Lebanon, Lib; Syria, Sy and Tunisia, Tu). Figures represent the number of DNA clones.

HSP70 gene of FLMaV-1 showed an internal variability at the nucleotide level ranging from 1% to 18%, whereas the maximum amino acid variability was 19% (Table 3). In the phylogenetic tree constructed with HSP70 nucleotide sequences the different isolates clustered in three distinct groups (Fig. 3A). In particular all Lebanese isolates were comprised in group A, whereas most of the isolates from Italy, Tunisia and Albania were in group B. The third group (C) consisted of only two isolates from Tunisia and Italy. The nucleotide variability within isolates of the same group was 7%, 12% and 10% for groups A, B and C, respectively (Table 3). SSCP analysis of FLMaV-2 isolates showed the following different groups of patterns: six from Lebanon, four from Syria, three from Albania, two from Tunisia and a single group from Italy and Algeria (Fig. 2). In total, fourteen different SSCP patterns were observed in all analysed samples, including an Italian isolate similar to the Albanian isolate denoted “Alb 8”. Sequence variability of the HSP70 gene of all FLMaV-2 isolates was slightly lower than that observed for FLMaV-1, ranging from 1% to 15% at the nucleotide level and from 1% to 6% at the amino acid level (Table 4). Phylogenetic analysis of the nucleotide sequences placed the viral isolates in two separate clusters. In particular, isolates from

Lebanon and Syria clustered in a single group (group A), while isolates from Albania, Algeria and Tunisia, clustered in group B, which comprised also the Syrian isolate Sy8 (Fig. 3B). Nucleotide variability within isolates of the same group was 11% and 10% for groups A and B, respectively (Table 4).

DISCUSSION

The present study expands the knowledge on the sanitary status of fig crops in some Mediterranean countries for which only scanty information was available. Although the sanitary assessment was limited to 415 trees, still it gives both an insight in the sanitary status of the orchards and an indication on the geographical distribution and incidence of FLMaV-1 and FLMaV-2 in the surveyed countries. Thus, FLMaV-1 was consistently detected in Lebanon, Albania, Italy and Tunisia but not in Algeria and Syria, whereas FLMaV-2 was present in all the countries surveyed with an infection level ranging from 4% (Italy) and 40 % (Syria). Interestingly, the comparative analysis of RT-PCR and dsRNA results showed that five of the 25 dsRNApositive samples were apparently free from FLMaV-1


25-06-2009

12:22

Pagina 429


Elbeaino et al.

429

Table 3. Nucleotide and amino acid (shadowed numbers) identity levels of the HSP70 gene sequences of various isolates of FLMaV-1 from the Mediterranean region. FLMa V1

Lib1 2

Lib1 4

Lib3 8

Lib4 5

Lib4 6

Alb 1

Alb 3

Alb 6

Alb 7

Alb1 1

It2 8

It3 0

It3 5

It4 3

Tu 6

Tu8 4

Tu1 28

88

99

99

99

99

96

96

94

91

95

95

97

95

87

93

98

96

89

88

89

88

87

87

85

83

87

88

89

85

81

83

89

87

99

98

99

95

95

94

92

94

96

98

94

86

92

97

95

99

99

96

96

94

91

95

95

97

94

87

93

98

96

99

97

97

95

92

96

96

98

95

88

94

99

97

96

96

94

91

95

95

97

94

87

93

98

96

96

97

94

97

95

97

95

85

94

96

96

94

94

95

95

95

98

87

96

96

94

94

95

94

95

95

83

94

94

94

92

93

94

95

82

98

91

91

94

96

94

85

94

96

95

96

94

84

93

95

95

94

86

94

97

97

85

97

94

94

83

88

85

93

93

FLMa V1 Lib12

93

Lib14

99

94

Lib38

99

93

98

Lib45

98

93

98

98

Lib46

99

94

99

99

99

Alb1

89

84

89

89

89

89

Alb3

90

86

89

90

90

90

91

Alb6

89

84

88

88

89

88

89

89

Alb7

90

86

90

90

90

90

89

89

90

Alb11

93

89

93

93

93

93

90

91

89

89

It28

89

85

89

89

89

89

89

88

88

91

89

It30

91

87

91

91

92

91

89

89

92

90

91

91

It35

89

84

88

88

89

89

91

91

89

91

89

89

89

It43

90

87

89

90

91

90

83

84

82

84

86

82

84

82

Tu6

91

86

90

90

91

90

89

90

90

99

89

91

90

92

85

Tu84

95

92

95

94

95

95

90

90

90

90

92

88

90

90

90

90

Tu128

89

86

89

89

90

89

90

91

90

90

91

90

91

91

84

91

96 91

The identification of numerous distinct groups of SSCP variants in each of the two viruses under study conforms to their quasi-species nature. Given the high intraspecific variability, FLMaV-1 and FLMaV-2 isolates of different origin grouped in different clusters in the phylogenetic trees with no apparent relation with the geographical origin, except for the isolates coming from the eastern Mediterranean countries (Lebanon and Syria). Whether the reasons for this rest on the way in which fig varietes are propagated and disseminated in these countries remains to be determined. The nucleotide variability of the HSP70 gene within FLMaV-1 and FLMaV-2 populations is in line with that registered for the same gene of other members of the

and FLMaV-2 when tested by RT-PCR, suggesting that hitherto unidentified viruses may occur in fig trees. The SSCP analysis of amplified HSP70 fragments of several FLMaV-1 and FLMaV-2 isolates showed a wide array of patterns, consequent to the molecular variation existing within the populations of these two viral species. This molecular variability was confirmed by comparing nucleotide and amino acid sequences of the same genomic portion of representatives for each different SSCP pattern of the two viruses. The highly discriminatory ability of this technique resulted in the production of different patterns when only a single nucleotide variation occurred between isolates, as confirmed by sequence analysis.

Table 4. Nucleotide and amino acid (shadowed numbers) identity levels of the HSP70 gene sequences of various isolates of FLMaV-2 from the Mediterranean region. FLMaV2 FLMaV2 Alg3 Alb8 Alb12 Tu58 Sy6 Sy8 Sy9 Lib53 Lib56 Lib57 Lib58 Lib68 Lib80

99 98 99 92 93 97 92 95 97 91 93 94 94

Alg3 99 98 99 92 93 96 92 95 97 91 92 94 94

Alb8 97 98 98 90 91 95 91 94 96 90 91 92 92

Alb12 98 99 99 92 92 96 91 95 96 91 92 93 93

Tu58 95 95 94 95 86 90 85 87 89 85 86 86 86

Sy6 99 98 96 97 95 93 99 93 93 90 93 93 91

Sy8 99 98 96 97 95 99 92 94 97 92 92 93 93

Sy9 98 97 95 96 94 99 99 92 92 89 93 92 91

Lib53 99 98 96 97 95 98 98 97 95 91 96 94 95

Lib56 98 97 95 96 94 97 97 96 97 92 92 93 93

Lib57 98 97 95 96 94 97 97 96 97 97 91 92 90

Lib58 98 97 95 96 95 98 98 97 97 96 96 92 95

Lib68 99 99 97 98 95 99 99 98 99 98 98 98 92

Lib80 98 97 95 96 94 98 98 97 97 96 96 98 98


430

25-06-2009

12:22

Pagina 430



Fig. 3. Phylogenetic trees constructed with nucleotide sequences from the HSP70 genes of FLMaV-1 (A) and FLMaV-2 (B) isolates from different Mediterranean countries. Numbers are bootstrap values for 100 replicates.


25-06-2009

12:22

Pagina 431


family Closteroviridae, i.e. Beet yellows virus (BYV), Citrus tristeza virus (CTV), Grapevine leafroll-associated virus 1 (GLRaV-1), -2 (GLRaV-2), -3 (GLRaV-3) and Olive leaf yellowing-associated virus (OLYaV) (Fadel et al., 2005; Turturo et al., 2005; Kominek et al., 2005; Essakhi et al., 2006). It ensues that the groups of isolates differentiated by SSCP and sequencing represent molecular variants of FLMaV-1 and FLMaV-2 rather than different virus species. Finally this study represents a first report on the presence of FLMaV-1 and FLMaV-2 in Tunisia and Syria, while both viruses are reported for the fist time from Albania.

REFERENCES Anonymous 2005. Food and Agriculture Organization of United Nations, Statistical Data (FAOSTAT). [Last date of consultation: 1st Feb. 2006]. Dodds J.A., 1993. dsRNA in diagnosis. In: Matthews R.E.F. (ed.). Diagnosis of Plant Virus Diseases, pp. 273-294. CRC Press, Boca Raton, FL, USA. Elbeaino T., Digiaro M., De Stradis A., Martelli G.P., 2006. Partial characterization of a Closterovirus associated with a chlorotic mottling of fig. Journal of Plant Pathology 88: 187-192 Elbeaino T., Digiaro M., De Stradis A., Martelli G.P., 2007. Identification of a second closterovirus in mosaic-diseased figs. Journal of Plant Pathology 89: 119-124. Essakhi S., Elbeaino T., Digiaro M., Saponari M., Martelli G.P., 2006. Nucleotide sequence variation in the HSP70 gene of Olive leaf yellowing-associated virus. Journal of Plant Pathology 88: 285-291. Fadel C., Digiaro M., Choueiri E., Elbeaino T., Saponari M., Savino V., Martelli G.P., 2005. Sanitary status of olive viruses in Lebanon. Bulletin OEPP/EPPO Bulletin 35: 3336. Felsenstein J., 1989. PHYLIP-phylogeny inference package (version 3.5). Cladistics 5: 164-166. Foissac X., Svanella-Dumas L., Gentit P., Dulucq M.J., Candresse T., 2001. Polyvalent detection of fruit tree Tricho-, Capillo- and Foveavirus by nested RT-PCR using degenerated and inosine containing primers (DOP RT-PCR). Acta Horticulturae 550: 37-43. Kominek P., Glasa M., Bryxiova M., 2005. Analysis of the molecular variability of grapevine leafroll-associated virus 1

Received January 21, 2009 Accepted March 5, 2009

Elbeaino et al.

431

reveals the presence of two distinct virus groups and their mixed occurrence in grapevines. Virus Genes 31: 247-255. Kuden A.B., 1996. Plant Genetic Resources of Fig, Mediterranean Selected Fruits Intercountry Network (MESFIN) under the aegis of FAO, Rome, Italy. Marck C., 1988. “DNA Strider”: a “C” programme for the fast analysis of DNA and protein sequences on the Apple Macintosh family computers. Nucleic Acids Research 16: 1829-1836. Markoff A., Savov A., Vladimirov V., Bodganova N., Kemensky I., Ganev V., 1997. Optimization of single strand conformation polymorphism analysis in the presence of polyethylene glycol. Clinical Chemistry 43: 30-33. Martelli G.P., Agranovsky A.A.., Bar-Joseph M., Boscia D., Candresse T., Coutts R.H.A., Dolja V.V., Falk B.W., Gonsalves D., Hu H.S., Jelkmann W., Karasev A.V., Minafra A., Namba S., Vetten H.J., Wisler C.G., Yoshikawa N., 2005. Family Closteroviridae. In: Fauquet C.M.,. Mayo M.A., Maniloff J., Desselberger U., Ball L.A. (eds), Virus Taxonomy. Eight Report of the International Committee on Taxonomy of Viruses, pp. 1077-1087. Elsevier-Academic Press, Amsterdam, The Netherlands. Martelli G.P., 2009. Fig mosaic and associated viruses. In: Hadidi A., Barba M., Candresse T., Jelkmann W. (ed.), Virus Diseases of Stone and Pome Fruits. APS Press, St. Paul, MN, USA (in press). Martins-Lopes P., Zhang H., Koebner R., 2001. Detection of single nucleotide mutations in wheat using single strand conformation polymorphism gels. Plant Molecular Biology Report 19:159–162 Saitou N., Nei M., 1987. The neighbour-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology Evolution 4: 406-425. Saldarelli P., Minafra A., Martelli G.P., Walter B., 1994. Detection of grapevine leafroll associated closterovirus 3 by molecular hybridization. Plant Pathology 43: 91-96. Sambrook J., Fritsch E.F., Maniatis T., 1989. Molecular cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York, USA. Thompson J.D., Gibson T.J., Plewniak F., Jeanmougin F., Higgins D.G., 1997. The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25: 48764882. Turturo C., Saldarelli P., Yafeng D., Digiaro M., Minafra M., Savino V., Martelli G.P., 2005. Genetic variability and population structure of Grapevine leafroll-associated virus 3 isolates. Journal of General Virology 86: 217-224.


25-06-2009

12:22

Pagina 432