icvg 2009 part I pp 1-131.pdf - Cornell University

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— 38 — GRAPEVINE VIRUSES IN CHILE: MULTI-PARALLEL DETECTION BASED ON METAGENOMIC STRATEGIES Esteban A. ENGEL 1,2* , Paula F. ESCOBAR 1 , Paulina RIVERA 1 , Pablo D.T. VALENZUELA 1,2 1 Fundación Ciencia para la Vida and MIFAB, Zañartu 1482, SANTIAGO, Chile 2 Universidad Andrés Bello, Facultad de Ciencias de la Salud, República 252, SANTIAGO, Chile *Corresp. author: Esteban A. Engel, Telephone: 56-2-3672014, Fax: 56-2-2372259, e-mail: eaengeli@yahoo.com - eengel@bionova.cl Summary At least 58 viruses have been reported to infect grapevines causing important economic damage all over the world (Martelli & Boudon-Padieu, 2006). Conventional detection strategies mainly based on serological assays, biological indexing and RT- PCR are widely used, although they target one or in the best case few viruses in each assay. Grapevines are prone to contain mixed infections of several viruses, making the use of these techniques time consuming. We report here a comprehensive 70-mer oligonucleotide microarray able to simultaneously detect a broad spectrum of viruses with total or partial genomic sequence available up to now. The array contains 570 unique probes designed against both, highly conserved and specific regions of 44 plant viral genomes in addition to plant housekeeping genes. The microarray accuracy was validated in the detection of 10 grapevine viruses present in Chile. Among them, 3 Closteroviridae members GLRaV-4, -7 and -9 were reported for the first time in Chile with this method. The different probe hybridization patterns obtained by each virus makes this approach a powerful tool that may be used for highly parallel certification purposes and for virus discovery if the genome sequence has partial similarity with the printed probes. Furthermore, we sequenced several hundred segments from four grapevine libraries to define the spectrum of viruses (virome) present on each sample without initial requirements such as primers or antibodies. Using this approach, we identified known grapevine viruses and also sequences from putative new viruses with scarce similarity to viruses present in GenBank. In addition, we were able to identify sequences from mycoviruses indicating that fungal species were also infecting some of the samples analyzed. Thus the metagenomic approaches shown here may be useful to simultaneously detect all the known and eventually unreported viruses present in plant samples. INTRODUCTION Efficient and early detection of grapevine viral pathogens is critical to diminish losses due to dissemination of infected material, the main cause of disease spreading in viticultural countries. ELISA and RT-PCR are the most common and widely used techniques for routine screening. Nevertheless they have limitations such as the maximum number of viruses detectable in a single assay. Grapevines samples are complex biological matrices with different co-infectants, making these techniques time-consuming and labor intensive. Recently, several studies have reported to successfully detect or discover, human, environmental, fungal and plant pathogens using DNA microarrays. The technique is based on the interrogation of labeled samples with thousands of unique immobilized probes. Alternately the advance of high throughput sequencing techniques raises the possibility to use sequencing as a routine viral detection/discovery approach. In the present paper, we explore these techniques to obtain insights in the comprehensive detection of grapevine viruses. MATERIAL AND METHODS Total RNA or double stranded RNA (dsRNA) was extracted from fresh bark scrapings as described elsewhere (Chang et al., 1993; Valverde et al., 1990). Seventy mer oligonucleotides derived from 44 fully or partially sequenced plant virus genomes taken from GenBank were designed against both, highly conserved regions within each viral family as well as specific genomic regions for each virus species and synthesized over 384 well plates. A total of 570 oligonucleotides were printed in duplicates over different areas of Poly-L-Lysine pretreated microscope slides. Microarrays were hybridized for 12 hours at 65ºC and imaged with a PerkinElmer ScanArray Gx instrument. Normalized background-subtracted Cy3 pixel intensity was analyzed by hierarchical clustering (Eisen et al., 1998) to plot microarray probes as horizontal stripes showing the Cy3 intensity as a red linear scale. Black stripes corresponded to probes with Cy3 intensity below the threshold. A spot was considered positive only if both duplicates printed in the array Cy3 intensities were above the threshold. dsRNA obtained from infected grapevines was reverse transcribed and randomly amplified by RT- PCR (Bohlander et al., 1992). PCR products were cloned into pCR4 using Invitrogen TOPO cloning kit and transformed into Top10 bacteria. Positive colonies were sequenced (both Sanger and Pyrosequencing methods may be used) and unique high quality sequences were phylotyped according to the best tBLASTx hit as viral, bacterial, fungal, plant and unassigned. RESULTS AND DISCUSSION The microarray was validated after several grapevine samples collected from all the Chilean growing regions were hybridized. Additionally, viral genomic libraries were also used as part of the validation process. The technique showed to be a powerful and fast diagnostic method when compared with traditional systems (Engel et al., 2006). By analyzing the hybridization patterns of the array in a clustogram, we were able to detect the expected viruses in the samples and for the first time in Chile the presence of GLRaV-4, -7 and -9 in grapevines with single or mixed infections (Engel et al., 2008; Escobar et al., 2008). Since
— 39 — glass planar microarrays can fit 30.000 or more probes, it is realistic to think of a large generic plant virus microarray. Finally, more than 1.500 sequence reads were obtained from 4 grapevine samples. Comparison with databases identified more than 10 viral genomes among the expected grapevine viruses (Fiore et al., 2008) plus fungal viruses such as Botryotinia fuckeliana partitivirus 1. The partial genome sequence of a putative new Closteroviridae member was identified and is currently being fully sequenced. Considering that approximately 1.200 different viruses as full or tentative species have been described to infect plants (Boonham et al., 2007) and near 60 have been reported solely to infect grapevines (Martelli & Boudon- Padieu, 2006), multi-parallel detection systems like the ones presented here, could avoid time consuming monodetection approaches. Hence, these might be suitable detection methods for plant diseases caused by complexes of viruses. LITERATURE BOLHANDER, S.K., ESPINOSA, R., 3rd, LE BEAU, M.M., ROWLEY, J.D. & DIAZ, M.O. 1992. A method for the rapid sequence-independent amplification of micro dissected chromosomal material. Genomics 13, 1322-4. BOONHAM, N., TOMLINSON, J. & MUMFORD, R. 2007. Microarrays for rapid identification of plant viruses. Annual Review of Phytopathology 45, 307-28. CHANG, S., PURYEAR, J. & CAIRNEY, J. 1993. A simple and efficient method for isolating RNA from pine trees. Molecular Biology Rep 11, 113-116. EISEN, M.B., SPELLMAN, P.T., BROWN, P.O. & BOTSTEIN, D. 1998 cluster analysis and display of genome-wide expression patterns. Proceedings of the National Academy of Sciences USA 95, 14863-8. ENGEL, E., ARREDONDO, V., GIRARDI, C., GONZALEZ, A., ESCOBAR, P., FIORE, N., ARCE, P. & VALENZUELA, P.D.T. 2006. Grapevine viruses in Chile: Genomics and detection based on immunocapture and microarray technologies. Extended Abstracts 15th Meeting of ICVG, Stellenbosch, South Africa, 118-119. ENGEL, E.A., ESCOBAR, P., MONTT, C., GOMEZ-TALQUENCA, S. & VALENZUELA, P.D.T. 2008. First report on the occurrence of Grapevine leafroll-associated virus 7 and 9 in Chilean grapevines. Plant Disease 92, 1252. ESCOBAR, P.F., FIORE, N., VALENZUELA, P.D.T. & ENGEL, E.A., 2008. First detection of Grapevine leafroll-associated virus 4 in Chilean grapevines. Plant Disease 92, 1474. FIORE, N., PRODAN, S., MONTEALEGRE, J., ABALLAY, E. & PINO, A.M., 2008. Survey of grapevine viruses in chile. Journal of plant pathology 90, 125-130. MARTELLI, G.P. & BOUDON-PADIEU, E. 2006. Directory of infectious diseases of grapevines and Viroses and virus-like diseases of the grapevine: Bibliographic Report 1998-2004. Options Méditerranéennes B55. VALVERDE, R.A., NAMETH, S.T. & JORDAN, R.L. 1990. Analysis of double-stranded rna for plant virus diagnosis. Plant Disease 74, 255-258. ACKNOWLEDGEMENTS We thank Dr. Nicola Fiore for providing part of the plant infected material, and MECESUP(2), CORFO-INNOVA 204-4040, UNAB DI-02- 06/I, PFB-16 and MIFAB P04-071-F for partial funding of this research. Progrès Agricole et Viticole, 2009, Hors Série – Extended abstracts 16 th Meeting of ICVG, Dijon, France, 31 Aug – 4 Sept 2009
Page 3 and 4: 16th Meeting of the International C
Page 5 and 6: AVANT-PROPOS Nous sommes très heur
Page 7 and 8: Content - Sommaire Page Partners an
Page 9 and 10: Session IV - Epidemiology - Survey
Page 11 and 12: Poster 51 Poster 52 Poster 53 Poste
Page 13 and 14: Identification and distribution of
Page 15 and 16: — 15 — GRAPEVINE VIROLOGY HIGHL
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Page 19 and 20: — 19 — Likewise, an antibody bi
Page 21 and 22: — 21 — LITERATURE ABOU GHANEM-S
Page 23 and 24: — 23 — ORECCHIA, M., NOELKE, G.
Page 25 and 26: — 25 — immunoassays, either ELI
Page 27 and 28: — 27 — ENGELBRECHT, D.J. & KASD
Page 29 and 30: — 29 — NEW DEVELOPMENTS IN UNDE
Page 31 and 32: — 31 — LIU, Y.-P., PEREMYSLOV,
Page 33 and 34: — 33 — RESULTS AND DISCUSSION O
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Page 47 and 48: — 47 — result suggests that the
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Page 53 and 54: — 53 — identity at the protein
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Page 69 and 70: — 69 — IDENTIFICATION OF PLASMO
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Page 73 and 74: — 73 — RECOMBINATION EVENTS IN
Page 75 and 76: — 75 — THE SPECIFICITY OF GFLV
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Page 79 and 80: — 79 — CHARACTERIZATION OF RASP
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— 89 — transgenic grape lines,
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— 91 — highest values were obse
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— 93 — Imura et al., 2008, Loud
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— 95 — GFLV-F13. Low levels of
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— 97 — This is probably due to
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— 99 — variation was distribute
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— 101 — Figure 1. Proportion of
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— 103 — were produced. The prod
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— 105 — 2007 positive results w
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— 107 — dehydrogenase (GAPDH) t
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— 109 — detected, either as sin
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— 111 — basal leaf tissue inclu
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— 113 — GLRaV-3 incidence cover
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— 115 — Figure 2. Frequency of
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— 117 — Ljutun, Mladenka, Nincu
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— 119 — ELISA reagent used in t
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— 121 — Due the late season the
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— 123 — RT-PCR & PCR: Primers f
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— 125 — Table 2. Sanitary statu
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— 127 — SANITARY STATUS OF AUTO
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— 129 — THE VIRUS STATUS OF GRA
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— 131 — PRESENCE OF GLRAV 1, 2,
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