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— 32 — VIRUSES OF NATIVE VITIS GERMPLASM IN THE SOUTHEASTERN UNITED STATES Sead SABANADZOVIC Department of Entomology and Plant Pathology, Mississippi State University, Mississippi State, MS 39762, USA Phone +.1.662.325.9322, Fax +.1.662.325.8837; Email: ss501@msstate.edu Summary A survey, aimed at the identification of viruses present in native Vitis germplasm in the southeastern USA was carried out in 2007 and 2008. The randomly selected pool of samples included native grapevines grown under cultivated conditions (muscadines) as well as samples collected from environments with minimal or no human impact (forest). Sequencing of randomly amplified cDNA clones generated on reverse-transcribed dsRNAs, and/or PCR products using genera-specific primers, revealed the presence of a panel of as yet undescribed viruses belonging to different taxa of ssRNA and dsRNA viruses. In general, the community of phytoviruses detected so far in native Vitis spp differs significantly from viruses reported to date from cultivated grapevines. The possible importance and impact of these viruses for Vitis vinifera and related rootstocks is yet to be studied. The study is still on-going. INTRODUCTION It is the consensus among scientists that the approximately 2,000 viral species currently recognized by the International Committee on Taxonomy of Viruses (ICTV; Fauquet et al., 2005) represent a gross underestimate of the total viral diversity on the planet Earth (Wren et al., 2006; Melcher et al., 2008; Swanson et al., 2009). Such a relatively small number of recognized viral species is mainly due to traditionally “biased” studies towards the virus communities of certain interest for humankind which, as a consequence, left other habitats/ecosystems very poorly investigated (i.e. marine, forest, soil, etc). Plant viruses account for almost half of the recognized species by the ICTV (Fauquet et al., 2005). Studies in plant virology, with rare exceptions, have traditionally focused on viruses of economically important crops. As a result, ca 77% of known phytoviruses have been described from cultivated plant species or agricultural weeds (11%) although they represent just a small portion of the terrestrial flora (Wren et al., 2006). Grapevines are intrinsically prone to infections by intracellular pathogens. More than 70 graft-transmissible agents/diseases have been reported from this crop to date (Martelli & Boudon-Padieu, 2006; Martelli, these proceedings). Viruses account for the majority of the infectious agents reported (circa 60), and are known to be potentially detrimental to the quality and quantity of grape production in any growing area of the world. The currently known viruses are reported from cultivated grapevine species, mainly Vitis vinifera. Apart from some isolated, virus-specific surveys (i.e. Soule et al., 2006), viruses of Vitis species other than V. vinifera have not been extensively studied. However, some virological projects have recently focused on plant viruses present in a wild and native flora in order to study complexity of viruses present in natural ecosystems (see among others: Fraile et al., 1997; Ooi et al., 1997; Raybould et al., 1999; Robertson, 2005; Melcher et al., 2008; Muthukumar et al., 2009; Sabanadzovic & Abou Ghanem-Sabanadzovic, 2009). The objective of this work is to shed light on the virus community present in the native grape germplasm in the Southeastern United States and to compare it with known viruses reported from cultivated Vitis spp. Results presented here are a combination of two independent and still ongoing studies. While one research line focused specifically on viruses of muscadines in Mississippi, the second study had the broader goal of characterizing plant viruses in natural environments (in our case Great Smoky Mountains National Park - GSMNP). In the latter study, specimens of wild Vitis spp. were collected and processed as a part of a much more complex sample pool. MATERIALS AND METHODS Virus sources. Virus sources used in this work belonged to various Vitis genotypes, including muscadines [Muscadinia (Vitis) rotundifolia Michx.], summer grape (Vitis aestivalis Michx) and sand grape (Vitis rupestris Scheele), as well as several specimens of uncertain botanical identity. Samples were collected from production plots, backyards and natural ecosystems during 2007 and 2008. Cloning, sequencing and data analyses. Double stranded RNAs (dsRNAs) extracted from phloem tissues (Valverde et al., 1990) were selectively treated with DNAse and RNAse (in high-salt conditions) prior to their use as a template for random-primer generated cDNAs according to the modified protocol of Froussard (1992). PCR-enriched complementary DNAs were digested with a proper restriction endonuclease, cloned into pUC118/EcoRI vectors and transferred to Escherichia coli Top10 competent cells. In average, twenty five clones/sample were selected and sequenced. Sequence analyses were performed with on-line resources (BLAST; Altschul et al., 1997), and with various software packages depending on the scope of analyses (i.e. Lasergene-DNAStar package, MEGA 4.0, TreeView). In an additional approach, reverse-transcribed dsRNAs were used in PCR with a panel of genus-specific primers described in literature (Tian et al., 1996; Sabanadzovic et al., 2000; Dovas & Katis, 2003; Digiaro et al., 2007) or designed in our laboratory (i.e. sobemovirus, umbravirus, luteovirus, endornavirus, etc). This approach allowed initial detection of some viruses (see Sabanadzovic et al., this volume for details) which were then completely sequenced applying the procedures described in Sabanadzovic et al., 2009c.
— 33 — RESULTS AND DISCUSSION Our approach of combining random-primer generated cDNAs and degenerate-primer RT-PCR allowed identification of dozens of viruses in tested native Vitis specimens to date. The majority of these viruses are as yet unreported plant virus species with ssRNA or dsRNA genomes. Viruses detected in Vitis spp. of the southeastern United States so far are briefly commented on below. Genus Potyvirus. Multiple overlapping clones containing potyvirus-like sequences were generated from a muscadine specimen collected from Southern Mississippi. A contig counting almost 1,500 nt was translated and compared with viral sequences available in NCBI. Results showed that clones belonged to an isolate of Bean common mosaic virus (BCMV). Curiously, the “muscadine isolate” shared 99% amino acid identity with the “peanut stripe” strain of BCMV (previously known as Peanut stripe mosaic virus) and less (93%) with the other isolates of the same virus. Considering that BCMV is one of the most widespread viruses in peanut fields in Mississippi (S. Sabanadzovic and D. Ingram, unpublished data) and that the majority of peanut production is located in the Southern part of the state, it is likely that this virus was transmitted from neighboring peanut plantations. Genus Marafivirus. A new marafivirus, denominated Grapevine virus Q and characterized by unique organization of the palm sub-domain of RNA-dependent RNA polymerase was detected in multiple samples of muscadines in Mississippi, as well as in summer grapes and Vitis vinifera (for more extensive explanations about this phenomenon see Sabanadzovic et al., 2009a and Sabanadzovic et al., 2009b). Surprisingly, the same virus was found in wild Rubus spp. making it the first tymovirid capable of infecting blackberries and grapevines. The genome of the muscadine isolate of this virus was completely sequenced at Mississippi State University as well as several additional isolates including the one from wild blackberry. The same virus was characterized by application of new generation of sequencing methodologies at the University of California and reported under the name Grapevine Syrah virus 1 (Al Rwahnih et al., 2009). Genus Oryzavirus. Cloning of a reverse-transcribed complex dsRNA pattern recovered from petioles of a summer grape specimen collected in a natural ecosystem (GSMNP) generated a library of clones with sequences reminiscent of reoviruses. Analyses of extensive nucleotide sequence data generated for multiple viral segments revealed the presence of a new viral species in the genus Oryzavirus. The highest identities (approx. 40%) between this virus and Rice ragged stunt virus (RRSV), the type species of the genus, were observed in viral segment 4 encoding a putative polymerase. Interestingly, this is only the second virus species described in this genus. To our knowledge, oryzaviruses were limited to rice in Southeastern Asia. Our work, together with a report of association of an oryzavirus with crumbly fruit disease of red raspberry in the Pacific Northwest (Quito et al., 2009) gives new insights into the host range and geographical distribution of this type of viruses. The exchange of initial sequence data between the two research groups revealed high amino acid identity levels (80-85%) between oryzaviruses from summer grape and raspberry indicating that they may represent distinct isolates of the same, novel species in the genus Oryzavirus. Genus Enamovirus (family Luteoviridae). Analyses of sequence data originally generated from a specimen of wild summer grape (Vitis aestivalis) collected from Great Smoky Mountains National Park indicated the presence of a new member of the family Luteoviridae. Molecular data revealed the presence of five open reading frames, an organization resembling that of Pea enation mosaic virus 1 (PEMV-1), the type species and currently sole member of the genus Enamovirus in the family Luteoviridae. Virus from summer grape shared ca 55% and 44% identical amino acids in RNA-dependent RNA polymerase and coat protein cistrons. Phylogenetic analyses confirmed allocation of this virus in the genus Enamovirus. Virusspecific primers designed on viral coat protein allowed detection of this virus in a few additional Vitis specimens. “PEMV disease complex” is caused by an intimate and symbiotic relationship of two taxonomically unrelated viruses: PEMV-1 (gen. Enamovirus) and PEMV-2 (gen. Umbravirus). We are currently investigating the possible association of an umbravirus with the enamovirus from summer grape, although random primer cloning did not give any evidence for that. Genus Endornavirus. The presence of high molecular weight dsRNAs in several samples was ascertained to be due to endornaviruses. Amplicons of expected size were consistently generated in RT-PCR tests from dsRNApositive specimens using endornavirus-specific degenerate primers (Sabanadzovic & Valverde, 2009). Pairwise comparisons of generated clones showed that at least two distinct endornaviruses, with sequences differing as much as 40%, have been identified so far in autochthonous Vitis germplasm. Family Partitiviridae. Numerous clones with significant sequence similarities to known cryptoviruses were observed and analyzed during this study. In particular, a cryptic virus with sequence homologies with Raphanus sativus cryptic virus 3 and a putative cryptovirus similar to Beet cryptic virus 3 were both detected in muscadine samples. Unclassified dsRNA viruses. Curiously, a pool of clones generated from summer grape samples from GSMNP contained sequences similar (50%) to both genomic segments of the recently described Curvularia thermal tolerance virus (CThTV; Márquez et al., 2007), a virus that infects an endophytic fungus, Curvularia protuberata, and confers heat tolerance to the plant host and enables it to survive in some extreme environments such as the hot soils of the Yellowstone National Park. At this point we focus on the identification of the fungal host of this virus and plan to go beyond simple virus description. Clones with sequences related to another recently described and unclassified dsRNA virus, Southern tomato virus (STV; Sabanadzovic et al., 2009) were obtained from summer grapes. Curiously, similar sequences were found in multiple rhododendron and viburnum samples indicating that this type of virus is rather widespread in nature. Mycoviruses. A significant number of analyzed clones had significant identities with a range of mycoviruses (i.e. 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
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Page 21 and 22: — 21 — LITERATURE ABOU GHANEM-S
Page 23 and 24: — 23 — ORECCHIA, M., NOELKE, G.
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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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