This 14th International Symposium on Virus Diseases of Ornamental Plants is jointly organized and sponsored by Agri-food and Veterinary Authority Singapore (AVA), Department of Biological Sciences (DBS), Faculty of Science, National University of Singapore (NUS), National Parks Board (NParks) Singapore, Plant Protection Society (Singapore), and Temasek Life Sciences Laboratory (TLL), under the International Society of Horticultural Science (ISHS) symposium series.

Local organizing committeeSek-Man Wong (convenor)Yok King FongZhong Chao YinLi-Huan KohAnn Nee YongPing Lee ChongSay Tin Kho

Guest-of-Honor Mr Desmond LeeSenior Minister of State, Ministry of National Development and Ministry of Law

Sponsors and Partners

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TENTATIVE PROGRAM14th International Symposium on Virus Diseases of Ornamental Plants26-29 June 2016 | Lecture Theatre 32, Faculty of Science, National University of Singapore

Day 1 (26 June 2016)

Arrival and Registation at Rochester Park Hotel.

Day 2 (27 June 2016)

Time Activity

8:00 am Bus leaves from Rochester Park Hotel to NUS

8:15 am Registration & Poster set up

8:45 am Participants to be seated

9:00 am Welcome Address by Sek-Man Wong, Convenor of symposium

9:10 am Plenary Lecture – Shyi-Dong Yeh01_A MARKER-FREE TRANSGENIC APPROACH FOR GENERATING CONCURRENT RESISTANCE TO A DNA GEMINIVIRUS AND A RNA TOSPOVIRUS

10:00 am Coffee/Tea Break & Poster Viewing

Session 1 : Virus Detection, Characterization And Diagnostic TechniquesChair: J. Hammond

10:30 am 02_DETECTION OF A NON-DESCRIBED VIRUS IN DRACAENA SURCULOSA?C.E. de Krom*, J.Th.J. Verhoeven, A.M. Dullemans, R.A.A. van der Vlugt, J.W. Roenhorst

10:45 am 03_SEQUENCE VARIABILITY BETWEEN PLANTAGO ASIATICA MOSAIC VIRUS ISOLATESJ. Hammond and M.D. Reinsel

11:00 am Introduction to access to NUS internet using wifi

12:00 am Lunch Break

13:20 pm Leave for Singapore Botanic Gardens - first UNESCO World heritage site in Singapore

14:00 pm Guided-tour at National Orchids Garden, Singapore Botanic Gardens

16:00 pm Visit to the Flower Dome and Cloud Forest at Gardens-by-the-Bay

18:00 pm Dinner (seafood steamboat & BBQ) at Satay-by-the-Bay

20:00 pm Light show at Gardens-by-the-Bay

21:30 pm Leave for Rochester Park Hotel/Bouna Vista MRT Station

End of Day 2

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Day 3 (28 June 2016)

Session 2: Epidemiology And Disease ControlChair: Scott Adkins

9:15 am 04_VIRAL SPREAD AND DIVERSITY IN ANTHROPOCENEK. R. Richert-Poeggeler and B. Lockhart

9:30 am 05_EPIDEMIOLOGY OF AND RESISTANCE TO ROSE ROSETTE VIRUSPatrick L. Di Bello, Thanuja Thekke-Veetil, Tobiasz Druciarek and Ioannis E. Tzanetakis*

9:45 am 06_REDBUD YELLOW RINGSPOT DISEASE: ANOTHER EMARAVIRUS ASSOCIATED WITH A DISEASE OF ORNAMENTAL PLANTSPatrick L. Di Bello, Alma G. Laney, Tobiasz Druciarek, Thien Ho, Rose C. Gergerich, Karen E. Keller, Robert R. Martin and Ioannis E. Tzanetakis*

Section 3: New and Emerging DiseasesChair: C.A. Chang

10:00 am 07_VIRUSES OF ORNAMENTALS EMERGING IN FLORIDA AND THE CARIBBEAN REGIONScott Adkins, Carlye A. Baker, Colleen Y. Warfield, Consuelo Estévez de Jensen, Ismael Badillo-Vargas, Craig G. Webster, Galen Frantz, H. Charles Mellinger, Joseph E. Funderburk and Rayapati Naidu

10.15 am Coffee/Tea Break and Poster Viewing

10:45 am 08_EVIDENCE OF NEW VIRUSES INFECTING FREESIA HYBRIDS SHOWING NECROTIC DISEASEA.M. Vaira, L. Miozzi, M. Vallino, A. Carra, R. Lenzi, D. Salvi, J. Hammond and H.R. Pappu

11.00 am 09_CHARACTERIZATION OF A NUCLEORHABDOVIRUS FROM PHYSOSTEGIAWulf Menzel, Dennis Knierim, Katja Richert-Pöggeler, Stephan Winter

11.15am 10_IDENTIFICATION OF NARCISSUS YELLOW STRIPE VIRUS AND A CLOSELY-RELATED POTYVIRUS ISOLATE IN PLANTS OF ALLIUM CARINATUMD. Bampi, M.D. Reinsel, and J. Hammond

11:30 am 11_NEW VIRUS DISEASES OF LISIANTHUS IDENTIFIED IN TAIWANY.K. Chen, H.Y. Chao, and P.J. Shih

11:45 am 12_CHARACTERIZATION AND ITS ETIOLOGY OF TWO APHID TRANSMISSIBLE VIRUSES FROM JASMINE IN TAIWAN.S.M. Wang, Y.Y. Lin, Y.C. Lin, and C.A. Chang

12:00 noon 13_CHARACTERIZATION AND GENOME SEQUENCE ANALYSIS OF LYCHNIS RINSSPOT VIRUSYoon-Hyun Bang, Eun-Gyeong Song, Ji-Yeon Kwon, Eun-Kyoung Kim, Jin-Sung Hong and Ki-Hyun Ryu*

12:15 noon Lunch Break & Poster Viewing

13:45 pm Leave for Lee Kong Chian Natural History Museum, NUS

14:00 pm Guided-tour at Lee Kong Chian Natural History Museum, NUS

15:30 pm Tea/Coffee break at Lee Kong Chian Natural History Museum, NUS

16:00 pm Visit to Temasek Life Sciences Laboratory (limited to 30 participants)

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17:00 pm Visit to Shaw Alumni Foundation House

18:00 pm Symposium dinner at The Scholar Chinese Restaurant at NUSS Guild House

21:00 pm Bus return to Rochester Park Hotel/Bouna Vista MRT Station

End of Day 3

Day 4 (29 June 2016)

Session 4: Viroids and PhytoplasmasChair: Dag-Ragnar Blystad

9:00 am Coffee/Tea Break/Viewing of Posters

9:15 am 14_CHRYSANTHEMUM STUNT VIROID IN ARGYRANTHEMUM---DISTRIBUTION AND ELIMINATIONZhibo Zhang, YeonKyeong Lee, Carl Spetz, Jihong Liu Clarke, Astrid Sivertsen, Gry Skjeseth, Sissel Haugslien, Qiaochun Wang and Dag-Ragnar Blystad*

9:30 am 15_MULTILOCUS TYPING FOR CHARACTERIZATION OF ‘CANDIDATUS PHYTOPLASMA ASTERIS’-RELATED STRAINS IN SEVERAL ORNAMENTAL SPECIES IN ITALY S. Paltrinieri, M.G. Bellardi, F. Lesi, E. Satta, S. Davino, G. Parrella, N. Contaldo and A. Bertaccini

9:45 am 16_RAPID SCREENING FOR PHYTOPLASMA PRESENCE IN FLOWER CROPS USING TUF GENE BARCODEN. Contaldo, S. Paltrinieri, M.G. Bellardi, F. Lesi, E. Satta, and A. Bertaccini

Session 5: Virus Resistance Through Breeding Or Transgenic MethodsChair: Pattana Srifah Huehne

10:00 am 17_SILENCING THE CymMV COAT PROTEIN GENE BY RNAi AND RECOMBINANT miRNA TECHNOLOGY IN TRANSGENIC DENDROBIUM ORCHIDPattana Srifah Huehne, Udomporn Petchthai, Anchalee Chuphromp, Kisana Bhinija

10:15 am 18_TRANSGENIC NICOTIANA BENTHAMIANA RESISTANCE TO SYNERGISTIC INFECTION OF TWO ORCHID VIRUSES CymMV AND ORSVU. Petchthai, Z. Xie, and S.M. Wong

10.30 am Coffee/Tea Break and Viewing of Posters

Section 6: Virus Detection And Diagnostic TechniquesChair: R.J. McGovern

11:00 am 19_FAST, EASY AND HIGHLY SENSITIVE DETECTION OF TOMATO CHLOROTIC DWARF VIROID (TCDVD) IN TOMATO AND ORNAMENTAL CROPS USING AGDIA’S TCDVD AMPLIFYRP® ACCELER8™Janet Lamborn, Rosemarie W. Hammond and Shulu Zhang

11:15 am 20_EVALUATION OF A SIMPLIFIED METHOD OF PLANT VIRUS INCLUSION VISUALIZATION BY LIGHT MICROSCOPYR.J. McGovern, P. Khampirapang, R. Cheewangkoon, L.H. Koh, C. To-anun, and S.M. Wong

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11:30 am Closing Remarks

12:00 noon Lunch

13:00 pm Bus return to Rochester Park Hotel/Bouna Vista MRT Station

End of Day 4: Bon Voyage

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ORAL ABSTRACTS

01_A MARKER-FREE TRANSGENIC APPROACH FOR GENERATING CONCURRENT RESISTANCE TO A DNA GEMINIVIRUS AND A RNA TOSPOVIRUS

Ching-Fu Yang1,3, Wei-Yu Lin1, Ying-Hui Cheng2, Kuan-Chun Chen3, Ya-Ling Huang1, Wan-Chu Chien1

and Shyi-Dong Yeh 1,,3, 4

1Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan 2Devision of Plant Pathology, Taiwan Agriculture Research Institute, Wufeng, Taichung, Taiwan3 Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan4NCHU-UCD Plant and Food Biotechnology Center, National Chung Hsing

Ｗ hitefly-borne ssDNA geminiviruses and thrips-borne ss(-)RNA tospoviruses cause severe damages on many important crops worldwide, including ornamentals. Transgenic approach based on RNA silencing is not effective against gemiviruses due to different replication strategy and high variability. Thus, we attempted to develop a novel transgenic approach to provide effective resistance to a geminivirus and a tospovirus at the same time. A two-T DNA vector carrying a hairpin of the intergenic region (IGR) of Ageratum yellow vein geminivirus (AYVV), residing in an intron inserted in two exonic sequences containing untranslatable nucleocapsid protein (NP) fragments of Melon yellow spot tospovirus (MYSV), was constructed. Transgenic Nicotiana benthamiana lines highly resistant to AYVV and MYSV were generated. Accumulation of 24-nt siRNA, higher methylation levels on the IGR promoters of the transgene, and suppression of IGR promoter activity of invading AYVV indicate that AYVV resistance is mediated by transcriptional gene silencing. Lack of NP transcript and accumulation of corresponding siRNAs indicate that MYSV resistance is mediated through post-transcriptional gene silencing. After selfing of selective lines, marker-free progenies with concurrent resistance to both AYVV and MYSV, stably inherited as dominant nuclear traits, were obtained. Hence, we provide a novel way for concurrent control of noxious DNA and RNA viruses with less biosafety concerns. Currently, this approach is being extended to tomato, with a new marker-free construct containing a hairpin construct with IGRs of two major leaf curl geminiviruses residing in a tomato intron inserted in between exonic sequences containing the highly conserved domain of tospoviral replicases to provide broad-spectrum resistance to different tospoviruses at the genus level. The marker-free transgenic tomato lines thus generated are expected to concurrently confer broad-spectrum resistance to tomato leaf curl geminiviruses and various tospoviruses.

02_DETECTION OF A NON-DESCRIBED VIRUS IN DRACAENA SURCULOSA?

C.E. de Krom¹, J.Th.J. Verhoeven¹, A.M. Dullemans², R.A.A. van der Vlugt², J.W. Roenhorst¹,

1 National Plant Protection Organization. P.O Box 9102, 6700 HC Wageningen, The Netherlands2 Wageningen University and Research Centre, P.O. Box 16, 6700 AA Wageningen, The Netherlands

The ornamental Dracaena surculosa is produced because of its decorative chlorotic rings and spots on the leaves. Since these symptoms resemble virus symptoms, occasionally samples of D. surculosa are submitted for virus diagnosis. These samples have been tested by various methods including electron microscopy, mechanical inoculation to indicator plants, DAS-ELISA and RT-PCR. So far, no virus has been detected. Last year, export of D. surculosa plants was impeded, because tospovirus infections were reported in this plant species. However, in samples of these plants no tospovirus was

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detected by DAS-ELISA for three tospoviruses and RT-PCR for generic detection of all known tospoviruses (Hassani-Mehraban et al., 2016) and also other virus tests were negative. Therefore further analysis was continued by application of next generation sequencing on RNA isolated from roots of a symptomatic plant. The obtained sequence data revealed a sequence of possible viral etiology. A contig sequence of 8.6 kb was assembled, which contains an ORF of 7.9 kb. This contig sequence showed no significant identity with any virus sequence in the GenBank database. However, deduced amino acid sequences showed low levels of homology (20-25 %) with polyproteins of several members of ssRNA positive strand viruses from the family Secoviridae. Whether this sequence indeed represents a so far non-described virus and possibly even a new genus remains to be established. Further work is in progress to complete the genome sequence and to examine whether this potentially new virus is indeed responsible for the chlorotic rings and spots on the leaves of D. surculosa.

Hassani-Mehraban A, Westenberg M, Verhoeven JTJ, Van de Vossenberg BTLH, Kormelink R, Roenhorst JW (2016) Generic RT-PCR tests for detection and identification of tospoviruses. J Virol Meth, http://dx.doi.org/10.1016/j.jviromet.2016.03.015

03_SEQUENCE VARIABILITY BETWEEN PLANTAGO ASIATICA MOSAIC VIRUS ISOLATES

J. Hammond and M.D. Reinsel

Floral & Nursery Plants Research Unit, USDA-ARS, USNA, 10300 Baltimore Avenue, Beltsville, MD 20705, USA

Plantago asiatica mosaic virus (PlAMV) was described four decades ago from the weedy species Plantago asiatica in the Russian Far East, but has also been reported from lilies (Lilium spp.) and primrose (Primula seiboldii) in Japan. More recently PlAMV has been reported in the Netherlands and elsewhere in Europe; in Taiwan; and in the United States in bulbs imported from the Netherlands. An isolate from heavenly bamboo (Nandina domestica) was initially described from California, USA as Nandina mosaic virus at about the same time as PlAMV was described from Russia, and was recognized as an isolate of PlAMV (PlAMV-NMV) only when the full sequence of the Nandina mosaic isolate was determined; however, Nandina is the only reported natural host of PlAMV-NMV. Lily isolates of PlAMV cause significant economic damage in lilies, especially Asiatic and Oriental hybrid types grown for cut-flower production. We have therefore determined the 3'-terminal sequence including the coat protein (CP) gene of multiple isolates from different imported lily cultivars, and compared these sequences to those of isolates from lilies and other hosts from different countries. To date the CP sequences of all isolates from imported lilies fall within the same clade as isolates from Europe, whereas lily and primrose isolates from Japan form a second clade; PlAMV-Type (from P. asiatica) and PlAMV-NMV form monotypic clades, as does the closely-related Tulip virus X (TVX), also reported from Japan. PlAMV-Type and PlAMV-NMV share multiple N-terminal CP residues that distinguish them from all other available PlAMV isolates; several other variant N-terminal residues are uniquely conserved within the ‘European’ clade, and others within the ‘Japanese’ (lily and primrose) clade. These results support the serological differentiation of ‘European’ lily isolates from PlAMV-NMV, and suggest that ‘Japanese’ isolates may also be serologically distinct.

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04_VIRAL SPREAD AND DIVERSITY IN ANTHROPOCENE

K. R. Richert-Poeggeler1, B. Lockhart2, J. S. Heslop-Harisson3 and T. Schwarzacher3

1 Institute for Epidemiology and Pathogen Diagnostics, Julius Kühn-Institut, Braunschweig, Germany2 Department of Plant Pathology, University of Minnesota, St. Paul, Minnesota, USA3 Department of Genetics, University of Leicester, Leicester, UK

The term “anthropocene” coined by Paul J. Crutzen in 2002 (Geology of mankind: the anthropocene, Nature 415, 23) is referring to our current epoch and illustrates the manifold influences by human existence and actions on geology and evolution.

Ornamentals are plants solely produced to please the eye of the beholder. The EU Commission stated 2014 an increase in flower production and cultivation of ornamental plants. The collected data reveals that in the EU one of the world's highest densities of flower production per hectare exists that comprises 10% of total world area and 44% of world flower and pot-plant production. It is predicted that the market for ornamentals in the EU will further expand and grow to 37 billion Euros in 2016 (Swedish Chamber of Commerce, 2011).

Global production and trade pathways as well as the consumer’s growing demand for new species and cultivars and their availability in shorter periods of time open gateways for viruses. The dynamics in trade are accompanied by strong overall growth affecting virus spread and diversity. The mass production of naturally cloned material displaying a uniform phenotype has an inherently high risk for multiplying and spreading viruses and/or viroids unwillingly, especially in case of asymptomatic (latent) infection. Synergy of electron microscopy and molecular biology tools were used to resolve these diagnostic challenges. 20 plant families were included in the analysis. Special attention was paid to Carlaviruses which often are accompanied by an asymptomatic phenotype and tobamoviruses which are easily mechanically transmitted. For both virus genera a high diversity was observed in the family of Solanaceae. The spread of endogenous pararetroviruses in wild petunia representing the parental crossing partner of nowadays hybrid petunia will be illustrated by Petunia vein clearing virus (PVCV). Analysis of integration loci shows similar arrangements in both genomes. However, preservation of integrated PVCV sequences is different. Genome hybridization has led to tandem array structures of chromosomal PVCV insertions that allow direct transcription of full-length infectious viral genomes.

05_EPIDEMIOLOGY OF AND RESISTANCE TO ROSE ROSETTE VIRUS

Patrick L. Di Bello1, Thanuja Thekke-Veetil1, Tobiasz Druciarek1 and Ioannis E. Tzanetakis1*

1Department of Plant Pathology, Division of Agriculture, University of Arkansas, Fayetteville, AR 72701, U.S.A.*Presenting author

Rosette is the most important virus disease of rose in the United States. It is caused by Rose rosette virus (RRV), an emaravirus vectored by the eriophyid mite Phyllocoptes fructiphilus (Keifer). Because of the recent discovery and proof that RRV is the causal agent of the disease all assumptions are based on visual observations of material that may or may have not been infected by the virus. This work uses a systems-based approach to address virus epidemiology and disease. A new, sensitive, detection protocol was developed and used to verify that RRV moves systemically in rose. Several

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genotypes were screened for resistance to the mite and/or the virus. The mite acquisition and inoculation access periods were also evaluated. The outputs of this study will assist in the better management of the vector and the disease. As the virus moves systemically in rose pruning is not an efficient approach to combat the disease. A resistant genotype has been identified. This discovery is important as this cultivar could be used in areas with high disease presence, for identification of the resistance mechanisms or in breeding to incorporate resistance to the virus in new cultivars. Chemical control for the vector may be a challenging undertaking as the vector can retransmit the virus in less than 1h feeding on RRV-free material.

06_REDBUD YELLOW RINGSPOT DISEASE: ANOTHER EMARAVIRUS ASSOCIATED WITH A DISEASE OF ORNAMENTAL PLANTS

Patrick L. Di Bello1, Alma G. Laney1, Tobiasz Druciarek1, Thien Ho1, Rose C. Gergerich1, Karen E. Keller2, Robert R. Martin2 and Ioannis E. Tzanetakis1*

1Department of Plant Pathology, Division of Agriculture, University of Arkansas, Fayetteville, AR 72701, U.S.A.2 USDA-ARS Horticultural Crops Research Laboratory, Corvallis, OR 97330.*Presenting author

Yellow ringspot (YR) is the only virus-like disease reported in redbud. The disease is expressed as leaf vein clearing, chlorotic ringspots and oak-leaf pattern and there have been reports being associated with tree decline. In the process of characterizing the causal agent of YR, we identified a new member of the genus Emaravirus. A detection protocol has been developed and used to assess the association of the virus with the disease. All symptomatic plants were infected by the virus and the name redbud yellow ringspot associated virus (RYRSaV) is proposed. Several steps were taken to understand RYRSaV and YR disease. A diversity study, based on two genomic regions of the virus, point to a homogeneous population structure. Transmission using Aculops cercidis, the most abundant eriophyid mite species colonizing infected material was also evaluated. Based on the accumulated data on emaravirus evolution we propose that speciation in the genus is allopatric, governed primarily by vectors.

07_VIRUSES OF ORNAMENTALS EMERGING IN FLORIDA AND THE CARIBBEAN REGION.

Scott Adkins1, Carlye A. Baker2, Colleen Y. Warfield3, Consuelo Estévez de Jensen4, Ismael Badillo-Vargas1,5, Craig G. Webster1, Galen Frantz6, H. Charles Mellinger6, Joseph E. Funderburk5 and Rayapati Naidu7

1 USDA-ARS, U.S. Horticultural Research Laboratory, Fort Pierce, FL USA2 FDACS-DPI, Gainesville, FL USA 3 Ball Horticultural Company, West Chicago, IL USA4 University of Puerto Rico, Mayagüez, PR USA5 University of Florida, NFREC, Quincy, FL USA6 Glades Crop Care, Inc., Jupiter, FL USA7 Washington State University, IAREC, Prosser, WA USA

Historically, Tomato spotted wilt virus has been a significant constraint to ornamental and vegetable crop production. With the emergence of Tomato chlorotic spot virus (TCSV) and a natural Groundnut ringspot virus (GRSV) reassortant in Florida and the Caribbean region, the significance of these three

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tospoviruses in production of major solanaceous vegetables including tomato and pepper has increased. In addition, TCSV has been reported in common solanaceous weeds including American black nightshade (Solanum americanum) and jimsonweed (Datura stramonium), in Florida and/or Puerto Rico. Experimental host range studies demonstrated that TCSV and/or GRSV can also infect solanaceous (petunia and brugmansia) and non-solanaceous (garden impatiens) ornamentals. During 2014, the first natural TCSV infections of non-solanaceous ornamentals porcelainflower (Hoya wayetii), false Christmas cactus (Schlumbergera truncata) and annual vinca (Catharanthus roseus) were detected in Florida. Since then, TCSV has been documented in other important crop and weed species, indicating host and geographic range expansion of this tospovirus. While surveying for tospoviruses infecting ornamentals, several tobamoviruses have also been detected in plants with symptoms similar to those induced by TCSV. In view of projected climate change-driven shifts in cropping systems, tracking emerging viruses infecting ornamentals in Florida and the Caribbean region will help strengthen agricultural security.

Contact/Presenting Author informationScott Adkins, USDA-ARS, USHRL, Fort Pierce, FL 34945 USA, Phone: 772-462-5885, Fax: 772-462-5986, E-mail: scott.adkins@ars.usda.gov

08_EVIDENCE OF NEW VIRUSES INFECTING FREESIA HYBRIDS SHOWING NECROTIC DISEASE

A.M. Vaira1, L. Miozzi1, M. Vallino1, A. Carra1, R. Lenzi1, D. Salvi2, J. Hammond3 and H.R. Pappu4

1 Institute for Sustainable Plant Protection, CNR, Strada delle Cacce 73, 10135 Torino, Italy 2 Studio Ferrari Salvi, Via Quinto Mansuino 12, 18038 Sanremo (IM), Italy3Floral & Nursery Plants Research Unit, USDA-ARS, USNA, 10300 Baltimore Avenue, Beltsville, MD 20705, USA4Department of Plant Pathology, Washington State University, PO Box 646430, Pullman, WA 99164, USA

Necrotic disorder of freesia is a high impact disease for this attractive ornamental crop and is now widespread in temperate world regions, being reported and studied in Europe, United States, New Zealand and South Korea. The presence of Freesia sneak virus (FreSV, genus Ophiovirus, family Ophioviridae) has been widely associated with the necrotic disease but other viral agents might be involved in producing the heavy necrotic symptomatology of this vegetatively propagated bulbous floral crop, so severely threatened by the disorder. During the 2014 growing season, freesia plants of different cultivars with heavy necrotic symptoms were collected in the Sanremo area, Northern Italy. Symptomatic foliar tissue was used both for mechanical inoculation to the model plant Nicotiana benthamiana and for virus purification using a method applicable to multiple viruses. A total RNA extraction of the sample enriched in viral agents was tested by Sequence-Independent Amplification to get preliminary information about the presence of both known and previously undescribed viruses. In parallel, Next Generation Sequencing (NGS) of total RNA was performed on samples extracted from the inoculated N. benthamiana plants, to more easily identify unknown virus (es), due to the fact that, unlike the freesia genome, the genome of N. benthamiana is available. A total of almost 25 million reads were obtained, then assembled into about 33 thousand contigs. NGS data analysis is currently under elaboration and validation but preliminary results show possible occurrence of previously undescribed DNA and RNA viruses infecting diseased freesia. PCR tests are being implemented to validate detection of such novel virus sequences.

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09_CHARACTERIZATION OF A NUCLEORHABDOVIRUS FROM PHYSOSTEGIA

1Wulf Menzel, 1Dennis Knierim, 1Katja Richert-Pöggeler, 1Stephan Winter

1Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstraße 7 B, 38124 Braunschweig, Germany2Julius Kühn Institute, Institute for Epidemiology and Pathogen Diagnostics, Messeweg 11/12, 38104 Braunschweig, Germany

In July 2014, a Physostegia plant showing severe chlorotic mottle symptoms and leaf deformation was received from Austria. Following mechanical inoculation, the virus systemically infected Nicotiana occidentalis 37b. Bullet shaped particles were found in electron microscopical observations indicating for presence of rhabdoviruses. As expected for negative strand RNA viruses, dsRNA extractions failed to extract viral replication intermediates from infected Physostegia or Nicotiana plants and RT-PCR using published sets of degenerate rhabdovirus oligonucleotide primers also did not result in amplification of viral sequences. This prompted us to use a total RNA extract as template for a next generation sequencing approach. Following selective depletion of ribosomal RNA (Invitrogen RiboMinus™ Plant Kit), a paired-end library was created from dscDNA (Illumina Nextera XT Library Preparation Kit) and sequenced on an Illumina MiSeq platform. De Novo assembly of the one million reads obtained was done using the Geneious software (ver. 9). A single sequence contig of 13,193 bases assembled from 40,376 reads was identified by blast as the putative genome (lacking the extreme 5’- and 3'-ends) of a nucleorhabdovirus, with highest nucleotide sequence identity of 70% to eggplant mottled dwarf virus and of 53% to a potato yellow dwarf virus. The nt identity values between assigned nucleorhabdoviruses range from 38.4% to 58.6%. Since no sequence identity threshold value has been defined for species demarcation within the genus Nucleorhabdovirus, it remains doubtful whether this isolate represents a deviant EMDV isolate or belongs to an independent species. Seedlings grow from seeds of infected Physostegia plants were symptomless and virus free, indicating the lack of seed transmissibility which is consistent with the lack of seed transmission in rhabdoviruses. Based on the symptoms observed on the original host, the isolate was named physostegia chlorotic mottle virus (PhCMoV) and is available at the DSMZ Plant Virus Collection under accession no. PV-1182.

10_IDENTIFICATION OF NARCISSUS YELLOW STRIPE VIRUS AND A CLOSELY-RELATED POTYVIRUS ISOLATE IN PLANTS OF ALLIUM CARINATUM

D. Bampi1,2, M.D. Reinsel1, and J. Hammond1

1 Floral & Nursery Plants Research Unit, USDA-ARS, USNA, 10300 Baltimore Avenue, Beltsville, MD 20705, USA2 Faculdade de Ciências Agronômicas, FCA-UNESP, Departamento Proteção Vegetal, Rua José Barbosa de Barros, 1780, CEP: 18610-307, Botucatu, SP, Brazil

A survey of varieties and species of ornamental Allium species revealed the presence of multiple viruses, including potyviruses, carlaviruses, and allexiviruses. Most of these viruses have been previously identified in garlic, onion, leek, and/or other edible Allium species. However, two samples of Allium carinatum were found by generic potyvirus PCR and transmission electron microscopy to be infected with potyvirus isolates which were not amplified with primers specific for Leek yellow

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stripe virus (LYSV) or Onion yellow dwarf virus (OYDV), the potyviruses most commonly found in cultivated Allium spp. These samples also yielded negative results in ELISA tests with LYSV- or OYDV-specific antibodies. Broad spectrum PCR primers for several regions of the potyvirus genome (Helper component-Proteinase, HC-Pro; Cytoplasmic inclusion, CI; and the Nuclear inclusion b to 3’ end, NIb-3') were then used to amplify and clone portions of the genome of the potyvirus isolates from A. carinatum; isolate-specific primers from the sequences obtained were then used to bridge gaps between the initial PCR products. The sequences generated were used to identify one isolate as a strain of Narcissus yellow stripe virus (NYSV), and the other as a potyvirus most closely related to NYSV, but differing sufficiently to be regarded as either a potential novel species based on current criteria for species boundaries, or possibly a recombinant isolate between NYSV and an unknown parent. However, divergent isolates of NYSV and Narcissus late season yellows virus have been reported from narcissus in Australia, suggesting that an intermediate between these species occurs, which might be either be regarded as a third species, or the two species collapsed into one by broadening the species demarcation criteria. Independent of the eventual status of the second isolate, Allium carinatum is a new host of NYSV, which has previously been reported only from Narcissus spp. and Nerine bowdenii.

11_ NEW VIRUS DISEASES OF LISIANTHUS IDENTIFIED IN TAIWAN

Y.K. Chen, H.Y. Chao, P.J. Shih, Y.S. Chang, and J.Y. Lee Dept. Plant Pathology, National Chung Hsing University, Taichung 402, Taiwan

Lisianthus (Eustoma grandiflorum) is one of the important ornamental crops in Taiwan. At least 20 viruses including two begomoviruses, Tomato yellow leaf curl virus (TYLCV) and Ageratum yellow vein virus (AYVV), have been reported to infect lisianthus. Lisianthus plants showing leaf curl and enation on cup-shaped upper leaves and flower petals were found in central Taiwan in 2015. Symptoms and the presence of whiteflies on the infected plants suggests a possible infection by a geminivirus. Degenerate primers for Begomvirus DNA-A, DNA-B and associated satellite DNA were used in PCR to detect possible Begomovirus. A 1.5 kb cDNA of Begomovirus DNA-A, but not DNA-B and satellite DNA, were consistently amplified from 17 out of 18 samples collected. Forward and reverse primers were further designed to amplify complete viral DNA-A. Three clones were sequenced and deposited to Genbank under accession numbers of LC089013, LC089014, and LC089766, respectively. All three clones are 2372 bp in length, containing typical genome organization of monopartite begomoviruses, and sharing more than 99% sequence identities. Sequence comparison revealed that the 3 isolates were most closely related to Papaya leaf curl Guangdong virus (PaLCuGdV)-PF1 (KC161184) and -BXG4 (KP876482) with 93.3% identities. This indicated that the lisianthus-infecting virus was PaLCuGdV. Two more virus diseases, caused by Pothos latent virus (PoLV) and Bidens mottle virus (BiMoV), in lisianthus will be reported and discussed.

12_ CHARACTERIZATION AND ITS ETIOLOGY OF TWO APHID TRANSMISSIBLE VIRUSES FROM JASMINE IN TAIWAN.

S.M. Wang1, Y.Y. Lin1, Y.C. Lin1, and C.A. Chang1

1 Dept. of Applied Chemistry, Chaoyang University of Technology, Taichung 41349, Taiwan.

Jasmine (Jasminum sambac) was widely grown as ornamental plants and for extraction of essential oil in Taiwan. In 2003, a potyvirus, designated as Jasmine virus T (JaVT) was isolated from jasmine

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exhibiting severe yellow mosaic symptom. However, due to jasmine’s vegetative propagation nature we were unable to conclude this yellow mosaic disease was exclusively due to JaVT’s infection. JaVT were later confirmed by sequence comparison as a new Potyvirus species (accession no. EF535842). Several potyviruses later reported from India and China were all found sharing more than 90% CP gene sequence identities with JaVT. In 2010, some JaVT isolates were obtained from a Jasmine plant showing only mild mottling symptom, indicating that JaVT might not be the real cause for the yellow mosaic symptom in jasmine. In 2012, we detected a carlavirus from yellow mosaic jasmine plants by the use of a degenerated carlavirus-specific primer pair. Full length sequence of this carlavirus was later revealed and the genome structure was identical to that of Carlavirus. Sequence comparison with other known species confirmed that it was a new carlavirus species and thus named as Jasmine virus C (JaVC). Subsequent studies confirmed that JaVC could only be detected in jasmine plant with severe yellow mosaic symptom but not in those bearing mild mottle symptoms. Antibodies against JaVC’s coat protein (CP) was successfully prepared by bacteria cloning and expressing methodology. By the use of ELISA, the presence of JaVC was also confirmed exclusively associating with severe yellow mosaic symptoms. Altogether, we believed that yellow mosaic symptom of jasmine was a synergistic effect of mixed infection by JaVT and JaVC.

13_CHARACTERIZATION AND GENOME SEQUENCE ANALYSIS OF LYCHNIS RINSSPOT VIRUS

Yoon-Hyun Bang1, Eun-Gyeong Song1, Ji-Yeon Kwon1, Eun-Kyoung Kim2, Jin-Sung Hong2 and Ki-Hyun Ryu1*

1Plant Virus GenBank, Seoul Women's University, Seoul, 01797 Republic of Korea2Kangwon National University, Chuncheon-si, Gangwon-do, 24341 Republic of Korea*Corresponding author: ryu@swu.ac.kr

Lychnis ringspot virus (LRSV), genus Hordeivirus, isolated from Lychnis spp. can cause systemic mosaic and severe necrosis in N. benthamiana. In this study, sequences of tripartite genomes (RNA a, RNA β and RNA γ) of LRSV were determined and analyzed. The open reading frames (ORFs) of LRSV were compared with the other viral genera. Coding proteins of helicase subunit of RNA-dependent RNA polymerase (αa), coat protein (βa), triple gene block (βb, βd and βc), polymerase subunits of replicase (γa), pathogenicity-related protein (γb) were compared with the other hordeiviruses and their similarities were 40.1 – 80.6% at nucleotide levels and 28.7 – 68.4% at amino acid levels, respectively. LRSV belongs to the lychnis infecting hordeivirus and phylogenetic analysis revealed that when LRSV-encoded proteins are compared with the other virus genera, this virus was closely grouped with the other hordeiviruses. In conclusion, the results indicate that this virus can be considered to be new strains of LRSV, and belong to the same species within the genus Hordeivirus.

14_CHRYSANTHEMUM STUNT VIROID IN ARGYRANTHEMUM---DISTRIBUTION AND ELIMINATION

Zhibo Zhang1, YeonKyeong Lee3, Carl Spetz2, Jihong Liu Clarke2, Astrid Sivertsen3, Gry Skjeseth3, Sissel Haugslien1, Qiaochun Wang1and Dag-Ragnar Blystad2*1Norwegian Institute of Bioeconomy Research, Ås, Norway2State Key Laboratory of Crop Stress Biology for Arid Areas, Key Laboratory of Genetic Improvement of Horticultural Crops of Northwest China, College of Horticulture, Northwest A&F University, Yangling, Shaanxi, P.R. China3Department of Plant Sciences, Norwegian University of Life Science, Ås, Norway.

Email: zhibo.zhang@nibio.no, dag-ragnar.blystad@nibio.no

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Chrysanthemum stunt viroid (CSVd) has to date been found widespread in the world wherever chrysanthemum is grown. In Norway, CSVd causes serious economic problems in the production of Argyranthemum, a genus closely related to Chrysanthemum. Use of viroid-free plants is pivotal for a sustainable production of these plants and allows exchange of the materials all over the world. In our study, we have two objectives: 1) eliminate CSVd from Argyranthemum, and 2) study distribution patterns of CSVd in Argyranthemum. After 5oC treatment combined with meristem tip culture, two CSVd-free ‘Border Dark Red’ plants were achieved, while no CSVd-free ‘Yellow Empire’. Using in situ hybridization of CSVd, we found that CSVd distribution patterns in the SAM showed no changes in diseased ‘Yellow Empire’ during 5oC treatment compared with the SAM at room temperature. However, the CSVd-free area in SAM was enlarged in diseased ‘Border Dark Red’ during prolonged 5oC treatment. At room temperature, in the diseased ‘Yellow Empire’ and ‘Butterfly’, CSVd was found in all tissues including the uppermost cell layers in the apical dome (AD) and the youngest leaf primordia 1 and 2. In diseased ‘Border Dark Red’ and ‘Border Pink’, CSVd was detected in the lower part of the AD and elder leaf primordia, leaving the upper part of the AD, and leaf primordia 1 and 2 free of viroid. No differences were found in the ability of CSVd to infect shoots and flower organs between different cultivars. Callose (β-1, 3-glucan) particles deposited at plasmodesmata in meristem is most likely responsible for the differences in ability of CSVd to invade meristem among Argyranthemum cultivars. The study contributed to give better understanding for the distribution of CSVd in systemically infected Argyranthemum and potential of using low temperature treatment and meristem tip culture to achieve viroid-free plants.

15_MULTILOCUS TYPING FOR CHARACTERIZATION OF ‘CANDIDATUS PHYTOPLASMA ASTERIS’-RELATED STRAINS IN SEVERAL ORNAMENTAL SPECIES IN ITALY

S. Paltrinieri1, M.G. Bellardi1, F. Lesi1, E. Satta1, S. Davino2, G. Parrella3, N. Contaldo1 and A. Bertaccini1

1Department of Agricultural Sciences, Plant Pathology, Alma Mater Studiorum University of Bologna, viale G. Fanin 42, 40127 Bologna, Italy2Dipartimento di Scienze Agrarie e Forestali, University of Palermo, Viale delle Scienze, Ed. 4, 90128 Palermo, Italy3Institue for Sustainable Plant Protection, CNR, Portici Unit, Via Università 133, 80055 Portici (Napoli), Italy

Different ornamental plants showing symptoms referable to phytoplasma presence and collected between 1993 and 2015 in various floricultural areas in north and south of Italy, enclosing Sicily, resulted to be infected by ‘Candidatus Phytoplasma asteris’-related strains, and after PCR/RFLP identification on 16Sr gene were assigned to 16SrI-B subgroup. These infected samples were employed for phytoplasma strain differentiation on tuf, groel, rp and amp genes. In particular, the 23 phytoplasma strains employed were from hydrangea (5), primula (3), gentian (2), petunia (2) and gerbera (1) samples showing flower virescence; from gladiolus samples both in vivo and in micropropagation (2) showing the “germs fins” symptomatology, from statice (2) with stunting and lack of flower production, from ranunculus (2) and carnation (4) with virescence and malformation of flowers. All the genes were amplified in nested PCR except amp gene. On tuf gene all the samples were amplified, and Tru1I RFLP analyses confirmed identical profiles with those of 16SrI group phytoplasmas, however on the other genes only samples from ranunculus, gladiolus in vivo, statice and hydrangea were amplified. On these genes the phytoplasmas were identical to each other and to reference strains belonging to 16SrI-B subgroups, and after RFLP analyses carried out with Tru1I and AluI they were further enclosed in the rpI-B and GroELI-III groups. Considering that these samples were collected in different Italian regions during 22 years, the relevant conservation in the studied genotypes can be perhaps linked to the presence of common leafhopper vectors, not always identified nor detected in the cultivation areas where the diseased plants were collected. It is

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important to underline that ‘Ca. P. asteris’ is the prevalent phytoplasma reported in flower cultivations worldwide, and its lack of genetic polymorphisms may also indicate a globalized trading of the pathogen together with his propagation material.

16_RAPID SCREENING FOR PHYTOPLASMA PRESENCE IN FLOWER CROPS USING TUF GENE BARCODE

N. Contaldo, S. Paltrinieri, M.G. Bellardi, F. Lesi, E. Satta, and A. BertacciniDepartment of Agricultural Sciences, Plant Pathology, Alma Mater Studiorum University of Bologna, viale G. Fanin 42, 40127 Bologna, Italy

Several molecular markers are nowadays available for phytoplasma strains discrimination, however, they often cannot be used for identification of phytoplasmas belonging to different ribosomal groups or are not friendly for routine diagnostics. The DNA barcode amplicon based on the elongation factor Tu (Tuf) gene for universal phytoplasma identification (420-444 bp) was employed for verification of phytoplasma presence in samples from different plant species in PCR/RFLP analyses. Samples from 13 flower species showing symptoms referable to phytoplasma presence and from corresponding asymptomatic plants were tested. The symptomatology present in the tested samples ranged from virescence in orchid, narcissus, gentian, primula, gladiolus, surphinia and hydrangea to phyllody and or flower malformation in ranunculus, carnation, petunia, statice, helicrysum, and gerbera. PCR products of the expected length were obtained from all symptomatic samples and no amplicons were produced from negative controls devoid of DNA and from healthy plants of the same species. The RFLP analyses carried out with TruI, Tsp509I, TaqI restriction enzymes allowed the differentiation among phytoplasmas in agarose 3% gels and resulted useful for fast screening of large number of samples. The achieved phytoplasma differentiation is in agreement with published phytoplasma groupings based on 16S rDNA. The visualization of restriction profiles in agarose is a handily tool for large number of sample processing enclosing identification ability. In case of phytoplasmas relevant for quarantine, sequencing may be necessary for confirmation. Tuf reference barcodes are deposited in the NCBI GenBank and in the newly developed Q-bank (http://www.q-bank.eu/Phytoplasmas/), a freely available online identification tool for plant pests and pathogens of quarantine status.

17_SILENCING THE CymMV COAT PROTEIN GENE BY RNAi AND RECOMBINANT miRNA TECHNOLOGY IN TRANSGENIC DENDROBIUM ORCHID

Pattana Srifah Huehne1,2, Udomporn Petchthai1, Anchalee Chuphromp1, Kisana Bhinija2

1. Dept. Genetics, Fac. Science, Kasetsart University, Chatuchak, Bangkok, 10900, Thailand 2. Lab. Biotechnology, Chulabhorn Research Institute, Laksi, Bangkok, 10210, ThailandE-mail: fscipns@ku.ac.th; pattana@cri.or.th

More than 20 genera of orchids have been reported to be infected by viruses showing a variety of symptoms. The common virus diseases in orchids are caused by two kinds of viruses included cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) which are world widely distributed viruses. To eliminate virus infection by gene silencing technology, the RNAi construction of CymMV viral coat protein (CP) gene and the artificial orchid miRNA combined with the partial CymMV-CP gene were transferred to Dendrobium orchids by particle bombardtment. After 6-9 month transformation, the transgenic orchids were confirmed the stable integration of the transgenes and the expression of CymMV-CP gene were examined by RT-PCR. The contaminated CymMV in transgenic lines obtained from the CymMV-CP RNAi transformation was gradually decreased and completely eliminated from the transgenic lines. In another word, no CymMV viral

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genes and virus particles were detected in the contaminated transgenic lines. Also, the growth of transgenic lines obtained from the CymMV-CP RNAi transformation performed better than the transgenic lines carrying the recombinant miRNA construction. These results suggest that the CymMV infecting in orchids can be entirely eradicated by silencing its coat protein gene using CymMV-CP RNAi technology.

18_TRANSGENIC NICOTIANA BENTHAMIANA RESISTANCE TO SYNERGISTIC INFECTION OF TWO ORCHID VIRUSES CYMMV AND ORSV

U. Petchthai1, Z. Xie1, and S.M. Wong1,2, 3

1 Dept. Biological Sciences, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260. E-mail: dbsup@nus.edu.sg

2 Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore

3 National University of Singapore Research Institute (Suzhou), Suzhou Industrial Park, Suzhou, PRC

Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) have been reported as co-infection (or synergistic interaction) resulting in more severe symptoms in orchids. In this project, silencing dual RNA dependent RNA polymerase (RdRp) transcripts of CymMV and ORSV base on RNA interference (RNAi) including of amiRNA-CymMV-ORSV or antisense-CymMV-ORSV constructs were adopted to create transgenic plants that can resist to CymMV and ORSV at the same time. A susceptible host model to both viruses, Nicotiana benthamiana, was generated first to test the feasibility of the RNAi strategy and gene stacking also was applied to increase copy number in transgenic N. benthamiana by cross pollination. Successfully of viral resistant analyses were performed until T3 generation using Southern blot, real-time RT-PCR and western blot. Resistant T3

transgenic plants had a higher transgene expression as compared to non-resistant plants, coupled with the absence of CymMV and ORSV coat protein which suggests negligible CymMV and ORSV concentration in that resistant plant which allowed the plant to exhibit a normal phenotype after mixed inoculation for three weeks. We believe this is the first report on the transgenic N. benthamiana plants that can resist against both CymMV and ORSV concurrently.

19_FAST, EASY AND HIGHLY SENSITIVE DETECTION OF TOMATO CHLOROTIC DWARF VIROID (TCDVD) IN TOMATO AND ORNAMENTAL CROPS USING AGDIA’S TCDVD AMPLIFYRP® ACCELER8™

Lamborn, Janet Agdia, Inc., Hammond, Rosemarie W. USDA ARS and Zhang, Shulu Agdia, Inc.Pospiviroid infection has been an international problem for many years. With the increase of global movement of tomato seed and symptomless ornamental crops, such as Brugmansia sanguine and Petunia x hybrid spp., the problem has also increased. Tomato chlorotic dwarf viroid (TCDVd) is a serious threat to tomato production, leading to reduced vigor, small and/or deformed fruit and loss of yield. Through collaborative work, an assay utilizing reverse transcription-recombinase polymerase amplification was developed (AmplifyRP® Acceler8™). This assay utilizes crude extracts, needs no specialized equipment, runs at a constant 39°C, needs no technical experience to run, is specific to TCDVd detection, comparable to conventional RT-PCR, results in 30 minutes and can be completed in a lab setting or in the field.

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20_EVALUATION OF A SIMPLIFIED METHOD OF PLANT VIRUS INCLUSION VISUALIZATION BY LIGHT MICROSCOPY.

R.J. McGovern1,2, P. Khampirapang1, R. Cheewangkoon1, L.H. Koh3, C. To-anun1, and S.M. Wong4,5,6

1Dept. Entomology and Plant Pathology, Chiang Mai University, Huay Kaew Rd., Chiang Mai, Thailand2NBD Research Co., Ltd., 91/2 Rathburana Rd., Lampang, Thailand 3Agri-Food and Veterinary Authority of Singapore. Animal and Plant Health Center, Laboratory Dept., 6 Perahu Rd., Singapore4Dept. Biological Sciences, National University of Singapore, 10 Kent Ridge Crescent, Singapore 5Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore6National University of Singapore Research Institute (Suzhou), Suzhou Industrial Park, Suzhou, PRC

Many viruses produce nuclear or cytoplasmic viral aggregates (inclusions) that are diagnostic for virus groups, and, in some cases, specific viruses. Visualization of inclusions after staining by light microscopy has been used for many years in human pathology to diagnose infection by such viruses as the Cytomegalovirus and Human papilloma virus. Christie and Edwardson and others at the University of Florida adapted this approach for the diagnosis of plant virus infection. While very useful, their techniques used a number of hazardous reagents and a stain that is no longer manufactured. Our objective was to develop a safer and simpler method of detecting plant virus inclusions by light microscopy. This method involves: obtaining 20-30 µm sections or epidermal strips of appropriate plant tissue; removing chlorophyll with lactic acid; staining in an aqueous mixture of toluidine blue O and basic fuchsin; destaining (optional) in dilute ethanol; mounting in sterile water and viewing at 1000x. Thus far, detection of inclusions typical of Tomato yellow leaf curl virus and Cucumber mosaic virus in tomato and pepper, respectively, and virus confirmation by PCR has been accomplished. Presumed potyvirus and tospovirus infection of Hippeastrum sp. and Scadoxus multiflorus, respectively, has been detected. This is a preliminary study and we will continue to confirm light microscopy findings by molecular methods.

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POSTERS01_MOLECULAR DETECTION OF VIRAL DISEASES IN HYDRANGEA PLANTS

Eun-Gyeong Song, Hye-Min Lee, Hyeon-Soon Lee and Ki-Hyun Ryu*

1Plant Virus GenBank, Seoul Women's University, Seoul, 01797 Republic of Korea*Corresponding author : ryu@swu.ac.kr

Hydrangea plants include approximately 25 species of flowering ornamentals that are widely distributed in Asia, the Americas, Europe, and South Africa. Hydrangea plants have been reported as infected with various viruses including Alfalfa mosaic virus (AMV), Cucumber mosaic virus (CMV), Elm mottle virus (EMoV), Hydrangea chlorotic mottle virus (HdCMV), Hydrangea ringspot virus (HdRSV), Tobacco ringspot virus (TRSV), Tomato ringspot virus (ToRSV) and Tomato spotted wilt virus (TSWV). In order to investigate the viral infection in hydrangea plants, eight species-specific primers were designed and used for RT-PCR analysis. Of the 211 hydrangea plants examined, and two of the eight viruses, HdCMV and HdRSV, were detected. HdCMV was detected in 10, and HdRSV was detected in 33; two plants were co-infected with HdCMV and HdRSV. To our knowledge, this study is the first report of HdRSV and HdCMV infection in hydrangea plants in South Korea.

02_DEVELOPMENT AND CHARACTERIZATION OF POLYCLONAL AND MONOCLONAL ANTIBODIES TO ROSE ROSETTE VIRUS

Ramon Jordan, Mary Ann Guaragna, and John Hammond

US National Arboretum, Floral & Nursery Plants Research, Beltsville, MD 20705

Garden roses, which form the cornerstone of the multi-billion dollar landscape industry, annually generate wholesale US domestic production valued at ~ $400 million. Over the past few decades Rose rosette disease, caused by Rose rosette virus (RRV; genus Emaravirus), has become a major threat to the rose industry in the U.S. RRV is transmitted by wind-blown eriophyid mites, and can kill a rose within 2-3 years of infection. The long term goal of a recently-funded USDA-NIFA-SCRI Project (which includes 17 scientists in 6 states) is to develop roses resistant to this virus or mite vector. The only strategy currently available for disease management is early identification and eradication of the infected plants, thereby limiting its potential spread. Key to this effort is the development of efficient diagnostic tools to enable sensitive, rapid, user-friendly and accurate detection of the virus; currently limited to nucleic acid-based methods, such as PCR. With the aim of developing a serological diagnostic tool, rabbit polyclonal and several mouse monoclonal antibodies have been developed to the nucleocapsid protein (NP) of RRV. These antibodies were evaluated against NP peptides, cloned NP expressed in bacteria and plants, and RRV-infected plants. These results and those using the antibodies in various serological assay formats, including double- and triple-antibody sandwich ELISA and membrane-based assays, for lab and field detection of Rose rosette virus in infected sources, will be presented.

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03_A NEW VIROID DISCOVERED FROM LYCHEE BY DEEP SEQUENCING

Jihong Jiang1,4, Thierry Candresse2，Zhixiang Zhang1, Bing Hu3,Guibing Hu3, Hongqing Wang4, Shifang Li1

1 State Key Laboratory of Biology of Plant Diseases and Insect Pests, Institute of Plant Protection (IPP), Chinese Academy of Agricultural Sciences (CAAS), China.

2 UMR 1332 BFP, INRA, Univ. Bordeaux, Villenave d'Ornon, France.3 College of Horticulture, South China Agricultural University, Guangzhou, Guangdong, China.4 Department of Fruit Science, College of Horticulture, China Agricultural University, China.

Viroids are small, infectious, circular RNAs. Three viroid-like RNA contigs were identified by datamining RNASeq data from a lychee sample. According to the sequence information of these contigs, the full sequence of a novel viroid was confirmed by reverse transcription-polymerase chain reaction（RT-PCR）using two adjacent primers of opposite polarity. Denaturing PAGE followed by Northern-blot hybridization, with an RNA probe specific for the (+) strand revealed the characteristic circular and linear forms of viroid RNA in various lychee samples. The new agent, which shares maximum sequence identity with other viroids of less than 57.3% was named lychee viroid (LVd). LVd displays the characteristic features of the family Pospiviroidae: a predicted rod-like secondary structure of minimum free energy with a central conserved region (CCR) and terminal conserved regions (TCRs). Multiple alignment of LVd with all current Pospiviroidae species and phylogenetic analyses indicate that LVd is most closely related to members of the Apscaviroid genus and represents a new member of this genus. Using northern-blot hybridization, LVd was found to be present in 54 out of 71 samples from field-grown lychee plants in China (belonging to 68 cultivars) but was not detected so far in lychee genetic resources originating from other countries. To get a first insight into the experimental host range of LVd, mechanical inoculations of herbaceous plants by slashing their stems dipped in dimeric head-to-tail in vitro transcripts or agro-inoculation with Agrobacterium tumefaciens containing a plasmid for expressing a dimeric head-to-tail transcript of LVd were carried out. Analysis by Northern-blot hybridization showed that none of the tested plant species, known to be the herbaceous hosts of different viroids, became infected, indicating that LVd appears to have an unusually narrow host range. LVd is the first viroid identified in lychee trees. The potential pathogenicity of this new Apscaviroid on its lychee hosts is still unclear.

Email:Sfli@Ippcaas.Cn

04_B-FAST ELISA – A NEWLY DEVELOPED ELISA TECHNIQUE ALLOWING LARGE SCALE TESTING IN TWO HOURS

Wulf Menzel, Stephan Winter

Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstraße 7 B, 38124 Braunschweig, Germany

Intercontinental travel and trade have increased significantly in recent decades and resulted in the emergence of many plant diseases including those caused by viruses. Several new detection methods have been developed in the past to cope with these challenges in plant protection, but only very few got adopted for routine testing for various reasons. Enzyme-linked immunosorbent assay (ELISA) is still the most widely used method for large scale testing, despite being limited in its sensitivity compared to molecular methods, the fact that many separate operating steps have to be carried out and that results are usually available only the second day. Regarding the latter two

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shortcomings, a superior ELISA method was developed, allowing obtaining reliable results within just two hours with a sensitivity being comparable to standard ELISA. Furthermore, the convenient protocol contains only very few individual steps and hands-on time. Following the simultaneous incubation of a mixture of capture and detection antibody (conjugate) together with the test sample for 1 hour, the ELISA wells are washed and substrate is added without any further operating steps. This new ELISA is fully compatible with standard ELISA buffers and equipment. The availability of such a newly developed rapid diagnostic tool which is suitable for large scale testing based on standard ELISA platforms will allow a faster response to future challenges in plant virus detection in a globalized environment.

05_B-BANK FOR SHARING DATA AND INFORMATION ON PLANT VIRUS AND VIROID ISOLATES IN COLLECTIONS

Wulf Menzel1, Annelien Roenhorst2, Rene van der Vlugt3, Stephan Winter1

1Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstraße 7 B, 38124 Braunschweig, Germany2National Plant Protection Organization, PO Box 9102, 6700 HC Wageningen, the Netherlands3Wageningen University and Research Centre, Droevendaalsesteeg 1, 6708 PB Wageningen, the Netherlands

The availability of well characterized reference materials of viruses and viroids is crucial for research and diagnostic laboratories. To ensure the existence of collections, their growth and sustainability it is of utmost importance to collaborate at an international level particularly considering decreasing budgets and loss of experienced staff. Future efforts should focus on sharing data as well as expertise on reference collections. Q-bank, the comprehensive data-bases on plant pests and diseases, offers an excellent platform to share data on plant virus collections (http://www.qbank.eu/Virus). This web based information system informs on biological, serological and molecular characteristics of virus and viroid isolates available in public collections and indicates the collection from where it can be obtained. In addition, the database provides easy access to overviews of regulated species, test protocols and a sequence BLAST tool to assist diagnostic laboratories in species identification. Although most laboratories endorse the need for reliable reference isolates or materials that fulfil basic quality standards, they do not have the means to establish a 'certified reference collection' under official ISO standards like ISO Guide 34. Q-bank offers the opportunity to share data of characterised isolates that are available in collections for public use. Moreover, curators ensure that quality standards are met and conservation is safeguarded. To further strengthen the Q-bank database and collections, the EUPHRESCO project VirusCollect aims to extend the network of reference collections for regulated and other important viruses and viroids at the European level.

06_PARTIAL GENOMIC SEQUENCE OF A NEW TOBAMOVIRUS FROM PIPER

Wulf Menzel1, Stephan Winter1, Anja Westerman2, Jos Heldens2

1Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstraße 7 B, 38124 Braunschweig, Germany2Iribov SBW, Middenweg 591b, 1704 BH Heerhugowaard, the Netherlands

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Several Piper spec. of tropical origin are of economic importance. Besides those grown for agricultural black pepper production, some are used as ornamental plants. In spring 2014, several plants of an ornamental Piper variety were observed showing severe chlorosis. Electron microscopical examinations revealed the presence of rod shaped particles resembling those of tobamoviruses. Following mechanical inoculation, dsRNA of ca. 6.5 kbp could be extracted from systemically infected Nicotiana benthamiana plants exhibiting crinkling and mottling symptoms, which is within the size range of assigned tobamovirus species. Extracted dsRNA served as template for a random RT-PCR and cloning approach. Obtained sequences were assembled into a contiguous sequence, containing four putative open reading frames in an arrangement characteristic of members of the genus Tobamovirus. An almost complete coding region of the genome was obtained, lacking ca. 140 N-terminal amino acids of the replicase. The sequence showed the highest overall nucleotide (nt) sequence identity with 67,4% to an isolate of Odontoglossum ringspot virus. Even if not the entire sequence is yet available, this virus can be regarded as an isolate of a new tobamovirus species based on the molecular ICTV species demarcation criteria (<90% overall nt identity). The name piper chlorosis virus (PChV) is suggested for this isolate which is available at the DSMZ Plant Virus Collection under accession no. PV-1126.

07_MOLECULAR CHARACTERIZATION OF MaYVYV INFCETING MALVA PARVIFLORA L. IN SICHUAN PROVINCE OF CHINA

K. Li, J. Zhang, B. Z. Liu, C. C. Jing, G. T. Wu, X. C. Sun and L. Qing*

Chongqing Key Laboratory of Plant Disease Biology, College of Plant Protection, Southwest University, Chongqing, 400716, P.R. China *Corresponding author: L. QingFax number: +86-023-68251269E-mail address: qling@swu.edu.cn

Malva parviflora L. is a common ornamental plant in southern China. In 2010, samples of 10 symptomatic Malva parviflora L. plants showing yellow vein and leaf crinkling, and of two symptomless plants were collected in Sichuan province of China. A 500 bp DNA fragment was successfully amplified from the ten symptomatic samples with the degenerate primers PA/PB specific to begomoviruses (Geminiviridae). The fragment of sample SC224 was randomly selected for cloning and sequencing. Based on the sequence obtained, the specific primers MaYVYV-Y6F3 (5’-ACAGGATGTATAGAAGCCCTGA-3’) and MaYVYV-R285 (5’-ATCTGCAGGACGCTTCGACAT-3’) were designed to amplify the remaining DNA-A sequence. Sequence analysis showed that the full-length sequence of SC224 was 2747 nt long (JX679249) with the highest nucleotide sequence identity (99.7%) with isolate SC44 of Malvastrum yellow vein Yunnan virus (MaYVYV) (KC189891). Then a 1000 bp long fragment specific to MaYVYV was amplified from nine symptomatic samples using primers MYV-F (5’-GCCCACTAACTACTGTCTG-3’) and MYV-R (5’-TTAGAGGCATGGGTACATGC-3’). With the universal abutting primers for betasatellite, an amplicon of 1300 bp was obtained from sample SC224 and sequenced to be 1356 bp long (JX679254), which has a 99.4% identity with isolate 327 of Malvastrum yellow vein Yunnan betasatellite (MaYVYB) (KC427276). An amplicon specific to MaYVYB was detectable in five symptomatic samples.

Acknowledgements This research work was supported by Program for New Century Excellent Talents in University by the Ministry of Education in China (NCET-12-0931).

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08_INVESTIGATION OF GENE RESPONSE TO ETHEPHON IN PHALAENOPSIS ORCHID

Nunnaphat Punsiri1, Noppamart Lokkamlue2, Kisana Bhinija3, Pattana Srifah Huehne1,3

1. Dept. Genetics, Fac. Science, Kasetsart University, Bangkok 10900, Thailand2. Faculty of Liberal Arts and Science, Kasetsart University, Nakornpathom 73140, Thailand3. Laboratory of Biotechnology, Chulabhorn Research Institute, Bangkok 10210, ThailandE-mail: Nunnaphat.sci@gmail.com

Ethylene is a phytohormone which can induce programmed cell death and senescence. In particular during flower senescence, several phenomena such as colour fading (anthocyanin destruction) and stress responses are induced rapidly. When viruses infect plants, the ethylene signalling protein, ethylene receptor protein, acts as an oxidative stress sensor and negative regulators of the ethylene signal transduction pathway. This study is to invest the response of the ethylene response sensor (ers), 1-Aminocyclopropane-1-carboxylic acid oxidase (aco) and chalcone synthase (chs) gene in wild type Phalaenopsis and ers transgenic Phalaenopsis treated with ethephon. After 7M ethephon treatment, the ethephon significantly enhances the expression of the aco gene involving ethylene biosynthesis but less effect to ers and chs gene expression. This is a strong indication that the exogenous ethylene provided by ethephon was unable to up-regulate the ers and chs gene expression in Phalaenopsis, the ethylene sensitive orchid.

09_GENOMIC CHARACTERIZATION OF AN ISOLATE OF PEPPER MILD MOTTLE VIRUS IN NORTHWESTERN CHINA

Shuhua Wu, Tingfang Li, Wenhao Zhao,Yinghua Ji and Yijun Zhou

Institute of Plant Protection，Jiangsu Academy of Agricultural Sciences / Key Lab of Food Quality and Safety of Jiangsu Province – State Key Laboratory Breeding Base，Nanjing 210014，China

Pepper (Capsicum annuum) is one of the most economically important vegetable crops in China. In recent years, with the development of facilities vegetables, more and more viruses infecting pepper were reported. In 2014, an investigation on pepper virus in Qinghai was carried out and a tobamovirus was detected in almost all the pepper samples with leaf mottle symptoms. To clarify the possible virus, a pair of primers for full genomic amplification was designed and the full genome of the virus was cloned and sequenced. Sequence analysis showed that the full sequence of the isolate contains 6356 nucleotides and encode 4 ORFs. The full genomic nucleotide sequence of the isolate shared the highest sequence identity (99.7%) with Pepper Mild mottle virus (PMMoV). Polygenetic analysis revealed the isolate was clustered into PMMoV branch, which contained 3 clusters, and with isolates from Japan (C-1421, J, TPO-2-19, Pa18), Spain (S), China (CN), Brazil (BR-DF01) and India (HP1), the isolate belonged to cluster III. These results demonstrate that the virus identified from pepper with mottle symptoms is an isolate of PMMoV. To our knowledge, this is the first report of PMMoV in northwestern China in recent years.

Acknowledgment:This work was supported by National Natural Science Foundation of China (No. 31572074) and Jiangsu 333 High-level Talent Training Fund (BRA2013262).

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10_ROLE OF TOMATO YELLOW LEAF CURL VIRUS (TYLCV) V2 IN BLOCKING SYSTEMIC RNA SILENCING Yinghua Ji, Xiaowei Zhou, Hui Zhang, Wenhao Zhao, Shuhua Wu and Yijun Zhou

Institute of Plant Protection，Jiangsu Academy of Agricultural Sciences / Key Lab of Food Quality and Safety of Jiangsu Province – State Key Laboratory Breeding Base，Nanjing 210014，China

Tomato yellow leaf curl virus (TYLCV) is a major tomato pathogen, causing devastating losses worldwide, which contains only one genomic circular ssDNA molecule encoding six open reading frames. V2 is the protein reported inhibiting local RNA silencing of GFP. To characterize the role of V2 in inhabiting systemic RNA silencing, leaves of 16c transgenic N. benthamiana plants were co-infiltrated with various constructs (35S-GFP, 35S-V2, empty vector) and then monitored under a hand-held UV lamp for systemic GFP silencing. Systemic GFP silencing occurred in approximately 32% of the plants infiltrated with 35S-GFP and the empty vector, and 0% with 35S-GFP and 35S-V2 by 25dpi. The percentage of plants showing systemic GFP silencing for the treatment increased to about 68% by 40dpi. While only about 19% of the plants co-infiltrated with 35S-GFP and 35S-V2, developed systemic GFP silencing by 40dpi and the remaining plants (about 81%) continued to show strong green GFP fluorescence in their upper young leaves even at 40dpi, indicating that TYLCV V2 can prevent the spread of GFP gene silencing in the infiltrated transgenic N.benthamiana.

Acknowledgment:This work was supported by National Natural Science Foundation of China (No. 31572074) and Jiangsu 333 High-level Talent Training Fund (BRA2013262).

11_MULTIPLEX PCR ASSAY FOR SIMULTANEOUS DETECTION OF FIVE DIFFERENT POTEXVIRUES IN CACTUS PLANTS

Chung-Hwa Park, Eun-Gyeong Song, Ji-Yeon Kwon1, and Ki-Hyun Ryu*

Plant Virus GenBank, Seoul Women's University, Seoul, 01797 Republic of Korea

Five different potexviruses, Cactus virus X (CVX), Opuntia virus X (OVX), Pitaya virus X (PiVX), Schlumbergera virus X (SVX), Zygocatus virus X (ZVX), were previously reported in cactus plants. In this study, multiplex PCR assay using dual priming oligonucleotide (DPO) primers (Seegene) were developed for simultaneous detection of five different potexviruses in cactus plants. The multiplex PCR primers were designed to amplify 385 bp for CVX, 247 bp for OVX, 651 bp for PiVX, 812 bp for SVX and 507 bp for ZVX. These primers were tested using plasmid DNAs derived from the five viruses, and were successfully confirmed by amplification and sequencing of the expected DNA fragments. The five-targeted multiplex primers were also applied to virus-infected cactus plants, which could be confirmed single and mixed infections.

12_DEVELOPMENT OF A UNIVERSAL ONE-STEP REAL-TIME RT-PCR METHOD FOR RAPID DIAGNOSIS OF TOSPOVIRUSES

S.L. Li, J.H. Sun, C.H. Liao and T.C. Chen*

Department of Biotechnology, Asia University, Wufeng, Taichung 41354, Taiwan

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Tospovirus is the only plant-infecting genus in the family Bunyaviridae with quasi-spherical enveloped particles of 80-120 nm in diameter that possess a segmented tripartite single-stranded RNA genome, named L, M and S based on their molecular sizes. The S RNA-encoded nucleocapsid protein (NP) is the most important target for diagnosis and identification of tospoviruses. Based on the homology of NPs lower than 90% amino acid identity, 29 Tospovirus species are officially reported so far. Additionally, tospoviruses are divided into serogroups. Viruses in the same serogroup are difficult to distinguish by serological assays with the aid of antisera against the NPs. Reverse transcription-polymerase chain reaction (RT-PCR) with N gene-specific primers can be used to identify tospovirus species. Ordinarily, methods developed for detecting the NP or N gene are species-specific that are insufficient to diagnose uncertain virus species in field. The genus-universal degenerate primers designed from the consensus sequences of genomic RNAs are suitable for exploration of viruses. In this study, a new Tospovirus-universal degenerate primer set was designed from the L RNA for a SYBR Green I-based one-step real-time RT-PCR system to detect all tested greenhouse-cultured tospoviruses in plant total RNAs. Melting curve assays were conducted to indicate the specificity of amplifications. No non-specific signals were detected when outgroup viruses, such as Cucumber mosaic virus, Cymbidium mosaic virus, Tobacco mosaic virus and Zucchini yellow mosaic virus, were used for test. The real-time RT-PCR detection method was further applied to diagnose tospoviruses in field plant samples in Taiwan. All positive amplicons were directly sequenced to validate the identity of tospoviruses. The developed one-step real-time RT-PCR method has a genus-universal potential that can be used for rapid diagnosis of all tospoviruses whatever known and unknown.

*Corresponding author: Tsung-Chi ChenE-mail: kikichenwolf@hotmail.com | Tel: +886-4-23394362

13_SEROLOGICAL PLATFORM FOR PROMPT DETECTION AND DIAGNOSIS OF TOSPOVIRUSES

Y. C. Wang1, S.D. Yeh2 and T. C. Chen1

1 Department of Biotechnology, Asia University, Wufeng, Taichung 41354, Taiwan2 Department of Plant Pathology, National Chung Hsing University, Taichung 40227, Taiwan

Tospoviruses, belonging to the only plant-infecting genus of the family Bunyaviridae, are globally important due to the wide host range and persistent thrips transmissibility. The structural nucleocapsid protein (NP) and non-structural NSs protein encoded by S RNA are the major targets for serological detection of tospoviruses. Based on the sequence homology and serology of NP, the 29 current tospoviruses including 11 formal species and 18 tentative species are divided into 6 serogroups with Tomato spotted wilt virus (TSWV), Watermelon silver mottle virus (WSMoV), Groundnut yellow spot virus (GYSV), Iris yellow spot virus (IYSV), Soybean vein necrosis-associated virus (SVNaV) and Lisianthus necrotic ringspot virus (LNRV) as individual type members. In general, the members of a serogroup have cross reactions when detected by polyclonal antibodies (PAbs) against the NP but can be differentiated by monoclonal antibodies (MAbs). However, MAbs targeting the common epitopes of the NPs or NSs proteins can be group-universal. In our laboratory, 20 tospovirus species in 6 serogroups were collected from around the world, and 15 PAbs and 23 MAbs against their NPs or NSs proteins were produced. The specificity of individual antibodies was demonstrated by testing with different tospoviruses. The geographically distinct tospoviruses can be differentiated by the group-universal PAbs and MAbs. The combinations of these antibodies can be used to detect all tested tospoviruses that have a potential for rapid detection of known tospoviruses.

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14_CHARACTERIZATION OF A NEW MONOPARTITE BEGOMOVIRUS WITH A DISTINCT BETASATELLITE ASSOCIATED WITH ROSE MALLOW EXHIBITING LEAF CURL AND VEIN ENATION

C.-H. Tai1, N. Sharma1, C.-H. Chao2 and F.-J. Jan1*

1Department of Plant Pathology, National Chung Hsing University, Taichung 40227, Taiwan2Plant Protection Laboratory, Taichung District Agricultural Research and Extension Station, Council of Agriculture, Changhua 51544, Taiwan *E-mail: fjjan@nchu.edu.tw

In November 2013, rose mallow (Hibiscus rosa-sinensis Linn) plants with begomoviral disease-like symptoms of leaf curl and vein enation were collected from Changhua County in central Taiwan. Total DNA was extracted from plant tissues and used for polymerase chain reaction with the primers specifically for detecting begomoviral DNA-A, -B and associated satellite. A 1.5-kb fragment representing begomoviral DNA-A was detected from the symptomatic leaf tissue using the degenerate primers. The full length viral DNA-A genome was cloned and sequenced after rolling cycle amplification. The comparison of DNA-A genome sequence with those of the other geminiviruses available in GenBank indicated that the virus has the highest nucleotide identity 88.7% with Malvastrum yellow vein Baoshan virus (FN386460). Primers specific for begomoviral DNA-B failed to amplify any products from diseased samples. In contrast, virus-associated satellite DNA was detected in the leaf tissue with symptoms, confirming that the virus was the same as other monopartite begomoviruses. The sequence of associated satellite DNA was obtained and identified as a betasatellite DNA with one ORF in the virus complementary sense (βC1) and conserved nano-sequence as the identified DNA-A. The comparison of virus-associated betasatellite DNA sequence with those of others available in GenBank showed that the virus shared its highest nucleotide identity of 78.6% with Malvastrum yellow vein betasatellite (AJ971708). Both of the infectious DNA-A and betasatellite were constructed by cloning tandem repeats of viral genomes into a binary vector. Agroinoculation of infectious viral DNAs to Nicotiana benthamiana showed that the virus alone induced mild symptoms, whereas co-inoculation with betasatellite induced severe symptoms. Because of DNA-A nucleotides have less than 89% identity with other geminiviruses, the virus associated with rose mallow leaf curl and vein enation disease should be considered as a new monopartite begomovirus with a distinct betasatellite and was designated as Hibiscus vein enation virus.

15_EFFICIENCY ASSESSMENT OF GENETIC DESIGNS WITH COAT PROTEIN IN TRANSGENE-MEDIATED RESISTANCE AGAINST CHRYSANTHEMUM VIRUS B

T. Mitiouchkina1,3, A. Firsov1, S. Titova, O. Shulga2,3, S. Dolgov1,3

1 - Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry of Russian Academy of Sciences, Pushchino, Moscow region, Russia2 - Centre Bioengineering of Russian Academy of Sciences, Moscow, Russia3 - Nikitsky Botanical Gardens, Yalta, Russia

One of the efficient methods for control virus diseases is to create resistant cultivars. Plant genetic engineering provides new methods for developing virus resistant plants based on the integration of viral genes and sequences into plant genomes. We compared the resistance of transgenic chrysanthemum to Chrysanthemum Virus B (CVB). Transgenic chrysanthemum plants were generated via constitutive expression sequence of CVB coat protein gene in sense, double sense and antisense orientation. We also used another most promising strategy to create virus resistant plants

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- gene silencing technology RNA interference (RNAi). In this construction we inserted short (276 bp) fragment of CVB coat protein sequence. More than 10 transgenic lines for each of the four constructions were obtained. The resistances to CVB of transgenic lines have been tested in greenhouse. For this purpose plants were artificially inoculated by grafting of infected CVB cuttings. Virus levels in infected transgenic and non-transgenic plants were estimated by ELISA, Western and Northern blot analysis. We observed different level of resistance to CVB in transgenic chrysanthemum plants. Some of transgenic lines transformed with double sense construction were completely virus resistant. Three RNAi lines showed dramatic decrease in the levels of virus infection relative to the levels found in non-transgenic infected plants. Significant differences in level of infection we observed in few lines with expression of coat protein sequences in sense and antisense orientation.

16_SEQUENCE ANALYSIS OF SAGUARO CACTUS CARMOVIRUS ISOLATED FROM THE GRAFTED CACTUS IN KOREA

Mi-Sang Lim, Byoung-Eun Min, Ki-Hyun Ryu and Sun-Hee Choi

Plant Virus GenBank, Department of Horticulture Biotechnology and Landscape ArchitectureSeoul Women’s University, Seoul, Republic of Korea

Keywords: Saguaro cactus virus, Gymnocalycium mihanovichii, grafted cactus,

The partial sequences of the single-stranded, (+)-sense RNA genome of saguaro cactus virus (SgCV-kr) was determined. A Korean strain of SgCV-kr was isolated from the grafted cactus in Korea. SgCV-kr was detected by RT-PCR assay. SgCV-kr infection was asymptomatic in the cactus plant but induced local lesions when inoculated onto the leaves of Chenopodium amaranticolor. Total RNA was extracted from local lesions of virus-inoculated leaves and RT-PCR was performed using SgCV 750 and SgCV CP primers. The complete genome of SgCV-kr expected approximately 3.8 kb. And a 3,064-nucleotide sequences has been retained. The partial sequences of the virus contained four open reading frames. Phylogenetic analyses indicated that SgCV-kr is related to other Carmovirus species, and is also closely grouped with CarMV, HnRSV, PFBV, AFBV and SgCV-USA.

17_STUDIES OF FUNCTIONAL PROTEINS, HC-PRO AND CP FROM A NOVEL POTYVIRUS, CALLISTEPHUS MOTTLE VIRUS.

E.-Y. Seo1, S. Lim2,3, S. Han1, J. Hammond4, J. S. Moon2,3 and H.-S. Lim1

1 Department of Applied Biology, Chungnam National University, Daejeon 305-764, Republic of Korea2 Molecular Biofarming Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea3 Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350, Republic of Korea4 United States Department of Agriculture-Agricultural Research Service, Floral and Nursery Plants Research Unit, Beltsville, Maryland, 20705, USA

Callistephus mottle virus (CalMV) was detected from Callistephus chinensis (china aster) in South Korea and reported as a novel putative member of the genus Potyvirus (Seo et al. 2016). The CalMV complete genomic sequence shared 67% nucleotide sequence identity and 54% polyprotein amino acid sequence identity to the most closely related potyvirus, Plum pox virus. CalMV contains the

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typical open reading frame of potyviruses, encoding a putative large polyprotein of 3,154 amino acids with an estimated molecular weight of 357.45 kDa. This polyprotein is expected to be cleaved into the functional mature proteins, P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa, NIb and CP by the virus-encoded proteases. A small ORF, PIPO protein embedded within the P3 cistron of CalMV is predicted to be expressed by frameshift at the sequence GA6U (nt 3106-3113). The CalMV polyprotein has some conserved motifs showing typical potyviral properties. The HC-Pro has the motifs 428KINC431 (instead of the KITC motif which is commonly found in potyviruses) and 632PTK634, which take the role of binding of HC-Pro to virions or CP, and the motif 557FRNK560 involved in RNA silencing suppressor function. The CP motif 2880DAG2882 is associated with aphid transmission. To reveal the biological characterization of CalMV, the functional mature HC-Pro and CP proteins were cloned into a GFP-fusion expression vector and evaluated for properties such as subcellular localization and interactions, and silencing suppressor function of HC-Pro. In addition, susceptibility of expressed CP was shown to degradation by co-agroinfiltration of the 25kDa protease of Pseudomonas oleovorans. These studies will be useful to investigate further properties of CalMV including host range and vector transmission.

18_IDENTIFICATION OF A CUCUMBER MOSAIC VIRUS FROM MALVA CRISPA IN SICHUAN, CHINA

C.Y. Fei, Z.B. Xu, L. Zhu, D.H. Xi

Ministry of Education Key Laboratory for Bio-Resource and Eco-Environment,College of Life Science, Sichuan University, Chengdu 610064, China

Malva crispa is a temperate horticultural and vegetable plant of the family Malvaceae, mainly grown in Sichuan, Yunnan, Guizhou provinces, southwest of China. In May 2015, Malva crispa plants showing severe mosaic symptom followed by growth reduction were observed in some green space in Chengdu, Sichuan. Symptomatic plants were collected, serological detection by Western blotting confirmed they were infected by Cucumber mosaic virus (CMV) (Agdia, Elkhart, IN). To further confirm the CMV infection, total RNA were extracted from infected plants using Trizol reagent (Invitrogen). Reverse transcription-PCR assay was performed with a pair of CMV consensus primers CMV-F (5’-ATGGACAAATCTGRATCWMCC-3’) and CMV-R (5’-CTGGATGGACAACCCGTTC-3’), which corresponding to the coat protein (CP) and partial 3’-UTR sequences of RNA3 of CMV. The obtained fragments were purified and cloned into pMD19-T vector (TaKaRa) and sequenced. Nucleotide BLAST analysis revealed that the objective 780 bp fragment shared the highest nucleotide sequence identity (99%) with the CMV strain Hnt (GenBank Accession No. KC407999.1), which belongs to subgroup II, and shared 95.43%-98.78% sequence identities with other isolates of CMVs belong to subgroup II. Phylogenetic analysis with the Neighbor Joining method based on the obtained sequence and other CMV isolates from China available in databases was performed. The sequence of the isolate clustered into the subgroup II clade which departed from the cluster of subgroup IA and IB clearly indicated that it belongs to CMV subgroup II. This is the first report of CMV subgroup II infecting Malva crispa in China, and its high incidence reflects the new trend of the occurrence of virus disease in Malvaceae plants.

19_OCCURRENCE AND COMPLETE NUCLEOTIDE SEQUENCE OF PLANTAGO ASIATICA MOSAIC VIRUS FROM LILIUM SPP. IN KOREA

Eun Kyoung Kim and Jin Sung Hong*

Department of Applied Biology, Kangwon National University, Chunchon 24341, Republic of Korea.

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Systemic severe necrotic symptoms on leaves and stem of diseased Lilium spp. plants were collected from a natural field in Heongseong and Injea, Korea, and their causal agent was investigated. Plantago asiatica mosaic virus (PlAMV) was identified by RT-PCR from the tested Lilium spp. and sequence determination were carried out to confirm the virus. An isolate of Plantago asiatica mosaic virus (PlAMV) named as PlAMV-Kr, was isolated from Lilium spp. The complete nucleotide sequences of the genome of PlAMV-Kr was determined. The genomic RNA sequence was 6102 nucleotides (nt) in length, excluding the poly (A) tail, containing the five ORFs (1-4 and the CP) typical of members of the genus Potexvirus. The genomic sequences showed 98% identical to that of PlAMV-Netherland strain at nucleotide level. Multiple alignment and phylogenetic analysis results indicated that PlAMV is most closely related to the PlAMV-Netherland strain. To our knowledge, this is the first report of complete genome sequence information of PlAMV from Lilium spp. in Korea.

20_VIRUS DETECTION IN NATURALLY INFECTED PLANTS USING HIGH-THROUGHPUT SEQUENCING F. Gawehns, E.T.M. Meekes, I. Stulemeijer, M. de Kock, M.J.M. Ebskamp

Despite the wide acceptance and robustness of traditional screening methods like ELISA and PCR, viruses can be missed using targeted detection. The number of unidentified viruses in plants are estimated relatively high due to a fast mutation rate, host specificity or low abundance. They can cause novel disease symptoms, can be part of a virus complex or can be present without showing any symptoms. Using the non-targeted High-throughput sequencing of RNA from infected leaves, both known and unknown viruses can be detected. RNAs, including small RNAs, were isolated from different ornamental plants that were naturally infected and showed clear disease symptoms. The total RNA fraction (>200 nt) or the small RNA fraction (~21-24 nt) were sequenced and virus identification was performed using an in house pipeline and virus database. All known viruses (including a viroid) could be identified with both the methods. However, a by far higher mapping percentage and sensitivity were reached with small RNA sequencing. Furthermore, we also show that small RNA sequencing is suitable to detect mixed infections by a virus complex or different virus strains and to identify unknown viruses de novo. In conclusion, we prove that RNA sequencing is a valuable method for the Dutch Inspection services and can be used as a safety net or to discover unknown viruses. Here work on Freesia viruses will be presented.

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CONTACT US

Convenor: Sek-Man Wong - Mobile 98566431 or email: dbswsm@nus.edu.sgOther contact numbers for assistance: Prem Das – 86738447Song Guo – 84225184Jonathan Lim – 92206909For emergency: Police: 999Taxi booking - 65521111

TRANSPORT ARRANGEMENTS for Participants staying at Rochester Park Hotel

Date Departure time from Rochester Park Hotel LobbyMonday, 27 Jun 2016 8.00amTuesday, 28 Jun 2016 8.30amWednesday, 29 Jun 2016 8.30am

For participants who do not stay at Rochester Park Hotel or missed the group departure, please make your own way to the department by public bus (number 95) opposite the hotel or by taxi.

HOW TO GET TO NUS CAMPUS AND SYMPOSIUM VENUE (LT 32)

Public Transportation:

1. By Bus No. 95 (opposite Buona Vista MRT Station) and alight at the University Hall Bus Stop (at the sixth bus stop).

2. By MRT – From Buona Vista station to Kent Ridge Station (located next to NUS Hospital). Follow the crowd to the NUS internal bus stop. Take internal shuttle bus A1 or D1 and alight at University Hall Bus Stop (at the second bus stop).

3. By Taxi – Address: Department of Biological Sciences, Science Drive 4, Lower Kent Ridge Road, NUS, just before the University Hall.

4. By Rochester Park Hotel shuttle bus. Hotel provides free shuttle service to a NUS bus stop which is about 500 meters from our symposium venue. It takes ~10 minutes to walk. Please check with the hotel reception desk for the time schedule.

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