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 committee
Sek-Man Wong (convenor)
Yok King Fong
Zhong Chao Yin
Li-Huan Koh
Ann Nee Yong
Ping Lee Chong
Say Tin Kho
Sponsors and Partners
TENTATIVE PROGRAM
14th International Symposium on Virus Diseases of Ornamental Plants
26-29 June2016 | 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 / Activity8:00 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 Yeh
01_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 Techniques
Chair: 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 ISOLATES
J. 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
Day 3 (28 June 2016)
Session 2:Epidemiology And Disease ControlChair: Scott Adkins
9:00am / 04_VIRAL SPREAD AND DIVERSITY IN ANTHROPOCENE
K. R. Richert-Poeggeler and B. Lockhart
9:15am / 05_OCCURRENCE AND COMPLETE NUCLEOTIDE SEQUENCE OF PLANTAGO ASIATICA MOSAIC VIRUS FROM LILIUM SPP. IN KOREA
Eun Kyoung Kim and Jin Sung Hong*
9:30 am / 06_EPIDEMIOLOGY OF AND RESISTANCE TO ROSE ROSETTE VIRUS
Patrick L. Di Bello, Thanuja Thekke-Veetil, Tobiasz Druciarek and Ioannis E. Tzanetakis*
9:45 am / 07_REDBUD YELLOW RINGSPOT DISEASE: ANOTHER EMARAVIRUS ASSOCIATED WITH A DISEASE OF ORNAMENTAL PLANTS
Patrick 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 Diseases
Chair: C.A. Chang
10:00 am / 08_VIRUSES OF ORNAMENTALS EMERGING IN FLORIDA AND THE CARIBBEAN REGION
Scott 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 / 09_EVIDENCE OF NEW VIRUSES INFECTING FREESIA HYBRIDS SHOWING NECROTIC DISEASE
A.M. Vaira, L. Miozzi, M. Vallino, A. Carra, R. Lenzi, D. Salvi, J. Hammond and H.R. Pappu
11.00 am / 10_CHARACTERIZATION OF A NUCLEORHABDOVIRUS FROM PHYSOSTEGIA
Wulf Menzel, Dennis Knierim, Katja Richert-Pöggeler, Stephan Winter
11.15am / 11_IDENTIFICATION OF NARCISSUS YELLOW STRIPE VIRUS AND A CLOSELY-RELATED POTYVIRUS ISOLATE IN PLANTS OF ALLIUM CARINATUM
D. Bampi, M.D. Reinsel, and J. Hammond
11:30 am / 12_NEW LISIANTHUS DISEASE CAUSED BY PAPAYA LEAF CURL GUANGDONG VIRUS
Y.K. Chen, H.Y. Chao, and P.J. Shih
11:45 am / 13_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 / 14_CHARACTERIZATION AND GENOME SEQUENCE ANALYSIS OF LYCHNIS RINSSPOT VIRUS
Yoon-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)
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 / 15_CHRYSANTHEMUM STUNT VIROID IN ARGYRANTHEMUM---DISTRIBUTION AND ELIMINATION
Zhibo Zhang, YeonKyeong Lee, Carl Spetz, Jihong Liu Clarke, Astrid Sivertsen, Gry Skjeseth, Sissel Haugslien, Qiaochun Wang and Dag-Ragnar Blystad*
9:30 am / 16_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 / 17_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. Bertaccini
Session 5: Virus Resistance Through Breeding Or Transgenic Methods
Chair:Pattana Srifah Huehne
10:00 am / 18_SILENCING THE CymMV COAT PROTEIN GENE BY RNAi AND RECOMBINANT miRNA TECHNOLOGY IN TRANSGENIC DENDROBIUM ORCHID
Pattana Srifah Huehne, Udomporn Petchthai, Anchalee Chuphromp, Kisana Bhinija
10:15 am / 19_TRANSGENIC NICOTIANA BENTHAMIANA RESISTANCE TO SYNERGISTIC INFECTION OF TWO ORCHID VIRUSES CyMMV AND ORSV
U. Petchthai, Z. Xie, and S.M. Wong
10.30 am / Coffee/Tea Break and Viewing of Posters
Section 6: Virus Detection And Diagnostic Techniques
Chair: R.J. McGovern
11:00 am / 20_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 / 21_EVALUATION OF A SIMPLIFIED METHOD OF PLANT VIRUS INCLUSION VISUALIZATION BY LIGHT MICROSCOPY
R.J. McGovern, P. Khampirapang, R. Cheewangkoon, L.H. Koh, C. To-anun, and S.M. Wong
11:30 am / Closing Remarks
12:00noon / Lunch
13:00 pm / Bus return to Rochester Park Hotel/Bouna Vista MRT Station
End of Day 4: Bon Voyage
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 Yeh1,,3, 4
1Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan
2Devision of Plant Pathology, Taiwan Agriculture Research Institute, Wufeng, Taichung, Taiwan
3 Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan
4NCHU-UCD Plant and Food Biotechnology Center, National Chung Hsing
Ｗhitefly-borne ssDNA geminiviruses and thrips-borne ss(-)RNAtospoviruses 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 Netherlands
2 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 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,
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.
04_VIRAL SPREAD AND DIVERSITY IN ANTHROPOCENE
K. R. Richert-Poeggeler1 and B. Lockhart2
1 Institute for Epidemiology and Pathogen Diagnostics, Julius Kühn-Institut, Braunschweig, Germany
2 Department of Plant Pathology, UniversityofMinnesota, St. Paul, Minnesota, USA
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.
05_OCCURRENCE AND COMPLETE NUCLEOTIDE SEQUENCE OF PLANTAGO ASIATICA MOSAIC VIRUS FROM LILIUM SPP. IN KOREA
Eun Kyoung Kimand Jin Sung Hong*
Department of Applied Biology, Kangwon National University, Chunchon 24341, Republic of Korea.
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-Krwas 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 showed98% identical tothat 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.
06_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 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.
07_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.