CRIS Project Titles:   
1.  Development of Detection and Protection Technologies for Viruses and Bacteria of Major Significance to Ornamental and Nursery Crops.
2.  Genetic Improvement of Floral Crops for Pest, Disease, and Stress Tolerance and Ornamental Qualities.
3.  Transformation for disease resistance in floral monocots.

INTRODUCTION:   
Conducts research on virus diseases of ornamental crops, including identification and characterization of newly occurring viruses, resistance to virus infection and replication in transgenic plants, and virus detection and strain differentiation by serology and nucleic acid hybridization.

PROGRESS:   
1.  Several newly reported viruses affecting ornamental crops have been identified and characterized. Phlox viruses. A potexvirus initially detected in creeping phlox (Phlox stolonifera) was shown to be an isolate of Alternanthera mosaic virus (AltMV), previously reported only from a weed species in Australia. The complete genome of one AltMV isolate from creeping phlox was cloned and sequenced, and AltMV shown to be most closely related to Papaya mosaic virus (PaMV). The coat protein gene and 3’ non-coding region of two additional isolates of AltMV from phlox, and one isolate from trailing portulaca (Portulacca grandiflora) were also determined; AltMV has also been detected in Phlox divaricata and P. pilosa by PCR using virus-specific primers. A novel carlavirus from Phlox stolonifera, a distinct carlavirus from Phlox divaricata, and other viruses infecting phlox have also been examined. The carlavirus from P. stolonifera was found to be serologically related to Potato virus S and Kalanchoe latent virus, but the nucleotide sequence was distinct; thecomplete genome has been cloned and sequenced, and shown to be distinct from previously characterized carlaviruses. The name Phlox virus S is proposed.  The carlavirus from P. divaricata was not found to be serologically related to any other carlaviruses tested so far, and to date about 5kb of the 3’ region of the genome has been cloned and sequenced. The sequence obtained is also distinct from that of other characterized carlaviruses. At least two distinct potyviruses have been detected in various cultivars of P. stolonifera, P. divaricata and P. pilosa. Tobacco ringspot virus was also detected in P. stolonifera, and a portion of the genome cloned and sequenced.Angelonia. A novel carmovirus causing flower break in angelonia was purified and partially characterized, in collaboration with Scott Adkins (USDA-ARS, Florida) and Abed Gera (ARO, The Volcani Center, Israel). The coat protein gene of the Beltsville isolate was cloned and sequenced, and compared those of the Florida isolate (fully sequenced) and the Israeli isolate (coat protein gene sequenced). Antisera produced to purified virus from Israel and Florida were used to verify the experimental host range of Angelonia flower break virus, as the virus has been named. Ornithogalum and Lachenalia viruses. The 3’-terminal region of several potyvirus isolates from the bulbous crops Ornithogalum and Lachenalia have been cloned and sequenced. Isolates similar to Ornithogalum mosaic virus and at least two other sequence types have been detected. An ophiovirus has been detected in Lachenalia, and portions of the genome cloned and sequenced, in collaboration with Anna Maria Vaira (CNR, Istituto Virologia Vegetale, Italy). Lolium latent virus. A flexuous virus was detected in a ryegrass germplasm accession in quarantine, and determined to be Lolium latent virus (LLV), in collaboration with Ruhui Li (USDA-ARS) and Clarissa Maroon-Lango (formerly FNPRU, now USDA-APHIS, PPQ). Most of the genome of LLV has been cloned and sequenced. LLV was also detected for the first time in ryegrass germplasm within the U.S., and a significant portion of the genome of a U.S. isolate also cloned and sequenced. Bacopa chlorosis virus. A novel ilarvirus was detected in bacopa (Sutera cordata) plants showing chlorosis and leaf dimpling. In collaboration with Hei-ti Hsu (FNPRU) and Clarissa Maroon-Lango (formerly FNPRU, now USDA-APHIS, PPQ), a significant portion of the genome has been cloned and sequenced, and shown to be most closely related to Parietaria mottle virus. However, the sequence is distinct, and the name Bacopa chlorosis virus proposed. Bean yellow mosaic virus. The complete genomic sequence of an isolate of Bean yellow mosaic virus (BYMV) from gladiolus was determined (with Rosemarie Hammond, USDA-ARS, MPPL). The coat protein gene and antisense RNA constructs have been used to create transgenic gladiolus with partial resistance to BYMV infection (with Kathryn Kamo, FNPRU). Beet mosaic virus. The complete sequence of an isolate of Beet mosaic virus was determined (with Rosemarie Hammond and Lev Nemchinov, USDA-ARS, MPPL; and Ramon Jordan, FNPRU).

2.  Effects of virus infection on floral pigment expression. The effects of infection with various viruses on gene silencing and differential expression of genes in the anthocyanin biosynthetic pathway is being examined in collaboration with Rob Griesbach. The Petunia ‘Star’ mutant is being used as a model system; the ‘Star’ pattern results from differential gene silencing that can be reversed by infection with any of several viruses. This is a very useful model for examining the interactions between virus infection and normal patterns of gene silencing, and for understanding floral pigmentation patterns. Virus resistance in gladiolus. Bean yellow mosaic virus (BYMV) coat protein and antisense RNA constructs that conferred effective resistance to BYMV infection in Nicotiana benthamiana were used to transform gladiolus in collaboration with Kathryn Kamo. Transformed gladiolus lines showed short-term, but not long-term resistance to BYMV.

PLANS:   1.  To evaluate the level and specificity of virus resistance in the transgenic plants produced, and to produce further plants expressing untranslatable RNAs or modified CPs designed to destabilize subunit:subunit or subunit:RNA interactions; obtain further insight into the mechanisms of virus resistance in transgenic plants to enable design of constructs conferring broader specificity of effective resistance to potyviruses. To examine the use of potyvirus coat protein as a carrier for vaccine-significant epitopes.

2.  To create an infectious clone of bean yellow mosaic virus to allow examination of the basic biology of the virus, including sequences necessary for replication and encapsidation. To determine the influence of different hosts and partial resistance in transgenic plants on potyvirus sequence stability after multiple passages. Also to examine the determinants of host range and symptom expression, by exchange of sequences from isolates and other potyviruses with different host ranges. To obtain cDNA clones of potyvirus isolates from Ornithogalum and Lachenalia, for sequence comparison and for future transformation; to obtain cDNA clones from turnip mosaic isolates from tulip, Tigridia, orchids, and other sources for sequence and phylogenic comparison. Continue strain differentiation of Botryosphaeria isolates and species, and develop methods for application to field samples.

RECENT PUBLICATIONS:

1994. Stein, A., Rosner, A. and Hammond, J. Detection of bean yellow mosaic virus in gladioli corms. Acta Hort. 377:209-220.

1994. Hammond, J., and Rosner, A. The application of molecular techniques for the detection and control of viruses of ornamental plants. Acta Hort. 377:223-239.

1994. Hammond, J., and Kamo, K. K. Potyvirus resistance derived from antisense RNA and native or chimeric coat protein gene. Acta Hort. 377:259-266.

1995. Hammond, J., and Kamo, K. K. Effective resistance to potyvirus infection in transgenic plants expressing antisense RNA. Mol. Plant-Microbe Interact.8: 674-682.

1995. Hammond, J. Replication of viruses and movement in the plant. Chapter 12 in:G. Loebenstein, R.H. Lawson and A.A. Brunt (eds.) Virus and Virus-like Diseases of Bulb and Flower Crops, John Wiley & Sons/Balaban, Chichester, United Kingdom.

1995. Hammond, R. W., and Hammond, J. Plant Viruses and Viroids. Chapter 11, pp.229-249, in: J. Hunter and A. Bolt (eds.) Maintaining Cultures For Biotechnology and Industry, Academic Press, New York.

1995. Hammond, J., and Kamo, K.K. Resistance to bean yellow mosaic virus (BYMV) and other potyviruses in transgenic plants expressing BYMV antisense RNA, coat protein, or chimeric coat proteins. pp. 369-389 in: D.D. Bills and S.-D. Kung (eds.) Biotechnology and Plant Protection: Viral Pathogenesis and Disease Resistance, World Scientific, Singapore.

1995. McGarvey, P.B., Hammond, J., Dienelt, M. M., Hooper, D. C., Fu, Z. F., Dietzschold, B., Koprowski, H. and Michaels. F. H. Expression of the rabies virus glycoprotein in transgenic tomatoes. Bio/Technology 13:1484-1487.

1996. Hammond, J.  Resistance to various potyviruses in transgenic plants expressing different forms of potyvirus coat protein gene. Acta Horticulturae 432:278-284.

1996. Hammond, J.  Biotechnology and resistance.  Acta Horticulturae 432:246-256.

1997. Hammond, J., and Dienelt, M.M.  Encapsidation of potyviral RNA in various forms of transgene coat protein is not correlated with resistance in transgenic plants. Mol. Plant-Microbe Interact. 10:1023-1027.

1997. Hammond, J.  Repelling plant pathogens with ribonuclease. Nature Biotechnology 15:1247.

1997. Hu, Y., Feldstein, P.A., Hammond, J., Hammond, R.W., Bottino, P.W., and Owens, R.A. Destabilization of potato spindle tuber viroid by mutations in the left terminal loop. J. Gen. Virol. 78:1199-1206.

1997. Kamo, K.K., Hammond, J., and Roh, M.  Transformation of Gladiolus for disease resistance. J. Korean Soc. Hort. Sci. 38:188-193.

1998. Tabler, M., Tsagris, M., and Hammond, J.   Antisense RNA- and ribozyme-mediated resistance to plant viruses. Chapter 7, pp.79-93 in: Plant Viral Disease Control; A. Hadidi, R.K. Khetarpal, and E. Koganezawa (eds.), APS Press, St. Paul. 684pp.

1998. Hammond, J., Pühringer, H., da Câmara Machado, A., and Laimer da Câmara Machado, M.  A broad-spectrum PCR assay combined with RFLP analysis for detection and differentiation of plum pox virus isolates. Acta Horticulturae 472:483-490.

1998. Hammond, J.  Serological relationships between the cytoplasmic inclusion proteins of potyviruses. Phytopathology 88:965-971.

1998. Nemchinov, L., Hammond, J., and Hadidi, A.F.  Nucleotide sequence of the putative replicase gene of the sour cherry strain of plum pox virus. Arch. Virol. 143:2247-2252.

1998. Hammond, J.  Resistance to plant viruses - an overview. pp.163-171 in: Plant Viral Disease Control; A. Hadidi, R.K. Khetarpal, and E. Koganezawa (eds.), APS Press, St. Paul. 684pp.

1999. Hammond, J.  Overview: The Many Uses and Applications of Transgenic Plants. Chapter 1, pp.1-19 in: Plant Biotechnology: New products and Applications; J. Hammond, P.M. McGarvey and V. Yusibov, (eds.), Current Topics in Microbiology and Immunology v. 240, 1999.

1999. Hammond, J., Lecoq, H., and Raccah, B.  Epidemiological Risks from Mixed Infections and Transgenic Plants Expressing Viral Genes. Advances in Virus Research 54:189-314.

2000. Berger, P.H., Barnett, O.W., Brunt, A.A., Colinet, D., Edwardson, J.R., Hammond, J., Hill, J.H., Jordan, R.L., Kashiwazaki, S., Mallouk, K., Morales, F.J., Rybicki, E., Spence, N., Ohki, S.T., Uyeda, I., van Zaayen, A., and Vetten, H.J. 2000. Family Potyviridae. Pp. 703-724 in: M.H.V. van Regenmortel, C.M. Fauquet, D.H.L. Bishop, E.B. Carsten, M.K. Estes, S.M. Lemon, J. Maniloff, M.A. Mayo, D.J. McGeoch, C.R. Pringle, and R.B. Wickner (eds.), Virus Taxonomy: Seventh Report of the International Committee on Taxonomy of Viruses. Academic Press, New York.

2000. Hammond, J. Potyviruses.  Pp. 810-817 in: Encyclopedia of Microbiology, Vol. 3. Academic Press, New York..

2000.  Zhao, Y., Hammond, J., Tousignant, M.E., and Hammond, R.W. Development and evaluation of a complementation-dependent gene delivery system based on cucumber mosaic virus. Archives of Virology 145:1-11.

2001. Hammond,J., and Jordan, R.L. Potyviruses. Pp. 792-800 in: Encyclopedia of Plant Pathology. John Wiley & Sons, New York.

2003. Farzad, M., Griesbach, R., Hammond, J., Weiss, M., and Elmendorff, H.G.  Differential expression of three key anthocyanin biosynthetic genes in a color-changing flower, Viola cornuta cv. Yesterday, Today and Tomorrow. Plant Science 165:1333-1342.

2003. Hammond, J., and Hammond, R.W.  The complete nucleotide sequence of isolate BYMV-GDD of Bean yellow mosaic virus, and comparison to other potyviruses. Arch. Virol. 148: 2461-2470.

2004. Nemchinov, L.G., Hammond, J., Jordan, R.L., and Hammond, R.W.  The complete nucleotide sequence, genome organization, and specific detection of Beet mosaic virus. Arch. Virol. 149:1201-1214.

2004. Chen, Y.P., Zhao, Y., Hammond, J., Hsu, H.T., Evans, J., and Feldlaufer, M.  Multiple virus infections in the honey bee and genome divergence of honey bee viruses. J. Invertebrate Pathol. 87:84-93.

2005. Kamo, K.K., Gera, A., Cohen, J., Hammond, J., Blowers, A., Smith, F., and van Eck, J. Transgenic Gladiolus plants transformed with either the bean yellow mosaic virus coat protein or antisense RNA. Plant Cell Reports 23:654-663.

2005. Maroon-Lango, C., Guaragna, M.A., Jordan, R.L, Hammond, J., Bandla, M., and Marquardt, S.  Two unique US isolates of Pepino mosaic virus from a limited source of pooled tomato tissue are distinct from a third (European-like) US isolate. Arch. Virol 150:1187-1201.

2006. Yusibov, V., Hammond, J., and Hammond, R.  Plant Viral Vectors for Vaccine Production: Proven Value and Potential. (Book Chapter; in review).

2006. Hammond, J., Hsu, H.T., Huang, Q., Jordan, R., Kamo, K., and Pooler, M. Transgenic approaches to disease resistance in ornamental crops. (invited review; J. Crop Improvement; in press).

2006. Hammond, J., Reinsel, M.D., and Maroon-Lango, C.J. Identification of Potexvirus Isolates from Creeping Phlox and Trailing Portulaca as Strains of Alternanthera Mosaic Virus, and Comparison of the 3’-Terminal Portion of the Viral Genomes. Acta Horticulturae (in press).

2006. Hammond, J. Current status of genetically modified ornamentals. Acta Horticulturae (in press).

2006. Hammond, J., Reinsel, M.D., and Maroon-Lango, C.J. Identification and full sequence of an isolate of Alternanthera mosaic potexvirus infecting Phlox stolonifera. Arch. Virol. 151:477-493.

2006. Maroon-Lango, C.J., Hammond, J., Warnke, S., Li, R., and Mock,R. First report of Lolium latent virus in ryegrass in the United States. Plant Disease 90:528.

2006. Li, R., Maroon-Lango, C.J., Hammond, J., and Mock, R. Lolium latent virus. Molecular Diagnosis of Plant Viruses. (Book chapter, in review).

2006. Adkins, S., Hammond, J., Gera, A., Maroon-Lango, C.J., Sobolev, I., Harness, A., Zeidan, M., and Spiegel, S.  Biological and molecular characterization of a novel carmovirus isolated from Angelonia. Phytopathology 96:460-467.

Patent applied for:
Hammond, R.W., Zhao, Y., and Hammond, J. (2000). Production of vaccines using transgenic plants or modified plant viruses as expression vectors and transcapsidated viral coat proteins as epitope presentation systems. U.S. Patent Application No. 09/603,997, June 6, 2000.

Non-peer-reviewed publications:

1995. Hammond, J., Pühringer, H., Steinkellner, H., da Câmara Machado, A., and Laimer da Câmara Machado, M. 1995. Transcapsidation in transgenic plants expressing potyvirus coat proteins. Phytopathology 85: 1151 (Abstr.).

1996. Hammond, J., Pühringer, H., da Câmara Machado, A., and Laimer da Câmara Machado, M. Combined RT-PCR and RFLP analysis to detect and differentiate field isolates of plum pox potyvirus. Phytopathology 86: (11) S13 (Abstr.).

1997. Hammond, J., Pühringer, H., da Câmara Machado, A., and Laimer da Câmara Machado, M.  Cloning of RT-PCR products from the NIb gene of plum pox potyvirus to further examine isolate differentiation and diversity. Phytopathology 87: S38 (Abstr.).
 
2001. Zhao, Y., Hammond, J., and Hammond, R.W. Development of a subgenomic promoter-driven Cucumber mosaic virus-based vector. Phytopathology 91:S99 (Abstr.).

2001. Hammond, J., Pühringer, H., da Câmara Machado, A., and Laimer da Câmara Machado, M.  Sequence analysis of PCR products from plum pox virus (PPV)-infected trees: presence of more than one PPV isolate in an individual tree. Phytopathology 91:S36 (Abstr.).

2003. Hammond, J., Reinsel, M.D., and Maroon-Lango, C.J.M.  Partial characterization and full sequence of a potexvirus from creeping phlox (Phlox stolonifera) Phytopathology 93: (6)S32 (Abstr.).

2004. Hammond, J., Reinsel, M.D., and Maroon-Lango, C.J.M.  Identification of potexvirus isolates from phlox and portulaca as strains of Alternanthera mosaic virus, and comparison of the genomic 3’ region. Phytopathology 94: (6) S38 (Abstr.).

2005. Adkins, S., Hammond, J., Maroon-Lango, C.J., Harness, A.. Kulemeka, B., Geister, R., Bandla, M., Spiegel, S., and Gera, A   Characterization of a carmovirus from angelonia. Phytopathology 95(6): S2 (Abstr.).

2005. Maroon-Lango, C., Guaragna, M.A., Jordan, R.L., Hammond, J., Bandla, M., Marquardt, S., and Stommel, J.  Pepino mosaic virus: variability in U.S. isolates. HortScience 40:1089 (Abstr.).

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