US National Arboretum

 

Floral and Nursery Plants Research Unit

Dr. John Hammond

Research Plant Pathologist
{Beltsville, MD}

e-mail: John.Hammond@ars.usda.gov

CRIS Project Titles:   
1.  New and Emerging Viral and Bacterial Diseases of Ornamental Plants: Detection, Identification and Characterization.

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. Development of a Universal Plant Virus Microarray for detection and identification of plant viruses.

PROGRESS:   
1.  Several newly reported viruses affecting ornamental crops have been identified and characterized.

Phlox viruses. A) 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 a Pennsylvania AltMV isolate from creeping phlox was cloned and sequenced, and AltMV shown to be most closely related to Papaya mosaic virus (PaMV). AltMV has also been detected in various types of phloxand other ornamentalspecies by PCR using virus-specific primers, and by Enzyme-Linked Immunosorbent Assay (ELISA). The Triple Gene Block 3 (TGB3) gene, coat protein gene and 3’ non-coding region of several additional isolates of AltMV from various types of phlox, trailing portulaca (Portulacca grandiflora), nandina, cineraria, and crossandra have also been determined. B) A novel carlavirus from Phlox stolonifera was found to be serologically related to Potato virus S and Kalanchoe latent virus, but the nucleotide sequence was distinct; the complete genome has been cloned and sequenced, and shown to be distinct from previously characterized carlaviruses; this virus has been named Phlox virus S. C) a distinct carlavirus from Phlox divaricata was found not to be serologically related to any other carlaviruses tested. The complete genome of this virus has been determined, is distinct from any previously characterized carlaviruses, and the virus named Phlox virus B. D) a third novel carlavirus from hybrid annual phlox have been identified and partially sequenced; this virus has been named Phlox virus M. E) other viruses infecting phlox have also been examined; two potyvirus isolates with distinct sequences, but both closely related to Spiranthes mosaic virus 3 have been detected in various cultivars of P. stolonifera, P. divaricata, P. subulata, P.paniculata, 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). The Lachenalia ohiovirus was identified as Freesia sneak virus (FreSV). FreSV was also identified in the United States for the first time in freesia from a nursery in Virginia.

Lolium latent virus. A flexuous virus detected in a ryegrass germplasm accession in quarantine, was 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 was cloned and sequenced. LLV was also detected for the first time in ryegrass germplasm within the U.S., and the full 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, now retired) 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.  Creation and use of infectious clones of Alternanthera mosaic virus and Lolium latent virus to examine host:pathogen interactions, and as vectors for protein expression and Virus-Induced Gene Silencing (VIGS).

Alternanthera mosaic virus. We have created four distinct infectious clones from a Maryland isolate of Alternanthera mosaic virus; these infectious clones differ in their level of replication and symptom severity. Differences in both the RNA-dependent RNA Polymerase (RdDp) and Triple Gene Block 1 (TGB1) proteins were found to affect the replication rate. The original phlox isolate was shown to have been composed of a mix of sequence variants that was maintained through multiple passages, with symptoms intermediate between the more severe and milder infectious clones. TGB1 was shown to localize to both the nucleolus and the cytoplasm, and a single amino acid variation in TGB1 demonstrated to have a significant effect on suppression of RNA silencing. This was exploited to create infectious clones suitable for high-level protein expression (having TGB1 effective for suppression of RNA silencing) or for VIGS (having the TGB1 variant with weak RNA silencing suppression activity). A novel bipartite delivery system for these plant viral vectors was also developed, increasing their potential as tools for high-throughput use in biotechnology applications. The TGB3 protein of AltMV was shown to target the chloroplast, and to be critical for the movement of AltMV from the epidermal layer into the mesophyll and beyond to establish systemic infection.

Lolium latent virus. Infectious clones of Lolium latent virus have also been developed, and used to demonstrate that two versions of the LoLV coat protein are both required for efficient systemic movement. LoLV is unique among characterized filamentous plant viruses in having two carboxy-coterminal variants of the coat protein that are incorporated into virus particles in essentially equimolar proportions. We have shown that the larger form of LoLV coat protein contains a chloroplast transit peptide, and that this form is absolutely required for virus particle formation. The smaller form of coat protein appears to be produced by more than mechanism, but preventing production of the smaller form of coat protein has a dramatic effect on systemic virus movement.

3.  Development of a Universal Plant Virus Microarray (UPVM), in collaboration with: David Henderson and Ramon Jordan (FNPRU); Claude Fauquet and Basavaraj Bagewadi (Danforth Plant Science Center, St. Louis, MO); Kael Fischer (University of Utah, Salt Lake City, UT); David Wang (Washington University, St. Louis, MO); Ulrich Melcher (Oklahoma State University, Stillwater, OK); and Keith Perry (Cornell University, Ithaca, NY). The UPVM utilizes 9556 oligonucleotide probes of 60 nt, designed from sequences conserved at three different taxonomic levels (within plant virus families, genera, and individual virus species) to detect, identify, and differentiate viruses infecting plants; an additional 44 control probes target sequences conserved in the host plant genomes.

To aid application of the UPVM, we have developed a protocol to allow purification of high quality nucleic acids from a very wide range of taxonomically diverse host plant materials, which contain various combinations of secondary metabolites such as oligosaccharides, phenolic compounds, latex, etc., which can interfere with extraction and enzymatic processing of total nucleic acids or RNA.

Validation of the UPVM with a range of plant viruses is in progress. At present, viruses that reach high titer in infected tissues can typically be detected and correctly identified with difficulty via direct cDNA labeling. Viruses occurring at low titer are more problematic, and are not always detected even following unbiased amplification of the samples prior to labeling. Methods for subtractive hybridization or other means of template normalization are currently under evaluation to increase sensitivity of detection of low-titered infections.

PLANS:   

To detect, identify, and characterize novel and emerging viruses affecting ornamental plants.  2. To develop broad-spectrum and virus-specific reagents for the detection and identification of viruses, including PCR, serological reagents, and the UPVM.  3. To apply Next-generation sequencing methods for characterization of novel viruses. 4. To further utilize infectious clones of Alternanthera mosaic virus to allow examination of the basic biology of the virus, including sequences necessary for host range, replication, systemic movement, and encapsidation. To examine the determinants of host range and symptom expression, by exchange of sequences from isolates or complementation of defective viruses with functional gene products from diverse isolates or other potexviruses with different host ranges. 5. To further utilize infectious clones of Lolium latent virus to examine the basic biology of the virus, including sequences necessary for, replication, systemic movement, and encapsidation. 6. To further utilize infectious clones of Alternanthera mosaic virus and Lolium latent virus to demonstrate their utility as viral vectors for protein expression and Virus-Induced Gene Silencing (VIGS).

RECENT PUBLICATIONS:

2012.  Henderson, D.C., and Hammond, J.  CKC: isolation of nucleic acids from a diversity of plants using CTAB and silica columns. Mol. Biotechnol. DOI 10.1007/s12033-012-9494-y.

2012. Vaira, A.M., Lim, H-S., Bauchan, G.R., Owens, R.A., Natilla, A., Dienelt, M.M., Reinsel, M.D., and Hammond, J. Lolium latent virus (Alphaflexiviridae) coat proteins: expression and functions in infected plant tissue.  J. Gen. Virol. 93:1814-1824.

2012. Valverde, R.A., Sabanadzovic, S., and Hammond, J. Viruses that Enhance the Aesthetics of Some Ornamental Plants: Beauty or Beast?  Plant Disease 96:600-611.

2011. Hammond, J., Jordan, R.L., and Vaira, A.M.  Virus Diseases of Ornamentals. Encyclopedia of Life Support Services, Section 6.24.30 In: Food and Agricultural Sciences, Engineering and Technology Resources, Encyclopedia of Life Support Systems (EOLSS), Developed under the auspices of the UNESCO, Eolss Publishers, Oxford, UK [http://www.eolss.net].

2011. Hammond, J.  Universal plant virus microarrays, broad spectrum PCR assays, and other tools for virus detection and identification. Acta Hort. 901:49-60.

2011.Hammond, J., and Reinsel, M.  Mixed infections and novel viruses in various species of Phlox. Acta Hort. 901:119-126.

2010. Lim, H-S., Vaira, A. M., Bae, H., Bragg, J.N., Ruzin, S. E., Bauchan, G. R., Dienelt, M. M., Owens, R.A., and Hammond, J.   Mutation of a chloroplast targeting signal in Alternanthera mosaic virus TGB3 impairs cell-to-cell movement and eliminates long distance virus movement. J. Gen. Virol. 91:2102-2115.

2010. Lim, H-S., Vaira, A. M., Domier, L. L.,  Lee, S.C., Kim, H.G., and Hammond, J.   Efficiency of VIGS and gene expression in a novel bipartite potexvirus vector delivery system as a function of strength of TGB1 silencing suppression. Virology 402: 149-163.

2010. Lim, H.-S., Vaira, A. M., Reinsel, M. D., Bae, H., Bailey, B. A., Domier, L. L., and Hammond, J. Localization of Alternanthera mosaic virus pathogenicity determinants to RdRp and TGB1, and separation of TGB1 silencing suppression from movement functions. J. Gen. Virol. 91:277 - 287.

2010. Hammond, R.W., Hammond, J.  Maize rayado fino virus capsid proteins assemble into virus-like particles in Escherichia coli. Virus Res. 147:208-215.

2009. Vaira, A.M., Hansen, M.A., Murphy, C., Reinsel, M.D., and Hammond, J.  First report of Freesia sneak virus in Freesia sp. in Virginia. Plant Disease 93:965.

2009.  Lim, H. S., Bragg, J. N., Ganesan, U., Ruzin, S., Schichnes, D.,  Lee, M. Y., Vaira, A. M., Ryu, K. H., Hammond, J., and Jackson, A. O.   Subcellular localization of the barley stripe mosaic virus triple gene block proteins. J. Virol. 83:9432-9448.

2008. Vaira, A.M., Maroon-Lango, C.J., and Hammond, J.  Molecular characterization of Lolium latent virus, proposed type member of a new genus in the family Flexiviridae. Arch. Virol. 153:1263-1270.

2008. Lim, H-S., Bragg, J.N., Ganesan, U., Lawrence, D.M., Yu, J., Isogai, M., Hammond, J., and Jackson, A.O.  Triple gene block protein interactions involved in movement of Barley stripe mosaic virus. J. Virol. 82:4991-5006.

2008. Jordan, R., and Hammond, J.  Bean common mosaic virus and Bean common mosaic necrosis virus. Encyclopedia of Virology, 5 vols. (B.W.J. Mahy and M.H.V. Van Regenmortel, Editors), pp. 288-295 Oxford: Elsevier.

2008. Li, R., Maroon-Lango, C.J., Hammond, J., and Mock, R.  Lolium latent virus. Molecular Diagnosis of Plant Viruses. Chapter 15, pp. 215-220 in: Characterization, Diagnosis & Management of Plant Viruses. Edited by G.P. Rao, C. Bragard, and B.S.M. Lebas. Studium Press, Houston, TX.
2007. Griesbach, R.J., Beck, R.M., Hammond, J., and Stommel, J.R.  Gene expression in the Star mutation of Petunia ×hybrida Vilm.  J. Amer. Soc. Hort. Sci. 132:680-690.

2007. Vaira, A.M., Kleynhans, R., and Hammond, J.  First report of Freesia sneak virus infecting Lachenalia cultivars in South Africa. Plant Disease 91:770.

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.

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. 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. Hammond, J. Current status of genetically modified ornamentals. Acta Hort. 722:117-128.

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 Hort. 722:71-77.

2006. Griesbach, R., Beck, R. and Hammond, J. Regulatory genes in creating flower color patterns. Acta Hort. 714:171-176.

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 17:155-210.

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.

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.

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.

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.

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.

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.

2001. Hammond,J., and Jordan, R.L. Potyviruses. Pp. 792-800 in: Encyclopedia of Plant Pathology. John Wiley & Sons, 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. Arch. Virol. 145:1-11.

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

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.

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.

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.

PATENTS APPLIED FOR:

Hammond, J., Lim, H.S., and Domier, L.L. (2009). An infectious plant viral vector and an artificial bipartite plant viral vector for gene expression or virus-induced gene silencing. U.S. Patent Application No. 12/645,027, December 22, 2009; International PCT/US/56953, November 17, 2010.

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.

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