CRIS Project Title:  
Development of Detection and Protection Technologies for Viruses and Bacteria of Major Significance to Ornamental and Nursery Crops

INTRODUCTION:  
Develops methods of eliciting specific immune response for production of polyclonal antisera and monoclonal antibodies and develops sensitive techniques for rapid and reliable identification of plant pathogens.  Conducts research on virus-vector-host plant interaction and explores vector cell culture system for studies of plant viruses.  Investigates induced-resistance to virus infection in transgenic plants containing single-chain variable fragment (scFv) antibody genes. 

PROGRESS:  
Developed a tissue blot immunoassay method for detection and identification of pathogens in infected plants.  Established an in vitro feeding method for the acquisition of tospoviruses by thrips vectors.  Described and formulated growth media for thrips monolayer cell cultures.  Investigated and characterized a serologically distinct high temperature gloxinia tospovirus (HT-1), now a strain or isolate of Capsicum chlorosis virus (CaCV), recovered from gloxinia infected with an Impatiens necrotic spot virus (INSV).  Also biologically, serologically and molecularly characterized a previously unreported new Calla lily chlorotic spot tospovirus.  Molecularly cloned nucleocapsid (NP) protein genes from various tospoviruses into a Zucchini yellow mosaic virus vector, and NP protein immunogens were expressed in and purified from zucchini squash, and antibodies produced in rabbits.  Identified and molecularly cloned a consensus region of the NSs gene shared by members of Watermelon silver mottle virus (WSMoV) serogoup and antibodies were produced.  Molecularly and serologically identified three new potyviruses, Calla lily latent virus, Amaryllis mosaic virus, and Lycoris mild mosaic virus.  Developed a biolistic method of inoculation for Cucumber mosaic virus in gladioli.  Characterized monoclonal antibodies produced to Cucumber mosaic virus (CMV) and tospoviruses including CaCV, INSV, and Tomato spotted wilt virus (TSWV).  Produced antisera to Lily symptomless virus (LSV) and developed an assay method for detection of LSV in lilies  Under Cooperative Research and Development Agreements with industries mouse monoclonal antibodies and rabbit antisera are now available to the public.

PLANS:  
Current research includes: 1) characterization of a new ilarvirus infecting Bacopa; 2) characterization of a new potyvirus infecting Lycoris; 3) finishing evaluation of  CMV resistance in transgenic gladioli containing CP, replicase and scFv genes; 4) concluding evaluation of a consensus sequence polypeptide on NSs of Tomato spotted wilt virus, Impatiens necrotic spot virus and Groundnut ringspot virus.  

RECENT PUBLICATIONS:

1994. Chiu, R. J., Chen, C. C. and Hsu, H. T. Purification and characterization of a tospovirus systemically infecting cucurbits in Taiwan. Plant Pathol. Bull. 3:198-208.

1994. Herman, E. M., Li, X. H., Su, R. T., Larsen, P., Hsu, H. T., and Sze, H. Vacuolar-type H+-ATPases are associated with the endoplasmic reticulum and provacuoles of root tip cells. Plant Physiology 106:1313-1324.
 
1994. Lawson, R. H., Dienelt, M. M., and Hsu, H. T. Influence of temperature on the ultrastructure and serological reactivity of two tospovirus isolates. Acta Hort. 337:149-158.

1995. Hsu, H. T., Kim, J. Y., and Lawson, R. H. Purification of lily symptomless carlavirus and detection of the virus in lilies. Plant Dis. 79:912-916.

1995. Hunter, W. B., and Hsu, H. T. A novel method for tospovirus acquisition by thrips. Phytopathology 85:480-483.

1995. Tsai, J. H., Yue, B., Webb, S., Funderburk, J. E., and Hsu, H. T. Effects of host plant and temperature on growth and replication of Thrips palmi (Thysanoptera: Thripidae). Environ. Entomol. 24:598-603.

1996. Lawson, R. H., Dienelt, M. M., and Hsu, H. T. Ultrastructural comparisons of defective, partially defective, and non-defective isolates of impatiens necrotic spot virus. Phytopathology 86:650-661.

1996.  Hunter, W. B., and Hsu, H. T.  Inoculation of thrips cell monolayers with a tospovirus.  Acta Hort. 431:359-414.

1996.  Hunter, W. B., and Hsu, H. T.  Formulation of an insect medium for thrips monolayer cell cultures (Thysanoptera: Thripidae: Frankliniella occidentalis).  J. Inv. Pathol. 67:125-128.

1996.  Hsu, H. T. Immunological detection and identification of tosposviruses.  Acta Hort. 431:109-121.

1996.  Lawson, R. H., Dienelt, M. M., and Hsu, H. T. The cytopathology of tospovirus-host-plant interactions.  Acta Hort. 431:267-290.

1996. Reddy, D. V. R., Satyanarayana, T., Dwivedi, S. L., Katana, A. S., Buiel, A. A. M., Ranga Rao, G. V., Nigam, S. N., Naidu, R. A., Hsu, H. T., and Demski, J. W. Diagnosis and resistance breeding of peanut bud necrosis virus. Acta Hort. 431:411-414.

1996. Hsu, H. T., and Liu, X. P.  Production of serological reagents and development of a test procedure for simultaneous detection and identification of tomato spotted wilt and impatiens necrotic spot tospoviruses.  Acta Hort. 432:140-147.

1997. Chen, C. C., Chen, M. J., Chiu, R. J., and Hsu, H. T. Rice ragged stunt virus (oryzavirus) possesses an outer shell and A-spikes.  Plant Prot. Bull. 39:383-388.

1998. Li, X., Su, T. C., Hsu, H. T., and Sze, H. The molecular chaperone calnexin associates with the vaculoar H+-ATPase from oat seedlings.  Plant Cell 10:119-130.

2000. Hsu, H. T., Ueng, P. P., Chu, F. H., Ye, Z., Subramaniam, K., and Yeh, S. D. Serological and molecular characterization of a high temperature-recovered virus belonging to tospovirus serogroup IV.  J. Gen. Plant Pathol. 66:167-175.

2000. Chen, C. C., Chen, Y. K., and Hsu, H. T. Characterization of a virus infecting lisianthus. Plant Disease 84:506-509. 

2000. Hsu, H. T., Barzuna, L., Hsu, Y. H., and Perry, K. L. Identification and subgrouping of cucumber mosaic cucumovirus with mouse monoclonal antibodies.  Phytopathology 90:615-620.  

2000. Gera, A., Hsu, H. T., Cohen, J., Watad, A., Beckelman, and Hsu, Y. H. Effect of  cucumber mosaic virus monoclonal antibodies on virus infectivity and transmission by aphids. J. Plant Pathol. 82:3-7.
 
2001. Chen, C.C., Tsai, S.L., and Hsu, H.T. Diagnosis of Lisianthus necrosis virus infection by light and electron microscopy and serological assays.  Plant Pathol. Bull. 10:105-114.

2002. Chang, C.A., Chen, C.C., and Hsu, H.T. Partial characterization of two potyviruses associated with golden spider lily severe mosaic disease.  Acta Hort. 568:127-134.

2002. Chen, C.C., Hu, C.C., Chen, Y.K., and Hsu, H.T. A fabavirus inducing ringspot disease in lisianthus.  Acta Hort. 568:51-57.

2002. Chen, C.C. and Hsu, H.T. Occurrence of a severe strain of Lisianthus necrosis virus in imported carnation seedlings in Taiwan.  Plant Disease 86:444.

2002. Chen, C.C., Ke, W.F., Lin, C.F., Jan, F.J., and Hsu, H.T. First report of Carnation mottle virus in calla lilies (Zantedeschia spp.).  Plant Disease 87:1539.

2004. Chen, C.C., Chang, C.A., Tsai, H.T., and Hsu, H.T. Isolation and Identification of a newly recognized potyvirus causing latent infection on calla lilies.  Plant Disease 88:1046.

2004. Chen, C.C., Chen, T.C., Lin, Y.H., Yeh, S.D., and Hsu, H.T. Manuscript: A chlorotic spot disease on calla lilies (Zantedeschia spp.) is caused by a tospovirus serologically but distantly related to  Watermelon silver mottle virus.  Plant Disease 89:440-445.

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

2005. Aebig, J., Kamo, K., and Hsu, H.T. Biolistic inoculation of gladiolus with Cucumber mosaic cucumovirus.  J. Virol. Methods 123:89-94.

2005. Chen, T.C., Hsu, H.T., Jain, R.K., Huang, C.W., Lin, C.H., and Yeh, S.D.  Purification and serological analysis of tospoviral nucleocapsid protein expressed by Zucchini yellow mosaic potyvirus vector in squash.  J. Virol. Methods 129:113-124.

2005. Chen, Y.K., Yang, C.T., and Hsu, H.T. Allamanda mosaic caused by Cucumber mosaic virus.  Plant Disease 89:259.

2005. Jan, F.Y., Chen, C.C., and Hsu, H.T. Identification of Tomato mosaic virus infection on lisianthus in Taiwan.  Plant Disease 87:1537.

2005. Lin, Y.S., Chen, T.C., Hsu, H.T., Chu, F.H., Chen, C.C., Lin, Y.Z., and Yeh, S.D.  Serological comparison and molecular characterization for verification of Calla lily chlorotic spot virus as a new tospovirus species.  Phytopathology 95:1482-1488.

2006. Aebig, J., Albert, H.H., Zhu, B.L., Hu, J.S., and Hsu, H.T. Cloning and construction of single-chain variable fragments (scFv) to Cucumber mosaic virus and production of transgenic plants.  Acta Hort. (in press).

2006. Chen, T.C., Huang. C.W., Kuo, Y.W., Liu, F.L., Hsuan Yuan, C.H., Hsu, H.T., and Yeh, S.D. Identification of common epitopes on a conserved region of NSs proteins among members of Watermelon silver mottle virus (WSMoV) serogroup.  Phytopathology (in press).

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

2006. Lawson, R.H., Quarantine viruses, viroids and phytoplasmas affecting movement of ornamental plants. Acta Hort. (in press).

2006. Xu, M.Q,  Li,  H.P., Wang, M., Wu, Z.C., Borth, W.B., Hsu, H.T., and Hu, J,S, Transgenic plants expressing a single-chain FV antibody to Tomato spotted with virus (TSWV) are resistant to TSWV systemic infection.  Acta Hort. (in press)

Reviews and Book Chapters:

1995. Hsu, H. T., Lawson, R. H., Lin, N. S., and Hsu, Y. H. Direct tissue blot immunoassay for analysis of plant pathogens. pp. 365-374. In: "Molecular Methods in Plant Pathology". Singh and Singh, eds. CRC Press, Inc.

1995. Hunter, W. B., and Hsu, H. T. Establishing thrips cell cultures to study tospoviruses. pp. 163-166. In: "Thrips Biology and Management". Parker, Skinner and Lewis, eds. Plenum, New York.

1995. Lawson, R. H., Gera, A., and Hsu, H. T. Transmission of Plant Viruses. pp. 117-133. In: "Virus and Virus-like Diseases of Bulb and Flower Crops". John Wiley Publisher.

1995. Gera, A., Lawson, R. H., and Hsu, H. T. Identification and Assay. pp. 165-180. In: "Virus and Virus-like Diseases of Bulb and Flower Crops". John Wiley Publisher.

1995. Lawson, R. H., and Hsu, H. T. Orchids. pp. 409-420. In: "Virus and Virus-like Diseases of Bulb and Flower Crops". John Wiley Publisher.

1995. Lawson, R. H., and Hsu, H. T. Impatiens. pp. 493-503. In: "Virus and Virus-like Diseases of Bulb and Flower Crops". John Wiley Publisher.

1998. Hsu, H. T. Molecular plant breeding for virus resistance in ornamental crops. pp. 137-145. In: "Frontiers in Biology: The Challenges of Biodiversity, Biotechnology and Sustainable Agriculture". Academia Sinica.

2001. Hsu, H.T.  Potato yellow dwarf Nucleorhabdovirus.  In: “Datasheet of Quarantine Pests for Eastern Africa. FAO-CABI”.

2001. Hsu, H.T.  Potato yellow dwarf virus.  In: “Crop Protection Compendium”.  CAB International.

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