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 Managing Tospoviruses through Biotechnology: Progress and prospects
By
H.R. Pappu
  
Keywords:  Genetic engineering; Disease/pest resistance. 
Correct citation: Pappu, H.R. (1997), "Managing Tospoviruses Through Biotechnology: Progress and prospects." Biotechnology and Development Monitor, No. 32, p. 14­17. 
Tospoviruses are a relatively new group of viruses, which attack many economically important crops world­wide. Genetic engineering is applied to develop tospovirus­resistant plants. Other efforts focus on diagnostics, and disease forecasting systems.

"Tospovirus" is an acronym derived from the name of the group's most prolific, and first recognized member, the tomato spotted wilt virus (TSWV) (see box). Earliest reports of diseases caused by tospoviruses were from Australia and South Africa in the 1910s. TSWV became well established in many parts of the world affecting crops such as potato, lettuce, tomato, pepper, groundnut, mungbean, and tobacco. Until the late 1980s, TSWV was considered to be the sole member of the group, and some of the diseases were believed to have been caused by TSWV. Increased interest in tospoviruses during the last ten years has led to the identification and characterization of several new tospoviruses from Brazil, Japan, India, Taiwan, and USA (see table).
Rough estimates calculate the world­wide loss due to tospoviruses at US$ 1 billion. In the state of Georgia, USA, spotted wilt causes a total annual loss of US$ 100 million in groundnut, vegetables and tobacco. Lettuce crop in Hawaii have suffered serious damage due to TSWV for several successive years, forcing the growers to switch to other crops. Annual losses due to peanut bud necrosis virus (PBNV) in Asia are estimated at more than US$ 89 million.
Since the 1980s, losses due to diseases caused by tospoviruses have risen, due especially to the introduction and/or proliferation of Franklienella occidentalis, the western flower thrips. Its wide host range has enabled this thrips species to establish itself quickly. Its distribution has likely been accelerated through the world­wide trade of planting material and through wind currents.

Management of tospoviruses
The extremely wide and overlapping host range of both TSWV and the thrips vector, and the fact that the virus can multiply in the insect vector, makes management of the disease difficult. Insecticides that control the thrips are largely ineffective in reducing disease incidence as is the case in most of the vector­borne viruses of plants. It is believed that the insect carrying the virus would introduce the virus into the host plant before the insecticide takes effect. Moreover, for reasons not fully understood, certain insecticides have increased the final disease incidence of TSWV in groundnut. Other cultural practices, such as adjusting the planting date have limited application, since no one planting date, is effective for all the crops that are susceptible to the virus.
The most effective way to manage tospoviruses is through growing resistant crops. However, natural resistance to these viruses is limited. Traditional plant breeding methods have been successful to some extent in utilizing naturally occurring host plant resistance to TSWV in tomato, groundnut and pepper, and to PBNV in groundnut. Another approach to introducing resistance to tospoviruses is through genetic engineering. This is especially useful in crops where natural sources of resistance are not available, or if available, are not amenable to transfer into agronomically superior cultivars by conventional breeding methods
.
Biotechnology in tospovirus control
Genetically engineered cross protection, i.e. the process by which plants transformed with portions of the viral genome resist further infection by homologous, and in some cases heterologous viruses, is effective in managing virus diseases in several crops. First demonstrated in the case of tobacco mosaic virus (TMV) in transgenic tobacco expressing the TMV­capsid protein, this type of engineered resistance has been demonstrated for a wide range of positive strand RNA viruses.
In 1991, researchers at the Wageningen Agricultural University (WAU), the Netherlands, first reported that the expression of TSWV NP gene in tobacco conferred resistance to challenge inoculation by TSWV. This was followed by several other reports of achieving transgenic resistance to TSWV in tobacco and tomato. The exact mechanisms of the protection are not well understood. Transgenic plants expressing TSWV NP gene sequences resisted infection by mechanical (manual) inoculation and thrips­mediated inoculation. Thus, this approach holds promise for the use of transgenic plants to minimize disease losses under the high thrip pressure often encountered in row crops.
The transgenic resistance achieved by introducing TSWV sequences, however, was mainly specific to TSWV, and in some cases to impatiens necrotic spot virus (INSV). In order to broaden the resistance to several tospoviruses, researchers at WAU transformed tobacco with NP genes of three tospoviruses: TSWV, groundnut ring spot tospovirus (GRSV), and tomato chlorotic spot tospovirus (TCSV). Several transgenic lines expressing all the three genes showed resistance to challenge infection by the respective tospoviruses. This possibility of developing crops with multiple virus resistance may have the greatest impact in regions where more than one tospovirus is present (see table).
While most efforts to introduce genetically engineered resistance to tospoviruses utilized tobacco, other crops of commercial value that have been studied include tomato, lettuce, groundnut, and ornamental crops (see table on page 17). Transgenic TSWV­resistant Oriental and Virginia types of tobacco were successfully field tested in Bulgaria. Projects are underway to introduce resistance to PBNV in groundnut in India, to watermelon silver mottle virus (WSMV) in watermelon in Taiwan, to TSWV in groundnut, and to Burley and flue­cured tobacco in the USA. Several laboratories in the USA and elsewhere have successfully transformed and regenerated selected groundnut varieties with TSWV sequences. TSWV­resistant transgenic groundnuts may become a reality in the near future.

Tospoviruses

The genus Tospovirus is placed in the family Bunyaviridae. Tospoviruses are the only members of this family that infect plants; other members of the virus family are arthropod­borne viruses that are serious pathogens of animals and humans. The viral genome consisting of three pieces of single stranded RNA can potentially code for six proteins that include two glycoproteins (G1 and G2), RNA polymerase, nucleocapsid protein (NP), and two non­structural proteins (NSs and NSm). The glycoproteins are involved in thrips transmission, and NSm in cell to cell movement. The role of NSs is not yet clearly established. Tospoviruses are differentiated by the variability in the NP sequences. 
The increased incidence of tospoviruses in several parts of the world since the 1980s has been attributed to the introduction and/or proliferation of its vectors: several species of thrips (family Thripidae). Unlike some other plant virus­vector relationships, the fidelity between tospoviruses and the thrips species that transmit them is limited. For example, tomato spotted wilt virus (TSWV) can be transmitted by at least seven different species of thrips, whereas peanut bud necrosis tospovirus (PBNV) and watermelon silver mottle tospovirus (WSMV) are transmitted by Thrips palmi.
Nevertheless, there is an innate biological association between tospoviruses and their thrips vectors. Once a virus is acquired by the thrips larvae, the virus replicates in the thrips and survives through the developmental stages of the insect. Only acquisition by first instar thrips larvae can lead to the adults that transmit the virus. A virus acquired by adults is not transmitted, due to the presence of a midgut barrier to virus acquisition in adult insects. Infective adults retain the ability to transmit the virus for life. However, the virus is not transmitted from an adult to its offspring. 
Tospoviruses as well as their thrips vectors have a wide host range. For example, TSWV is known to infect about 650 plant species in 45 families.


Recognized tospoviruses and their geographic distribution
 
Species Geographic Distribution
Tomato spotted wilt virus (TSWV) The Americas, Africa, Australia, and Europe 
Tomato chlorotic spot virus (TCSV)  Brazil
Groundnut ring spot virus (GRSV)  Brazil, South Africa
Impatiens necrotic spot virus (INSV)  North America, Europe
Watermelon silver mottle virus (WSMV)  Taiwan, Japan
Peanut bud necrosis virus (PBNV) India, Nepal, Pakistan, Sri Lanka, Thailand and China
Melon spotted wilt virus (MSWV)  Japan
Peanut yellow spot virus (PYSV) India, Thailand, and Myanmar
Isolates Chry1* and BR­11t*  Brazil
Isolate BR­09z*  Brazil
 *These isolates have not yet been named.

International collaboration
Close collaboration and exchange of information is essential among different countries to sustain a concerted effort in developing management strategies for tospoviruses. For example, PBNV is a major constraint to groundnut production in the Indian subcontinent. Collaborative research between the University of Georgia, USA, and the International Crops Research Institute for the Semi­Arid Tropics (ICRISAT) resulted in cloning and sequencing of portions of the viral genome and the subsequent development of constructs of the NP gene for plant transformation. Collaborative efforts between the University of Brasilia and EMBRAPA in Brazil, and the WAU resulted in the identification of several new tospoviruses. Cooperation between the National Chung Hsing University, Taiwan, and the US Department of Agriculture facilitated the identification of a new tospovirus in Taiwan. Other collaborative efforts, among different laboratories in Africa, Europe, Asia, and the Americas, have resulted in the production of high quality virus­specific antibodies and reliable diagnostic methodologies to detect and differentiate different tospoviruses.

Transgenic crops resistant to tospovirus infections
Crop Introduced sequence Greenhouse/Field Tested
Tobacco NP gene of TSWV Both
Tobacco NSm of TSWV Greenhouse
Tobacco NP genes of TSWV, TCSV, GRSV  Greenhouse
Tomato NP gene Both
Transgenic inbred tomato hybrid  NP gene of TSWV Both
Groundnut NP gene of TSWV Both
Chrysanthemum NP gene of TSWV Greenhouse
Lettuce NP gene of TSWV Both
NP gene: Nucleocapsid protein gene;
NSm: Nonstructural protein coded by the middle RNA of TSWV

The future
Research into exploring new ways of applying biotechnology is needed to deal with these emerging virus infections of crops. New paths might include the engineering of resistance to tospoviruses by expressing the tospovirus glycoproteins in transgenic plants to block virus acquisition by thrips, by expressing truncated or modified forms of movement protein(s) of heterologous viruses, or by expressing tospovirus­specific antibody genes.
It is imperative that a combination of conventional and biotechnological methods be deployed to minimize losses caused by tospoviruses. This combination includes the cultivation of virus resistant plants developed through conventional breeding or transgenic technology, use of appropriate cultural practices, and vector management. Additionally, due to the development of antibodies specific to the viral non­structural gene products, it is now possible to differentiate transmitters from non­transmitters in a thrip population. This can potentially be used to develop a disease forecasting system.
Since most of the progress made in our understanding of the biology and molecular biology of tospoviruses was accomplished through active collaboration among developed and developing countries, it is safe to expect that this cooperation should continue and is likely to result in a positive impact in managing diseases caused by tospoviruses.
H.R. Pappu

Assistant Professor, Department of Plant Pathology, University of Georgia, College of Agriculture and Environmental Sciences, Coastal Plain Experiment Station, Tifton, GA 31793­0748, USA. Fax (+1) 912 386 7285; E­mail hrp@tifton.cpes.peachnet.edu

Sources
M.E. Daub, R.K. Jones and J.W. Moyer (1997), "Biotechnological Approaches for Virus Resistance in Floral Crops." In: R.L. Geneve, J.E. Preece and S.A. Merkle (eds.), Biotechnology of Ornamental Plants. Wallingford: CAB International. pp.335­351.

R. Goldbach and D. Peters (1996), "Molecular and Biological Aspects of Tospoviruses." In: R.M. Elliott (ed.), The Bunyaviridae. New York: Plenum Press.

R.A. Mumford, I. Barker and K.R. Wood (1996), "The Biology of Tospoviruses." Annals of Applied Biology, vol. 128, pp.159­183.

H.R. Pappu, C.L. Niblett and R.F. Lee (1995), "Application of Recombinant DNA Technology in Plant Protection: Molecular approaches to engineering virus resistance in crop plants." World Journal of Microbiology and Biotechnology, vol. 11, pp. 426­437.

G. Kuo and R. Goldbach (eds.)(1995), "Proceedings of the International Symposium on Tospoviruses and Thrips of Floral and Vegetable Crops." November 7­10, 1995, Taiwan Agricultural Research Institute, Taiwan. ActaHorticulturae. Vol. 431.



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