Journal of Undergraduate Research
Volume 1, Issue 2 - November 1999

Evaluating Resistance to Tomato Spotted Wilt Virus in Arabidopsis thaliana

Heather Edwards

ABSTRACT

Tomato spotted wilt virus (TSWV), a member of the Bunyaviridae family, is an important plant pathogen that infects numerous species worldwide. Peanut, Arachis hypogaea L., grown in the southeastern United States is severely affected by TSWV, which is vectored by thrips. Arabidopsis thaliana should be a model plant for elucidating genetic mechanisms involved in plant resistance to TSWV. Ten ecotypes of A. thaliana were field tested in Marianna, FL for evaluation of genetic resistance. Symptoms of TSWV were seen in all of the ecotypes except for Nossen (No-0). These initial results were confirmed by ELISA analysis. Mechanical inoculation and thrips inoculations in the greenhouse are being initiated and the data from these experiments will be compared with the completed field experiment.

INTRODUCTION

Spotted wilt disease, caused by tomato spotted wilt virus (TSWV), a member of the Bunyaviridae family, is an important plant pathogen that infects numerous species throughout temperate and subtropical regions worldwide. Its large host range includes vegetable, ornamental, and field crops (Mumford et al., 1996). Symptoms of TSWV are concentric ring spots and speckling on leaves, stunting and premature death of susceptible plants. Spotted wilt was first reported in Australia in 1919 and has since been documented all over the United States and Canada (Johnson III et al., 1996). Since direct control of TSWV, and other closely related tospoviruses, is not possible, viral diseases of crops are extremely difficult to manage.

Heather EdwardsPeanut, Arachis hypogaea L., is severely affected by TSWV, which is mainly vectored by the tobacco thrips, Frankliniella fusca (Hinds), and the western flower thrips, Frankliniella occidentalis (Pergande). TSWV is acquired by thrips only in the larval stage and is transmitted primarily by adults. Spotted wilt was first reported on peanut in Texas in 1974 (Halliwell and Philley, 1974); it has spread into Mississippi, Alabama, Florida, and Georgia, which are major peanut producing regions. The northeastern United States and southeastern Canada were exposed to spotted wilt from importing Georgia-grown TSWV-infected transplants (Johnson III et al., 1996).

Culbreath et al. (1992) showed that final incidence of spotted wilt was lower in the peanut cultivar Southern Runner compared to Florunner. This difference was not correlated to the number of adult or larval thrips found on each cultivar. Therefore, it was concluded that Southern Runner possessed a degree of genetic resistance to TSWV. Consequently, Southern Runner has been used in peanut breeding programs to develop cultivars that display more resistance to TSWV; however no currently grown commercial peanut has immunity.

D. W. Gorbert, a University of Florida Institute of Food and Agricultural Sciences Agronomy Professor developed the SunOleic 95R (Gorbet and Knauft, 1997) and 97R peanut. These peanuts surpass olive oil in cholesterol lowering properties, have a 10-14% greater yield than the industry standard, Florunner, as well as a 3-15-fold increase in shelf-life. SunOleic 97R is grown in five major peanut producing states: Florida, Georgia, Alabama, South Carolina, and Texas. SunOleic 97R has decreased susceptibility to TSWV compared to SunOleic 95R; however, when disease pressure is high, significant yield losses are observed (Gorbet, personal communication).

The overall objective of our research is to improve TSWV-resistance in peanut. However, due to its tetraploid nature, relatively long life cycle, and narrow germplasm base, molecular genetic studies on peanut are arduous. Since Arabidopsis thaliana has a small genome size, short life cycle, and produces as many as 104 seeds, it has become the model plant for molecular genetic studies ( Meyerowitz, 1987). It has been used in recent years to begin defining host factors responsible for resistance to viruses. Leisner and Howell (1992) examined the relationship between Cauliflower Mosaic Virus (CaMV) and various ecotypes of A. thaliana. Systemic symptoms were influenced by the rate of plant development. Ecotype En-2 was an exception to this trend because it did not produce symptoms in response to any of the three CaMV viral isolates tested. Therefore, this ecotype is being examined further to define the genetic basis for resistance to CaMV. Martin et al. (1999) have examined resistance to turnip mosaic potyvirus in A. thaliana. Various forms of resistance were found in four ecotypes out of the 106 ecotypes evaluated. German et al. (1995) have shown that TSWV can replicate and systemically infect A. thaliana. The initial symptoms of TSWV became visible three weeks post-inoculation with the appearance of chlorotic spots on uninoculated leaves. As a first step to understanding resistance mechanisms involved in the TSWV infection process, we have screened ten ecotypes of A. thaliana for TSWV in field experiments.

MATERIALS AND METHODS

Heather EdwardsTen ecotypes of A. thaliana: Nossen (No-0), RLD, C24, Landsberg erecta, Wassilewskia, Columbia (Col-O), Columbia (Col), Columbia (Col-PRL), Niederzenz, and Bensheim were obtained (Lehle Seeds) for evaluation of TSWV resistance. Seeds were germinated on autoclaved soil ( Fafard Mix No. 2 ) in four inch pots within a plastic tray covered by a transparent lid. A. thaliana were subirrigated and placed in a growth room under a 16-h photoperiod with cool white flourescent lights (80 Fmol m-2 s-1) at 25EC. Plants were kept under these conditions until they were taken to the field; 8 to 10 days post-planting. Field-testing occurred at the North Florida Research and Education Center, Marianna, FL in May and June 1999 when high TSWV pressure was evident. Pots containing at least 10 A. thaliana plants of each ecotype were placed between rows of a highly TSWV-susceptible peanut genotype and remained in the field for 24 h. The experiment was replicated three times.

Plants were brought back to Gainesville, FL and maintained in a greenhouse until symptoms were visible. Plants were destructively sampled, and flowers, leaves and roots were tested for the presence of TSWV using enzyme-linked immunosorbent assay analysis (ELISA). Previously tested peanuts were used as positive and negative controls. TSWV antibody and conjugate were obtained from Agdia. Samples were read at 405 nm in an ELISA microplate reader (Bio-Rad Model 550).

RESULTS

TSWV symptoms appeared as ring spots of light green on the leaves, as well as wilting of the leaves. Based on ELISA, all ecotypes were positive for the presence of TSWV except for Nossen (No-0) which tested negative for all three replications. However, even among the ecotypes that were TSWV-positive, the location of TSWV within the plant varied (Figure 1). Eight of the nine remaining ecotypes exhibited TSWV in their leaves. Approximately half were positive for TSWV in their flowers, and only two out of nine ecotypes had TSWV detected in their roots.

FUTURE RESEARCH

To confirm the resistance of Nossen to TSWV, and to extend the ecotype evaluation work, TSWV mechanical inoculations with various isolates will be initiated. In this experiment, careful observations will be made regarding the timing of symptoms, and sampling for ELISA anaysis relative to the rate of plant development for each ecotype. Data generated from these experiments will be compared with the field experiment. Time permitting, mutant A. thaliana will also be researched.

Photos by Jane Gibson

ACKNOWLEDGEMENTS

The authors thank the University Scholar Program for support, M. Murakami for assistance and guidance in ELISA analysis and Dr. A. Abouzid for technical advice in working with TSWV.

REFERENCES

  1. Culbreath A.K., Todd J.W., Demski, J.W., and Chamberlin J.R. 1992. Disease progress of spotted wilt in peanut cultivars Florunner and Southern Runner. Phytopath. 82:766-77.
  2. German T.L., Adkins S., Witherell A., Richmond K.E., Knaack W.R., and Willis D.K. 1995. Infection of Arabidopsis thaliana ecotype Columbia by tomato spotted wilt virus. Plant Mol. Biol. Rep. 13:110-117.
  3. Gorbet D.W., and Knauft D.A. 1997. Registration of 'SunOleic 95R' peanut. Crop Sci. 37:1392.
  4. Halliwell R.S., and Philley G. 1974. Spotted wilt of peanut in Texas. Plant Dis. Rep. 58:23-25.
  5. Johnson III W.C., Todd J.W., Culbreath A.K., and Mullinix Jr, B.G. 1996. Role of warm-season weeds in spotted wilt epidemiology in the southeastern coastal plain. Agron. J. 88:928-933.
  6. Leisner S.M., and Howell S.H. 1992. Symptom variation in different Arabidopsis thaliana ecotypes produced by cauliflower mosaic virus. Phytopath. 82:1042-1045.
  7. Martin A.M, Cabrere y Poch H.L., Herrera D.m., and Ponz F. 1999. Resistance to turnip mosaic potyvirus in Arabidopsis thaliana. Mol. Plant-Microbe Interact. 12:1016-1021.
  8. Meyerowitz E.M. 1987. Arabidopsis thaliana. Ann. Rev. Genet. 21:93-111.
  9. Mumford R.A., Barker I., and Wood K.R. 1996. The biology of tospoviruses. Ann. Appl. Biol. 128:159-183.

--top--

Back to the Journal of Undergraduate Research