An important disease of groundnuts in India has recently been shown to be caused by tomato spotted wilt virus (TSWV), which is transmitted by thrips. The results are given of further studies on Scirtothrips dorsalis Hood (which had earlier been shown to be a vector), on the role of Frankliniella schultzei (Tryb.) as a vector and on the application of the haemagglutination technique to detect viral antigen in thrips. Both species of thrips were able to transmit the virus from groundnuts to groundnuts and urd bean (Vigna mungo), but F. schultzei was the more efficient. Haemagglutination tests detected viral antigens in extracts from both species that had been exposed to infected leave

Amin, P W

Ghanekar, A M

Reddy, D V R

ICRISAT Open Access Repository

Transmission of Tomato Spotted Wilt Virus, the Causal Agent of Bud Necrosis of Peanut, by Scirtothrips dorsalis and Frankliniella schultzeiP. W. AMIN, D. V. R. REDDY, and A. M. GHANEKAR, Groundnut Improvement Programme, International Crops Research Institute for the Semi-Arid Tropics, Hyderabad 500 016, India; and M. S. REDDY, Department of Plant Pathology, Andhra Pradesh Agricultural University, Hyderabad 500 030, IndiaABSTRACTAmin, P. W., Reddy, D. V. R., Ghanekar, A. M .,and Reddy, M. S. 1981. Transmission of tomato spotted wilt virus, the causal agent of bud necrosis of peanut, by Scirtothrips dorsalis and Frankliniella schultzei. Plant Disease 65:663-665.Tomato spotted wilt virus, the cause of bud necrosis of peanut, was transmitted by Scirtothrips dorsalis and Frankliniella schultzei. S. dorsalis is a new vector of the virus, but it is a less efficient vector than F. schultzei. Hemagglutination tests detected viral antigens in extracts from both thrips speSesexppsSa to ihffctedleavesTBud necrosis is probably  the m ost im p o r ta n t  v ira l d isea se  o f  p e a n u t (Arachis hypogaea  L.) in India (3,6). U ntil 1977, the vector was unknown,, a lthough  several insects including A ph is  craccivora  Koch, E m poasca devastans Stal, Orosius spp., Bemisia tabaci Genn., and Tetranychus spp. had been tested (8,11,12,16, A m in unpublished).Initial investigations a t the International C rops R esearch Institu te  fo r the Semi- A rid Tropics research center showed tha t th rips were the m ost com m on insect pests a t the tim e o f disease developm ent, both  du ring  and after rainy seasons (Amin, unpublished). Recently, the disease was show n to  be caused by tom ato  spotted w ilt virus (TSW V), which is transm itted  by th rip s  (6,14). The m ost ab u n d an t th r ip s  on p ean u ts  in H y d erab ad  are S c i r t o t h r i p s  d o r s a l i s  H o o d  a n d  F r a n k l in ie l la  s c h u l t z e i  (T ry b o m ) , and prelim inary  tests showed th a t the form er transm its the causal virus o f bud necrosis (1).Approved as ICR1SAT journal article 142.Accepted fo r publication 25 November 1980.0191 -2917/81/08066303/$03.00/0®1981 American Phytopathological SocietyThis paper describes fu rther research on the transm ission of TSW V  by S. dorsalis, on the role of F. schultzei as ano ther vector, and on the application of the hem agglutination technique to  detect viral antigen in th rips,M A T E R IA L S  A N D  M E T H O D SP eanu t (cv. TM V-2) plants infected by sap  in o c u la t io n  (5) w ere u sed  fo r  acquisition  feeding tests. In prelim inary experim ents, term inal leaves of infected p lants were the best source of inociilum. P eanut seedlings in the second quadri- foliate stage were used as test plants in ' early experim ents, bu t u rd  bean-, ( Vigna m u n g o  L. cv. U P U r l )  w as la te r  substitu ted  fo r peanut since it was more susceptible and showed sym ptom s in 15-20 days com pared w ith 30-40 days fo r peanut.Virus-free colonies were in itiated  from  5—10 adu lt th rips collected in  the field and ra is e d  on  p e a n u t p la n ts .  A p e a n u t seedling, w ith the roo ts rem oved, was placed in a screw-capped 500-ml plastic ja r  containing w ater and  then  enclosed in a  lan tern  globe. Five to  ten th rips were released in to  each cage and  allowed to oviposit fo r 24 hr. The newly hatched nym phs were transferred  to  a new set of h e a lth y  p e a n u ts  en c lo se d  in  la n te rn‘ globes. A glass funnel w ith a sm all glass vial (3 X 1 cm) inserted in its stem was attached  to  the open end of the lan tern  globe (Fig. 1). The ad u lt thrips were collected in the glass vial and  used for starting virus-free colonies.A lternatively, the insects were raised on peanut leaves enclosed in 3. X 1 cmfrom  healthy colonies were frequently released onto  test plants to ascertain  tha t they were virus-free.Transm ission tests. Fine-tipped cam el’s h a i r  b ru s h e s  w ere u sed  to  t r a n s fe r  nym phs and adults. Test plants were covered by lan tern  globes.The nym phs were allowed to  acquire the virus by feeding on detached infected leaves floating  on w ater in a petri dish. Usually 20-25 nym phs were released on each leaflet and allowed to  feed for 2-3 days. A group of 10-15 nym phs was thenTable 1. Transmission of tomato spotted wilt virus by thrips*Acquisition accessTest Infected HealthyThrips plant leaves” leaves'Scirtothrips Peanut 2/206 0/200“dorsalis Urd bean 7/131 0/55Frankliniella Urd bean 26/60 0/60schultzei Urd bean' 25/102 0/60“Nymphs were given 2-3 days of acquisition access and 10-12 days of inoculation access feeding. Ten to 15 nymphs were fed on each test plant.bNymphs exposed to infected leaves.c Nymphs exposed to healthy leaves served as a control.“Number of plants infected/exposed.'  Single adult thrips fed on each plant.Plant Disease/August 1981 663transferred  to  a test p lan t and allowed to feed fo r 10-15 days.F or transm ission tests with single thrips, nymphs were allowed an  acquisition access feeding of 6-10 days, and single a d u lts  th a t  em erg ed  w ere u sed  in  transm ission tests. The inoculation access feeding period  was 4 -5  days. A sim ilar procedure was followed fo r the controls except th a t the th rips were exposed to healthy peanut leaves.A fter the inoculation  access period, the exposed p lants were sprayed with 0.025% d e m e to n  m e th y l (B ay er, In d ia )  an d  m aintained in a screenhouse. Peanut test plants were m aintained fo r 45 days andurd bean plants fo r 20 days. D uring this p e rio d ,, p la n ts  w ere  sp ra y e d  w ith  insecticide a t weekly intervals. A fter sym ptom s appeared, the presence of virus was confirm ed by assays on cow pea, Vigna unguicula ta  (L. cv. C-152), as described by G hanekar et al (6).H em agglutination  test. Samples of th rips were collected and held a t 13 C for ab o u t 1 hr. Insects were then  counted  and tr itu ra ted  in 500 times their weight of 0.05 M phosphate  buffer, pH  7.0, contain ing 0.02 M 2-m ercaptoethanol. The weight of an individual insect was assum ed to be 2.5 X 10~2mg (4), and the ex tract was considered to  be 1:500 dilu tion . Eachextract was clarified a t 2,000 g  fo r 5 min in a  refrigerated Remi K-24 centrifuge.T h e  h e m a g g lu t in a t io n  t e s t  w as perform ed as described by R ajeshw ari et al (10). G lutaraldehyde-treated tanned red blood cells were suspended in 0.15 M phosphate buffered saline, pH  7.0, a t a 3.0% concentration . One volum e of TSW V antibody suspension was added to nine volum es of tanned  red blood cells and  incubated a t 37 C in a w ater b a th  for 30 min. The coated cells were resuspended in phosphate buffered saline, pH  7.2, contain ing  0.5% bovine serum  album in, washed three tim es, and suspended in the same solution  to  give a concentration  of 3 .0%  p a c k e d  ce lls . S e r ia l tw o fo ld  dilutions of th rips ex tract were prepared in the phosphate  buffered saline w ith bovine serum  album in, and 0.5 m l was placed in each well o f a lucite plate. Later, 0.08 ml of sensitized cells (3.0%) was added to  each well, mixed gently, incubat­ed for 2 h r a t room  tem perature and then overnight at 4 C. In a  positive reaction, red cells form ed a sm ooth  m at (with serrated m argin) on the bo ttom  of the well; a  negative reaction  resulted in a discrete red ring a t the periphery of the well (Fig. 2).R E SU L T S A N D  D ISC U SSIO NBoth 5. dorsalis and  F. schultzei transm itted  TSW V , bu t S. dorsalis was a less efficient vector (Table 1). The virus was detected by the hem agglutination  technique in S. dorsalis in three separate 4«stS4isiB.g-80^MX,-an<L20jiise.cls..ajid.in_£L sc h u ltze i  in a te s t u sing  11 insects. E xtracts from  virus-free S', dorsalis and F. schultzei gave nonspecific agglutination a t dilutions of 1:500 to  1:1,000. The e x tra c ts  p re p a re d  fro m  e x p o se d  S. dorsalis gave hem agglutination  titers of 1:4,000 to  1:8,000 and th a t of F. schultzei gave titers o f 1:4,000.O ur results show th a t S. dorsalis and  F. schultzei can transm it TSW V , the causal agent of bud necrosis disease of peanut. S. dorsalis is a new vector o f TSW V (13), bu t it appears to  be less efficient th an  F. schu ltze i. O ther Sc ir to th r ip s  spp. (S. longipennis (Bagnall), 5. citri M oulton, and S. m an iho ti (B ondar)) have been reported  as crop pests from . different parts of the world (7).F. schultzei has no t previously been reported  on peanut although it occurs in In d ia  (2). H eav y  in fe s ta t io n s  o f F. schultzei were observed ofl’g roundnu t a t H yderabad and o ther areas and may explain the w idespread occurrence of TSW V in India. Peanuts are also infested by C alio th rip s  (H e lio th r ip s ) ind icus  (Bagnall), F. dam pfi Priesner, R etithrips . syriacus M ayet, M egalurothrips distalis K arny (2), Thrips hawaiiensis (M organ) (15), T. fla v u s  Schrank  (Amin, un­published), and T. tabaci (D eshm ukh, personal com m unication). Their role as possible vectors of TSW V, except T. tabaci (13), needs to  be.assessed.Fig. 1. Virus-free thrips colonies: (A) On detached shoot: (B) On a leaflet of peanut.•.■Fig. 2. Hemagglutination test showing positive reaction of: (A) Healthy leaf extract. (B) Extract from leaf infected with tomato spotted wilt virus. (C) Extracts from Scirtothrips dorsalis exposed to infected leaves. (D) Extract from thrips not exposed to infected leaves.664 Plant Disease/Vol. 65 No. 8H em agglutination  tests have been used to  detect TSW V  antigens in p lant extracts (6), and now the test has been applied to detect viral antigen in thrips. E xtracts from  thrips fed on healthy leaves gave h ig h e r  n o n sp e c if ic  h e m a g g lu tin a tio n  titers th an  those from  healthy  peanut leaf extracts. Nevertheless, the titers obtained with exposed colonies were a t least fou r to  eight times higher than  those of extracts from  healthy  thrips. Paliw al (9). dem onstrated  th a t viral antigen could be detected in  th rips by the m icroprecipitin test, using 50 insects a t a  d ilution 1:4.5. We used 11 F. schultzei adults and 20 S. dorsalis adu lts to  detect viral antigens by the hem agglutination test. Experim ents are under way to  ad ap t the test to detect viral antigens in individual thrips.ACKNOW LEDGM ENTSWe are grateful to  W. R. Richards, Biosystematic Research Institute, O ttawa K.IA 0C6, Canada; T. A. Ananthakrishnan, Director, Zoological Survey of India, Calcutta 700 001; and L. A. M ound, British M useum (Natural History), for identifying the thrips.We also thank G. V. Gooding, Jr., for providing TSWV antisera.LITERA TUR E CITED1. Amin, P. W., Reddy, D. V. R., and Ghanekar, A. M. 1978. Transmission o f bud necrosis virus (BNV) of groundnut by chilli thrips, Scirtothrips dorsalis  H ood ( T hysanop tera : Thripidae). (Abstr.) Indian Phytopathol. 31:118.2. A nanthakrishnan, T. N. 1973. Thrips: Biology and Control. Macmillan. India. 120 pp.3. Chohan, J. S. 1974. Recent advances in diseases of groundnut in India. Pages 171-184 in S. P. Rayachaudhari and J. P. Verma, eds. Current Trends in Plant Pathology. Lucknow University, Botany Department, Lucknow. 324 pp.4. D ay, M. F ., and  Irzyk iew icz, H. 1954. s  Physiological studies on thrips in relation totransmission of tom ato spotted wilt virus. Aust. J. Biol. Sci. 7:274-281.5. Ghanekar, A. M., and Nene, Y. L. 1976. A method of assaying groundnut bud necrosis virus. Trop. Grain Legume Bull. 5:39.6. Ghanekar, A. M., Reddy, D. V\ R., Iizuka, N., Amin, P. W., and Gibbons, R. W. 1979. Bud necrosis of groundnut (Arachis hypogaea) in India caused by tom ato spotted wilt virus. Ann. Appl. Biol. 93:173-177.y7. Lewis, T. R. 1973. Thrips: Their Biology, Ecology and Economic Im portance. Academic Press, New York. 349 pp.8. Nariani, T. K., and Dhingra, K. L. 1963. Amosaic disease of groundnut (Arachis hypogaea L.). Indian J. Agric. Sci. 33:25-27.9. Paliwal, Y. C. 1974. Some properties and thrip* transmission of tom ato spotted wilt virus in Canada. Can. J. Bot. 52:1177-1182.10. Rajeshwari, R., Reddy, D. V. R:, and Iizuka, N. ,1981. Improvements in the passive hemagglutina­tion technique for serological detection of plant viruses. Phytopathology. 71:306-308.11. Reddy, M., Reddy, D. V. R., and Appa Rao, A. 1968. A new record of virus disease of peanut. Plant Dis. Rep. 52:494-495.12. Reddy, D. V. R., H ariprasad Rao, M., Padma, M. C., and Gopal Rao, V. 1969. Haemagglutina- tion test to detect tobacco mosaic virus antigens. Indian J. Exp. Biol. 7:162-165.13. Sakim ura, K. 1963. Frankliniella fusca, an additional vector for the tom ato spotted wilt virus, with notes on Thrips tabaci, another vector. Phytopathology 53:412-415.14. Sakimura, K. 1969. A comment on the colour a forms of Frankliniella schultzei (Thysanoptera:Thripidae) in relation to transmission of the to m a to  sp o tte d  w ilt v irus. Pac. Insects 11:761-762.15. Sandhu, G. S., and Brar, K. S. 1977. Insect pests of groundnut and their control. Agric. and Agro- Ind. J. 10(11):7-10.16. Sharm a, D. C. 1976. Studies on bunchy top, chlorosis and ring mottle virus diseases of groundnut (Arachis hypogaea L.). Phytopathol. Z. 57:127-137.Plant Disease/August 1981 665

Transmission of tomato spotted wilt virus, the causal agent of bud necrosis of peanut, by Scirtothrips dorsalis and Frankliniella schultzei

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Transmission of tomato spotted wilt virus, the causal agent of bud necrosis of peanut, by Scirtothrips dorsalis and Frankliniella schultzei

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