Serology is indispensable for the detection

and identification of plant viruses.

Recently, the highly sensitive enzyme-linked

immunosorbent assay (ELISA) and immunosorbent

electron microscopy (ISEM) techniques

have been developed and may replace

some of the conventional serological methods.

The direct or standard double-antibody

sandwich (DAS) form of ELISA. as first

described by Clark and Adams (1977), has

wide applications in plant virology. An

indirect form of ELISA (I-ELISA) has recently

been developed by Barbara and Clark

(1982). The ISEM procedure developed by

Derrick (1973) combines the specificity of

serology with coventional electron microscopy.

DAS-ELISA, I-ELISA and ISEM have

been adapted for the detection and identification

of several groundnut viruses. This paper

will provide a description of each technique

and its application for the detection and characterization

of viruses occurring on groundnut

in India

Bharathan, N

Nolt, B L

Rajeshwari, R

Reddy, D V R

ICRISAT Open Access Repository

Reprinted from Proc. National Sem. Management of Diseases of Oilseed Crops, Madurai, 1983. pp. 1—6. TNSAU, Agric. Coll. & Res. inst. Improved Serological Techniques for the Detection and Identification of Groundnut Viruses B, L. Nol t , R. Rajeshwari, N. Bharathan, and D. V. R. Reddy G r o u n d n u t Improvement Program, Internat ional Crops Research Inst i tute for the Semi-Arid Tropics (ICRISAT), Patancheru P. O. A n d h r a Pradesh 502 324, India. CP 0125 Serology is indispensable for the detec-t ion and identification of plant viruses. Recently, the highly sensitive enzyme-linked immunosorbent assay (ELISA) and immuno-sorbent electron microscopy (ISEM) techni-ques have been developed and may replace some of the conventional serological methods . The direct or standard double-antibody sandwich (DAS) form of ELISA. as first described by Clark and Adams (1977), has wide applications in plant virology. An indirect form of ELISA (I-ELISA) has recen-tly been developed by Barbara and Clark (1982). T h e ISEM procedure developed by Derrick (1973) combines the specificity of serology with coventional electron micros-copy. DAS-ELISA, I-ELISA and ISEM have been adapted for the detection and identifica-tion of several groundnut viruses. This paper will provide a description of each technique and its application for the detection and char-acterization of viruses occurring on ground-n u t in India . M A T E R I A L S A N D M E T H O D S M a i n t e n a n c e o f v i r u s e s . Cowpea mild mott le (CMMV), peanut clump (PCV), peanut mott le (PMV) and tomato spotted wilt (TSWV) viruses were all isolated from groundnut in India a n d maintained on the following hosts : C M M V on soybean, PCV on Nicotiana glutinosa X N. Clevelandii ( N . hybrid), PMV on peas and TSWV on groundnut. A n t i s e r u m p r o d u c t i o n . I n the s tandard procedure, 1.0 ml of purified virus (ca. 1 mg/ ml concentrat ion) was emulsified with an equal volume of Freund ' s incomplete adjuvant and injected intramuscularly into New Zealand White rabbits at weekly intervals. Serum was collected 2 weeks after the fourth injection and t i t red by the precipit in ring (est. The titres for ant i sera used in this study were C M M V 1/2560, PCV 1/1280, PMV 1/2560 and TSWV 1/256. - g l o b u l i n e x t r a c t i o n a n d e n z y m e c o n -j u g a t i o n . The extract ion and conjugation of -globulins ( l g G ) was similar to that des-cribed by Clark and Adams (1977) a n d Lister (1978). The r-globulins were pre cipitated from crude serum with 18% sodium sulphate, washed with phosphate-buffered saline (PBS) and then dialyzed against PBS to remove residual sodium sulphate. E x t r a -cted -globulins were adjusted to a 1 mg/ml concentrat ion ( A 2 8 0 = 1.4) and conjugated with alkaline phosphate (type VII, Sigma Chemical C o . ; a t an enzyme to -globulin ratio 'of 2 : I. P r e p a r a t i o n o f i m m u n o g l o b u l i n f r a g -m e n t s . F(ab ' ) 2 fragments of I g G were prepared by pepsin digestion as described by Barbara and Clark (1982). IgG was adjusted to a 1 mg/ml concent ra t ion with 0.07 M sodium acetate buffer, pH 4.0, containing 0.05 M NaCl. Pepsin (Sigma 1 : 10,000) in distilled water, was added to a final concentrat ion of 45 μg/mg I g G a n d incubated overnight at 37°G. F ( a b ' ) 2 frag-ments were separated from excess enzyme and digest ion breakdown products by dialysis against three changes of PBS. DAS-ELISA P r o c e d u r e . T h e method emplo-yed was similar to that described by C l a r k and Adams (1977) a n d Lister (1978) ( F i g . l . ) . In the first s tep extracted -globulins (coating antibodies) were adsorbed to the surface of wells in a special microt i t re plate (Dynatech Laboratories) . Following incubation, any excess unadsorbed coat ing ant ibodies were removed from the well by thorough washing. Next was added the test sample which was a crude leaf, insect or seed extract or purified vi rus . If the test sample contained viral antigens serologically related to the coating ant ibody, they would be bound to the well surface by the coa t ing antibodies. After washing away any unbound material , trapped viral antigens were detected by adding alkaline phosphatase - label led- - globulins (detecting antibodies) . The -globulins used for labelling were the same as the coating antibodies. If viral antigens were bound to the surface of the well by the coating ant ibodies the detecting antibodies would bind to the trapped antigens. The presence of the enzyme-labelled detecting antibody can be assayed by the addit ion of the substrate p nitrophenyl phosphate (Sigma Chemical Co ). The product of the enzyme-substrate reaction is yellow in colour and can be assessed visibly or quant i ta t ively at A4o5 in a spectrophotometer since colour intensity is propor t ional to the virus concentration. I-ELISA procedure. The I-ELISA method employed was similar to that described by Barbara and Clark (1982) (Fig. 2) The surface 2 Fig. 1 DAS-ELISA Procedure Coat wells with -globulins (coating antibodies) Incubate 3 hrs at 37 C wash Add sample extracts to the coated wells Incubate overnight at 4 C wash Add, -globulins conjugated with the enzyme alkaline phosphatase (detecting antibodies) Incubate 3 hrs at 37 C wash Add enzyme substrate p-mtrophenyl phosphate Incubate 30 min at room temperature add 3N NaOH yellow colour = positive reaction Measure absorbance at 405 nm Fig. 2 I - ELISA Procedure Coat well with F (ab1) fragments (coating antibodies) Incubate 3 hrs at 37 C wash Add sample extracts to the coated wells incubate overnight at 4 C of wells in microti tre plates was coated with F ( a b 1 ) 2 fragments (coating antibodies . Test samples were added to the well after washing. If viral antigens in the test sample were related to the F (ab l ) 2 fragments coating the well they would be retained after washing. Next , unlabelled intact IgG(detecting antibo-dies) were added which would bind to trapped viral antigens. Crude serum can be used for detecting antibodies. The presence of the unlabelled detecting antibody is deter-mined by adding enzyme-labelled an t i - rabbi t r-globulins produced in goats which are specific for the Fc por t ion of the detect ing ant ibody The conjugated Fc - specific an t i -rabbi t r-globulins are commercially available (Cappel Laborator ies , USA) I m m u n o s o r b e n t e l e c t r o n m i c r o s c o p y procedure (ISEM). The method used is similar to that described by Derr ick (1973) and Milne and Luisoni (1977) (Fig. 3) , 3 wash Add crude serum or r - globuiins (detecting antibodies) incubate 3 hrs at 37 C wash Add commercially available F - specific anti-rabbit r-globulins conjugateed with the enzyme alkaline phoshatase Incubate 3 hr at 37 C wash Add enzyme substrate p - ni trophenyl phosphate Incubate 30 min at room temperature add 3N NaOH yellow colour = positive reaction Measure absorbance at 405 nm Fig. 3 ISEM Procedure Coat carbon-formvar grids by floating on drops of antiserum Incubate 5 min at room temperature wash with 20 drops 0.1 M K P O 4 , pH 7. 0 F loa t coated grids on drops of sample extract Incubate 15-30 min at room temperature wash with 40 drops distilled water Stain grids with 6 drops 2% aqueous uranyl acetate Formvar-coated copper grids were made hydrophi l ic by exposure for 10 sec to a carbon glow discharge. The grids were coated with virus-specific r-globulins by floa-ting film-side down on drops of ei ther crude ant iserum or extracted r-globulins. After washing away excess r -g lobul ins with drops of phosphate buffer, the coated grids were placed film-side down on drops of the test sample. If viral antigens in the test sample are serologically related to the coating antibodies, they will be bound to the surface of the grid. Any unre la ted , there-fore unbound, viral antigens or host plant const i tuents were washed from the surface of the grid with distilled water. The washed grids were negatively stained with 2% aqueous uranyl acetate and examined for the presence of trapped virus particles with the electron microscope. RESUSTS A N D DISCUSSION Virus d e t e c t i o n in s e e d us ing DAS -ELISA. The DAS-ELISA procedure has been adapted for rapid screening of large numbers of g roundnut seed samples for the presence of PMV Since P M V . is seed-transmitted in groundnuts , it is impor tan t to identify non-seed-transmitted lines. However, this requires the assaying of thousands of seeds each season which would be difficult with conventional serological techniques or by bioassaying for virus on indicator hosts . DAS-ELISA is sensitive enough to detect virus antigen up to a l0ng/ml concentrat ion ; therefore only a small por t ion of an individual seed is required for the assay. The test is reliable even when portions from five different seeds are assayed together in the same well. By testing five seeds/well, over 2500 seeds can be assayed in a single day. Every single seed which con-tained viral antigen as determined by ELISA also transmit ted the virus in growing-on tests. Several germplasm lines have been identified which do not t ransmit PMV through seed. Virus d e t e c t i o n in i n s e c t v e c t o r s us ing DAS-ELISA. The DAS - ELISA test can detect the presence of TSWV in groups of 5-10 thrip insects. The test can be used in studying the epidemiology of the thr ip-transmitted bud necrosis disease. The th r ip population can be sampled throughout the groundnut growing season for the presence of viruliferous thrips. This facilitates scheduling of chemical control measures at the t ime when the proport ion of viruliferous insects in the population reaches a critical level. Determin ing s e r o l o g i c a l re la t ionships . DAS-ELISA, I-ELISA and ISEM tests have been employed for determining the serologi-cal relationships of C M M V , PCV and PMV. All three tests were used to establish that CMV is related to carnat ion latent, chrysanthemum B, dulcamara, helenium S, hop mosaic and po ta to virus M viruses; but unrelated to lily, symptomless and po ta to virus S viruses Table 1. T a b l e 1 . T h e r e l a t i o n s h i p o f C M M V with o t h e r c a r l a v i r u s e s a s d e t e r m i n e d b y D A S - E L I S A , I - E L I S A a n d I S E M A n t i s e r a t e s t e d C a r n a t i o n l a t e n t C h r y s a n t h e m u m 8 C o w p e a m i l d m o t t l e D u l c a m a r a H e l e n i u m S H o p m o s a i c L i l y s y m p t o m l e s s P o t a t o V i r u s M P o t a t o v i r u s S D A L - E L I S A % R e l a t i v e a f f i n i t y 3 5 3 0 100 18 34 2 3 N o n e 24 N o n e I - E L I S A % R e l a t i v e a f f i n i t y 58 59 100 42 59 55 N o n e 67 N o n e I S E M % R e l a t i v e a f f i n i t y 4 2 34 100 29 31 35 N o n e 74 N o n e 4 Interestingly, these serological relationships could notbe established using the conventional microprecipitin test. DAS-ELISA and ISEM were used to show a serological relationship between PMV and several members of the potyvirus group. PMV is related to adzuki bean mosaic (ABMV), amaranthus leaf mot t le (ALMV), bean common mosaic (BCMV), clover yellow vein (CYVV), and soybean mosaic (SMV) viruses, but unrelated to groundnut eye spot, peanut green mosaic , pepper veinal m o t t l e , potato virus y, sugarcane mosaic and tu rn ip mosaic viruses. (Table 2.) These serological relationships could not be detected with the agar gel diffusion and microprecipi t in tests. Table 2. The relationships of PMV with other potyviruses as determined by DAS-ELISA and ISEM. Antisera tested Adzuki bean mosaic Amaranthus leaf mottle Bean common mosaic Clover yellow vein Groundnu t eye spot Peanut green mosaic Peanut mottle Pepper veinal mot t l e Potato virusy Soybean mosaic Sugarcane mosaic Turnip mosaic DAS-ELISA % Relative affinity 19 27 16 23 None N o n e 100 None None 20 None None ISEM % Relative affinity 68 56 17 94 N o n e None 100 N o n e 12 86 N o n e None Using I-ELISA and ISEM,no serological relationship could be detected between PCV and antisera to any of the soil-borne nematode-transmitted (tobacco ratt le P R N and C A M strains and pea early browning) and Polymyxa - transmitted (barley yellow mosaic, beet necrot ic yellow vein, rice necrosis mosaic, wheat mosaic and wheat yellow mosaic) viruses tested. Interestingly, PCV reported from West Africa is not sero-logically related to the PCV occurring in India. Studying morpholog ica l [character is t ics . The length distribution of PCV particles was established with the aid of ISEM. Insufficient numbers of particles are adsorbed on conven-tional leaf dip grids for accurate length measurements. However, by pre-coat ing grid with PCV antiserum over a 100-fold increase in the particle count is observed, thus facilitating an accurate assessment of the particle length distr ibution. The width of the PCV particle was determined to be 24 + 2 nm with modal lengths of 249 + 8 nm and 184 + 4 nm. DAS-ELISA has proved to be a sensitive and rapid test for the detection of g roundnut viruses in a large number of test samples. The test is especially amenable to screening seeds and insect vectors for the presence of viruses. As a result of successful app l ica t ion of DAS-ELISA for the detection of PMV in seed, several hundred seed samples can now be tested at one t ime for the presence of 5 PMV, thus facilitating the identification of n o n seed-transmit t ing genotypes and providing a useful tool for p lan t quarant ine programs. Breeding for high yielding, non seed-transmitt ing cultivars would greatly reduce the spread of PMV in the field. DAS-ELISA has also been used to detect TSWV in the thr ip vector of the economically impor tant bud necrosis disease The test can be employed to assess the proport ion of viruliferous insects in field samples. The I -ELISA test is an improvement over the conventional DAS-ELISA technique because higher dilutions of the coating and detect ing antibodies can be used thus con-serving antisera. Fur thermore I-ELISA obviates the need for a conjugated detecting antibody, DAS-ELISA, I -ELISA and ISEM have successfully been used to study the serological relationships of the carlaviruses, potyviruses and PCV. However, I-ELISA has been shown to detect broader serological reactions than DAS-ELISA and ISEM. REFERENCES 1. Barbara, D. J. and M. F. Clark, 1982. J. gen. Virol. 58 : 315-322, 2. Clark, M. F. and A. N. Adams, 1977. J. gen. Virol . 34 : 475-483. 3. Derrick, K. S. 1973. Virology 56 : 652-653. 4. Lister, R. M. 1978. Phytopathology, 68 : 1 3 9 3 - 1 4 0 0 . 5. Milne, R. G. and E. Luisoni, 1977. In Methods in Virology, K. Maramorosch and M. Koprowski , eds. Vol. 6: 265-281. Academic Press, New York and London. 6 

Improved Serological Techniques for the Detection and Identification of Groundnut Viruses

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