PURCIFULL & ZITTER: TEST FOR 143

2. Batiste, William C, Ray C. King and John Joos. 1970. Bui. 841:1-29. Field and laboratory evaluations of insecticides for control 5. Poe, S. L. 1973. The tomato pinworm in . of the tomato pin worm. J. Econ. Entomol. 63:1479-1486. Bradenton, AREC. Rept. GC1973-2. 4 p. mimeo. 3. Campbell, R. E. and J. C. Elmore. 1931. Damage to 6. Swank, George R. 1937. Tomato pinworm (Gnorimo- tomatoes in southern California by the tomato pinworm and schema lycopersicella (Busck)) in Florida. Florida Entomol. the potato tuber moth. Calif. Dept. Agric. Monthly Bulletin 20:33-42. 20:458-460. 7. Watson, J. R. and W. L. Thompson. 1932. Pinworm on 4. Elmore, John C. and A. F. Howland. 1943. Life History tomatoes. Fla. Entomol. 36:14. and control of the tomato pinworm. U. S. Dept. Agric. Tech.

A SEROLOGICAL TEST FOR DISTINGUISHING MOTTLE AND LETTUCE MOSAIC VIRUSES

D.E. PURCIFULL filamentous, with particles about 720-750 nm long (3, 12). The two viruses have different host ranges IF AS Plant Pathology Department and LMV is seed-borne in lettuce, whereas there Gainesville is no evidence that BMV is seed-borne. A rapid and means of distinguishing these two viruses in field T. A. Zitter camples was needed to evaluate and implement con trol measures. Serology was tested because of its IF AS Agricultural Research and Education Center general success for diagnosing other plant viruses Belle Glade (10, 11, 13), and because of preliminary tests which indicated that LMV and BMV are serolo- Abstract Lettuce mosaic (LMV) and gically distinct (5; Purcifull and K. A. Kimble, bidens mottle virus (BMV) both cause serious unpublished). mosaic diseases of lettuce and endive in Florida. To expedite identification of the viruses in field Materials and Methods samples, a rapid, specific assay technique was needed. For this purpose, antisera to each virus An isolate of LMV obtained from commercial were produced in rabbits and serological tests seed was used for preparation of antisera. This were conducted in agar gels. The BMV antiserum isolate was compared serologically to the American reacted positively with extracts from BMV-in- Type Culture Collection (ATCC) isolate of LMV fected plants, and the LMV antiserum reacted (PV-63) and to various field isolates from lettuce with extracts from LMV-infected plants, but and endive. For routine culture LMV was propa neither antiserum cross-reacted. The serological gated in garden pea (Pisum sativum L. cv. Little tests have been used to detect three new weed Marvel). For purification, the virus was cultured hosts of BMV, and to further assess the im either in pea or in Chenopodium quinoa Willd. portance of LMV and BMV in lettuce and endive Most of the work with BMV was donewith the production. ATCC isolate (PV-165) cultured in a Nicotiana hybrid (2). Virus diseases of lettuce (Lactuca sativa L.) LMV and BMV were purified by routine virus and endive (Cichorium endivia L.) are among the purification methods, involving clarification of leaf most important limiting factors in producing these homogenates withchloroform or n - butanol; pre crops in Florida (6). Two major viruses have been cipitation of virus with polyethylene glycol, fol implicated thus far: lettuce mosaic virus (LMV), lowed by differential and density-gradient cen- and bidens mottle virus (BMV). trifugation (Purcifull, E. Hiebert, and S. Christie, LMV and BMV are distinct, although they have unpublished). several properties in common. They both cause An antiserum to LMV was prepared by inject mosaics, mottling, and stunting of lettuce and en ing a rabbit intramuscularly with purified or pyrro- dive; they are borne (3, 5, 12), and both are lidine treated virus (11) emulsified 1:1 with Freund's incomplete adjuvant. Oneml of virus was injected initially, followed six weeks later by an Florida Agricultural Experiment Stations Journal Series No. 5132. injection of pyrrolidine-treated virus (1 ml)7 with We greatly appreciate the assistance of S. R. Christie and W. E. Crawford. a final injection ofpurified virus (0.5 ml) being 144 FLORIDA STATE HORTICULTURAL SOCIETY, 1973 administered 2 months after the first injection. Sera used in this study were collected perodically during a 3 month interval, starting 4 months after the first injection. Antigens from healthy pea tissue for absorption of the LMV antiserum were prepared as described elsewhere (7). Host proteins were mixed with antiserum (1:4, v/v) and incubated overnight at 4 C. The mixture was centrifuged at 66,000 g for 1 hour, and the supernatant containing the anti- sera was freeze-dried and reconstituted with de- ionized water prior to use. Fig. 1. Immunodiffusion patterns showing serodiagnosis The antiserum to BMV was prepared by in of lettuce mosaic (LMV) and bidens mottle viruses (BMV) in lettuce and endive. Specific precipitation lines appear as jecting a rabbit intramuscularly with 3 ml of white bands between certain peripheral wells and the center wells. The center wells were filled with LMV antiserum (L), purified virus (emulsified 1:1 with incomplete ad or BMV antiserum (B). The peripheral wells contain deterg ent treated extracts from: (1) BMV-infected lettuce; (2) juvant). Sera collected 4 to 5 months after the in LMV-infected lettuce; (3) BMV-infected endive; (4) LMV- jection were frozen and lyophilized until ready for infected endive; (5) Healthy lettuce; and (6) Healthy endive.

use. Immunodiffusion tests were done in Petri from BMV-infected plants, and not with extracts dishes in Noble agar (0.8%) containing sodium either from healthy plants or from plants infected azide (1.0%) and 0.5% sodium dodecyl sulfate with LMV alone. BMV was detected serologically (4), a detergent. Leaves to be tested were ex in lettuce, endive, the Nicotiana hybrid, C. quinoa, tracted by grinding tissue in water (g/ml), and sunflower (Helianthus annuus L.), zinnia (Zinnia then adding 1 ml of 3% sodium dodecyl sulfate/g elegans Jacq.), Bidens pilosaL., horseweed (Erig- tissue (7). The sap was expressed through cheese eron canadensis L.), american burnweed (Erech- cloth and added to reactant wells, which were tites hieracifolia (L.) Rab.) and Mexican prick- punched in the agar with a gel cutter (Grafar lepoppy ( L.). The latter three Corp., Detroit). Occasionally samples treated with species are reported as hosts of BMV for the first sodium dodecyl sulfate were lyophilized and re- time. suspended in water prior to use. Antisera were To assess the reliability of the serological tests added to central wells, and the plates were in for diagnosing the viruses in field samples, 31 let cubated 2 days at 25 C in a moist chamber. tuce and endive samples were indexed both by as Inocula for bioassays were prepared by grind say to indicator plants,and by serology. Each of ing leaf tissue in 0.5% sodium sulfite. A small the 23 samples which reacted positively with BMV amount of Celite was added as an abrasive, and antiserum also infected the Nicotiana hybrid inocula were applied to the leaves of test plants and/or sunflower, as is typical for BMV isolates with gauze pads. (3, 5). In turn, Nicotiana hybrids and sunflowers infected with field isolates reacted only with BMV Results antiserum. Of the 8 samples which failed to react Serological tests showed that the LMV and with BMV antiserum, none infected either the BMV antisera were highly specific and could be Nicotiana hybrid or sunflower. used for virus identification in naturally and ex Nine of the 31 samples infected pea and re perimentally infected samples. The LMV anti acted positively for LMV antiserum. Another 16 serum (Fig. 1, well L) reacted only with extracts samples reacted negatively by both techniques. Of from plants infected with LMV, but not with ex the remaining 6 samples, two reacted positively by tracts from healthy plants or from plants infected serology but failed to infect pea; the other four with BMV alone. LMV was specifically detected in reacted negatively by serology but did infect pea, lettuce, endive, zinnia, ( Jacq.), and were assumed to.be LMV based on symp garden pea, and C. qninoa. Immunodiffusion tests tomatology. • -:r showed that the ATCC isolate and field isolates Some lettuce and endive samples indexed posi gave identical reactions to the isolate used for im tively by serology for both LMV and BMV. These samples infected both pea and the Nicotiana hy munization. The BMV antiserum (Fig. 1, well B) also was brid, and were assumed to be doubly infected. highly specific, as it reacted only with extracts A total of 61 lettuce plants (iceberg, Boston, PURCIFULL & ZITTER: TEST FOR LETTUCE VIRUSES 145 and romaine types) from Belle Glade-South Bay symptoms in the lettuce cultivar "Gallega", a farms indexed positive by serology; 26 were in variety which carries tolerance to LMV (1, 9); fected with BMV, 28 with LMV, and 7 more were and three new natural weed hosts of BMV were doubly infected. Of 38 escarole samples from the found (American burnweed, Mexican pricklepoppy, Zellwood area, 30 were infected with BMV, 3 with and horseweed). BMV appears to be a threat to LMV, and 5 more indexed positive for both viruses. lettuce and endive production, and because it in fects several common weeds and is widespread, it Discussion may prove difficult to control. On the other hand, a significant advance toward the control of LMV The serological techniques described herein pro has been made during the past year as a result of vide a simple means for distinguishing BMV from establishing regulations requiring that lettuce seed LMV in field samples within 24-48 hours. The only planted in designated areas in Florida must be in other technique currently available for distinction dexed as 0 in 30,000 for LMV (8). of LMV from BMV is by assaying samples in in dicator plants, and it takes about two weeks to obtain results by this method. The serological tech Literature Cited nique was as reliable as the use of indicator plants 1. Bannerot, H., L. Boulidard, J. Marrou, and M. Duteil. 1969. Etude de '1 heredite de la tolerance au virus de , la for diagnosing BMV. Both methods usually gave mosaique de la laitue chez la variete gallega de invierno; positive results for LMV in field samples, al Ann. Phytopathol. 1:219-226. 2. Christie, S. R. 1969. Nicotiana hybrid developed as a though neither technique was sufficiently sensitive host for plant viruses. Plant Disease Reptr. 53:939-941. 3- , J. R. Edwardson, and F. W. Zettler. 1968.' to always detect this virus. There are various pos Characterization and electron microscopy of a virus isolated sible reasons for the differences in results obtained from Bidens and Lepidium. Plant Disease Reptr. 52:763-768. 4. Gooding, G. V., Jr., and W. W. Bing. 1970. Serological by the two methods with regard to LMV, but they identification of potato virus Y and tobacco etch virus using immunodiffusion plates containing sodium dodecyl sulfate are probably due to the fact that infectivity can Phytopathology 60:1293. be more sensitive for virus detection than serology 5. Purcifull, D. E., S. R. Christie, T. A. Zitter, and M. J. Bassett. 1971. Natural infection of lettuce and endive by (10), to tissue sampling errors, and to the ap bidens mottle virus. Plant Disease Reptr. 55:1061-1063. 6. , D. E.; and-T; A; Zitter. 1971. Virus diseases parently low concentration of LMV in lettuce (12). affecting lettuce and endive in Florida. Fla. State Hort. Soc. It is concluded that the serological tests are pre Proc. 84 :165-168. 7. , D. E., E. Hiebert, and J. G. McDonald. ferable to the use of indicator plants for routine 1973. Jmmunochemical specificity of cytoplasmic inclusions induced by viruses in the potato Y group. Virology 55:275- distinction of BMV and LMV, since serology re 279. quires much less time and space to perform. The 8. Rules of the Florida Department of Agriculture, Di vision of Plant Industry, Chapter 5B-38, Supp. No. 29, 1973, indicator plant method, however, is still a useful "Lettuce mosaic". 9. Ryder, E. J. 1970. Screening for resistance to lettuce confirmatory technique. mosaic. HortScience 5:47-48. Using serodiagnosis, some important new in 10. Sampson, P. J., and R. H. Taylor. 1968. A comparison of the electron microscope, microprecipitation tests, and in formation about the virus disease complex in let dicator plants for the detection of potato viruses S, X, and Y. Phytopathology 58:489-493. tuce and endive" has been obtained: (i) BMV ap 11. Shepard, J. F. 1970. A radial immunodiffusion test pears r. to be a more significant problem in both for the simultaneous diagnosis of potato viruses S and X. Phytopathology 60:1669-1671. crops than was previously surmised (6); (ii) in 12. Tomlinson, J. A. 1970. Lettuce mosaic virus. Com monwealth Mycological Institute Description of Plant Viruses dividual plants can harbor infection by both viruses No. 9. at the same/time; (iii) BMV causes mottling 13. Wetter, C. 1965. Serology in virus-disease diagnosis. Annu. Rev. Phytopathol. 3:19-42.