Latex Agglutination as a Rapid Detection Assay for Spiroplasma citri

Antiserum production. Antiserum was Fletcher, J., and Slack, S. A. 1986. Latex agglutination as a rapid detection assay for Spiroplasma produced in New Zealand white ra 3 citri. Plant Disease 70:754-756. against Illinois horseradish isolates and BR6 as described (5). Antiserum titer The latex agglutination test (LAT) was effective in detecting the wall-less mollicute Spiroplasma was 1: 16,000 by the spiroplasma citri. The minimum detection level in culture medium or turnip samples enriched with spiroplasma deformation test (18). cells was 107 cells per milliliter. Cross-reactions were not detected with corn stunt spiroplasma, LAT. The LAT was performed as honeybee spiroplasma strain AS576, or flower surface spiroplasma strain 23-6. S. citri was described by Khan and Slack (10) and accurately detected in preparations of some plant species, with negative results from uninfected modified by Slack et al (16). Antiserum controls. However, false positives occurred in other uninfected plant species, especially weeds. was serially diluted in twofold increments Spiroplasmas were detected in single leafhoppers (Circulifer tenellus) as well as in groups of 3, 5, 10, 25, and 41 insects, but false positives were also detected (in about 25% of cases) with healthy (1:100-800) in 100 mM Tris (hydroxyleafhoppers. LAT can be used as a rapid and simple qualitative assay for detecting and identifying methyl) aminomethane-HCd (Tris), pH S. citri in culture; its application to experimental uses with infected plant or insect tissue is limitedto 7.4, and conjugated with latex (polythose for which stringent controls are available, styrene) spheres 0.794 gm in diameter, 10% concentration, w/v (Sigma) diluted 1:50 (v/v) in 0.85% NaCl. Sensitized latex Since their discovery as a distinct type S. citri in cultured samples and many was washed twice in 0.02% polyvinylof microorganism in 1972 (3), spiroinfected plant species. False-positive reacpyrrolidone (mol wt 40,000) in 0.85% plasmas have been detected in plant hosts tions with some plant species limit its use NaCl and resuspended in Tris containing and insect vectors by symptomatology, to previously tested species or those for 0.02% sodium azide. Bovine serum light and electron microscopy, remission which adequate controls are available. A albumin (0.4%, w/v) was added to the of symptoms after tetracycline treatments, preliminary report has been published (7). suspension to reduce nonspecific backcultivation in artificial medium, and ground. Samples were tested in capillary serology. The latter approach has proven MATERIALS AND METHODS tubes by rotating at seven cycles per to be the most specific and reliable. Sources and maintenance of spirominute for 15 min before scoring. Enzyme-linked immunosorbent assay plasma cultures and infected tissues. S. For each assay, samples were tested (ELISA) has been shown to be reliable citri isolate BR6 was originally cultivated with two to four antiserum dilutions of and sensitive for detection of Spiroplasma from Illinois horseradish affected by sensitized latex and reactions were rated citri (1) and the corn stunt spiroplasma brittle root disease (8), and spiroplasma visually with a dissecting microscope on a (4) and useful for quantifying the strains AS576 and 23-6 were kindly scale of 0-5 (0 = no agglutination, 5 = pathogen in specific tissues (5,15). provided by R. E. Davis (USDA/ARS, complete agglutination). Values Ž2.0 However, a faster and simpler assay Beltsville, MD). These spiroplasmas were were considered positive (a rank of 1.0 would be welcome, especially where small maintained in LD8 broth (11) at 31 C. indicates clouding of the background, 2.0 numbers of samples are being evaluated Corn stunt spiroplasma was isolated represents clear agglutination). Positive and spiroplasma detection is the primary from infected corn leaves supplied by L. (S. citri cultures) and negative (no concern. R. Nault (Ohio Agricultural Research antigen) controls were included in each The latex agglutination test (LAT) has and Development Center, Wooster, OH) test. been reported to be a useful detection and was maintained under anaerobic Test sensitivity was determined by method for plant-pathogenic viruses conditions (Gas-Pak Anaerobic Jar, evaluating S. citri dilution series against (9,10,13,14) and bacteria (16). In this Baltimore Biological Laboratories, dilutions of sensitized latex. S. citri paper, we demonstrate that LAT is a Cockeysville, MD) at 31 Cin C3-G broth concentrations were determined by rapid, sensitive, and simple serological (2). Infected and healthy plant materials dilution plating on agar-solidified LD8. test for detecting the wall-less mollicute tested included horseradish (Armoracia Test antigens were prepared either rusticana Gaertn., Mey., & Scherb.), from spiroplasmas maintained in liquid turnip (Brassica rapa L.), shepherd's (LD8 or C3-G) culture, from leaf tissue of purse (Capsella bursa-pastoris (L.) plants, or from whole-body extracts of Journal Series No. 4915, Oklahoma Agricultural Medic.), wild mustard (B. kaber (DC.) insects. From culture, spiroplasma Experiment Station, Oklahoma State University, L. C. Wheeler), yellow rocket (Barbarea suspensions were diluted twofold in 0.1 Stillwater. vulgaris R. Br.), China aster (Callistephus M Tris, p H 7.4, containing 0.85% NaCl1 Mention of a trademark, proprietary product, or chinensis Nees), and periwinkle and 0.1% sodium azide (TBS). In cellvendor does not constitute a guarantee or warranty of (Catharanthus roseus (L.) G. Don), washing experiments, cultures were the product by Oklahoma State University and does prvided by C.EEat n(Iloi peeedt1450×gfr2mnad not imply its approval to the exclusion of other pr.E.Esmn(lios eltdat1,0Xgfr20m ad products or vendors that may also he suitable. Natural History Survey, Champaign, resuspended in an equal volume of LD8. IL), common bean (Phaseolus vulgaris Washing was repeated twice. Cells were Accepted for publication 4 March 1986 (submitted L.), a weed (Macroptilium lathyroides counted by direct dark-field microscopic for electronic processing). (L.) Urban) supplied by S. Haber observation. Plant leaf material (0.3 g) _____________________________ (University of Illinois), and a number of was minced with a sterile scalpel in 1-2 ml The publication costs of this articleweredefrayed in part weed samples collected from horseradish TBS except where indicated and either by page charge payment. This article must therefore be field borders in southern Illinois (Table ground with a mortar and pestle or hereby marked "advertisement" in accordance with 18 U.S.C. § 1734 solely to indicate this fact. 1). Infected and healthy beet leafhoppers homogenized using an electric tissue (Circuifer tenellus Baker) were supplied homogenizer (Rotary Mechanical © 1986 The American Phytopathological Society by C. E. Eastman. Homogenizer Model TSO-45, Heat 754 Plant Disease/Vol. 70 No. 8 System-Ultrasonics, Inc., Plainview, eliminate the false-positive reactions. turnip (B. rapa), which reach 10'°-10" NY). Samples were clarified for 2.5 min in When applied to individual leafhoppers cfu/g as described by Fletcher and a Microfuge (Beckman Model 11, (C. tenellus) fed on infected turnip plants, Eastman (5). Soluble antigens, which can Beckman Instruments, Palo Alto, CA). the test was positive for 19 of 36 samples. increase detection sensitivity considerably For some experiments, known quantities When leafhoppers were bulked in groups (S. A. Slack, unpublished), were not of cultured spiroplasma were added to of 3, 5, 10, 25 and 45, the ratios of detected in the cell-washing experiment. healthy plant samples before mincing to positives to numbers of samples tested Normal agglutination did not occur when simulate the effect of using infected were 12/20, 10/11, 6/11, 2/2, and 1/1, other spiroplasmas, even those in the samples. Leafhoppers, which were respectively. However, false positives same serogroup, were tested. provided frozen at -20 C, were placed (agglutination occurring on samples of We found the test, when used on plant singly or in groups into 0.25 ml of TBS, leafhoppers fed only on uninfected food samples, to be sensitive and dependable homogenized with the electric tissue plants) were frequent. In bulked groups for detecting S. citri in turnip, periwinkle, homogenizer, and clarified. Blind tests of 1, 3, 5, 10, 25, and 41, these uninfected and several weed species, with no were performed in which one investigator insect samples were positive, respectively, agglutination in healthy samples from prepared coded samples and a second in 4/16, 3/11, 4/10, 1/6, 0/2, 0/1, and these species. However, one cultivated investigator evaluated these samples. 0/1 cases. horseradish plant and several weed species, apparently not infected with S. RESULTS DISCUSSION citri, gave a positive agglutination. A number of serological procedures Spiroplasmas could not be cultured from LATm waslucessful ind etecing tissu.c have been used successfully to detect any of these plants. Nonspecific serological from culture medium and living tissue. Optimum antiserum dilutions varied spiroplasmas in plant and insect tissues. reactions are not unique to LAT; among preparations but were generally Each is useful in circumstances determined horseradish and many of the same weed best at 1:400-1:600 for about 8 wk after by factors such as need for speed, reliaspecies also produced false positives in preparation and 1:200-1:400 after 8 wk. bility, sensitivity, or lack of complexity. ELISA using IgG prepared from the Sensitized latex was stored at 4 C. Based LAT, though slightly less sensitive than same antiserum (6). Other researchers on four tests in which twofold dilutions of ELISA (13,14), has been used to detect (12) have also reported false positives in cultured material were tested, the plant viruses in plants (14) and insects ELISA when testing for the presence of minimum detection level for S. citri, (13). Slack et al (16) developed the test as viruses in many of the same weed species. using the optimum antiserum preparation, a diagnostic assay tool for detecting C. Thus LAT, like other serological tests, was 10 cells per milliliter. Washing cells sepedonicum, the bacterial pathogen should be used only on acceptable plant had no effect on detection levels, causing ring rot of potatoes. species or those for which adequate Test specificity was evaluated by We have shown that LAT is useful in controls are available. examining reactions of other spiroplasmas detecting the wall-less mollicute S. citri The LAT was tested on only one broth culture. Illinois horseradish under some conditions. Using latex beads leafhopper species, C. tenellus. Single frot sensitized with antiserum against S. citri, leafhoppers fed on infected turnip plants isolates of S. citri could not be distinguished from each other by the test. a minimum of 107 cfu/ ml was required for were rated positive in 53% of the cases, No agglutination was detected with the detection of this spiroplasma. This level is and detection success generally increased corn stunt spiroplasma, which is in the well below the peak titers of S. citri in as the number of insects in composite same serogroup as S. citri (17). With honeybee spiroplasma (strain AS576), also in the same serogroup as S. citri, Table 1. Use of latex agglutination for detection of Spiroplasma citri in plants occasional low agglutination values Samples positive/ (0.5-1.5) were recorded, but these values samples tested are not considered positive. Spiroplasma Plant Conditiona (no.) Mean av.b strain 23-6 from flower surfaces, which belongs to a different serogroup, caused Infected 3/3 3.6 significant agglutination in some tests but Wild mustard Healthy 0/2 0.0 not in others. The precipitant was Infected 3/3 3.2 associated with old cultures, and because Yellow rocket Healthy 0/5 0.0 it looked very different from the Infected 4/5 2.0 homologous reaction, it would not be Turnip Healthy 0/ 8c 0.4 mistaken for a normal positive reaction. Infected 6/6 2.8 Latex agglutination proved effective Horseradish Healthy 1/ 3d 1.5 with some limitations for detecting S. Infected 10/11 4.1 citri in infected plant hosts (Table 1). Periwinkle Healthy 0/8 0.0 Po i i e( 2 0 e e a s g e oInfected 11 /11 3.2 Poiieratings (>20weeasgdto Aster Healthy 0/2 0.3 almost all plants designated infected by Infected 4/4 4.0 visual evaluation and/or by ELISA or Weedse Field-collected, spiroplasma isolation. However, false presumed healthyf 8/15 1 .5g positives (agglutination with samples aConditionof plants was determined by visual rating and/or by ELISAor isolationof spiroplasmas. from which spiroplasmas had not been bMean agglutination value at latex dilution of 1 :400, 1:600, or 1:800, from a scale of 1-5, where 0= recovered in isolation attempts) occurred no agglutination and 5.0 = maximum agglutination. with one turnip sample when concentrated C Healthy turnip samples gave positive reactions on three occasions when a 1:200 dilution of latex (1:200) latex was used, with one of three was used; this dilution was later dropped. healthy horseradish leaf samples, and dA false-positive reaction occurred at a latex dilution of 1:400. with more than half of the weed samples e Ten weed species were collected adjacent to horseradish fields in southern Illinois, and each species collected from the field. Adding 0.2% was separately tested for S. citri. Included were Plantago lanceolata L. (plantain), Solidago L. sp. ovalbumin, 0.00 1 M dithiothreitol (goldenrod), Taraxacum officinale Weber (dandelion), Erigeron annuus (L.) Pers. (daisy fleabane), Cirsium Mill. sp. (thistle), Rumex L. sp. (dock), Vicia L. sp. (vetch), Hippuris vulgaris (Cleland's reagent), 0.02% polyvinylL. (mares' tail), and Chrysanthemum leucanthemum L. (daisy). pyrrolidone (mol wt 40,000), or 0.1% f None of the weeds showed characteristic symptoms. None yielded spiroplasmas when normal Tween 20 to the grinding buffer or isolation procedures were followed. grinding in 0.35 M sorbitol did not g~a average of the weeds is an average of the readings of all 15 weed samples tested. Plant Disease/August 1986 755 tests increased. Nonspecific agglutination Nault and of spiroplasma cultures from R. E. Davis. 9. Fribourg, C. E., and Nakashima, J. 1984. An was observed, but the percentage of We also thank Janet Leath, Susan Rheingans, Helen improved latex agglutination test for routine Fagbenle, and Jana Collins for excellent technical detection of potato viruses. Potato Res. positives from healthy leafhopper assistance. 27:237-249. samples was always less than half of that 10. Khan, M. A., and Slack, S. A. 1978. Studies on from infected insects. Omura et al (13) the sensitivity of a latex agglutination test for the found that adding Tween 20 to their LITERATURE CITED serological detection of potato virus S and 1. Archer, D. B., Townsend, R., and Markham, P. potato virus X in Wisconsin. Am. Potato J. insect-grinding buffer dispersed nonG. 1982. Detection of Spiroplasma citri in plants 55:627-637. specific aggregates, but in our experience, and insect hosts by ELISA. Plant Pathol. 11. Lee, I.-M., and Davis, R. E. 1984. New media for this did not solve the problem. Although 31:299-306. rapid growth of Spiroplasma citri and corn stunt sample treatments may be developed that 2. Chen, T. A., and Davis, R. E. 1979. Cultivation spiroplasma. Phytopathology 74:84-89. of spiroplasmas. Pages 65-82 in: The Myco12. Mink, G. I., Howell, W. E., and Fridlund, P. R. will minimize or eliminate nonspecific plasmas. Vol. 3. R. F. Whitcomb and J. G. Tully, 1985. Apple tip leaf antigens that cause spurious reactions, current data suggest that the eds. Academic Press, New York. reactions with tomato ringspot virus antisera in application of LAT to testing samples for 3. Davis, R. E., Worley, J. F., Whitcomb, R. F., enzyme-linked immunoassay. Phytopathology the presence of spiroplasma is limited and Ishijima, I., and Steere, R. L. 1972. Helical 75:325-329. filaments produced by a mycoplasma-like 13. Omura, T., Hibino, H., Usugi, T., Inoue, H., must include stringent control samples. organism associated with corn stunt disease. Morinaka, T., Tsurumachi, S., Ong, C. A., Although LAT lacks the precise, Science 176:521-523. Putta, M., Tsuchizaki, T., and Saito, Y. 1984. quantifiable features of some other 4. Eden-Green, S. J. 1982. Detection of corn stunt Detection of rice viruses in plants and individual serological techniques, it has become a spiroplasma in vivo by ELISA using antisera to insect vectors by latex flocculation test. Plant varlu iable aide simles ihad beconomical extracts from infected corn plants (Zea mays). Dis. 68:374-378. valuable aid for simple and economical Plant Pathol. 31:289-297. 14. Polak, J. 1980. A leaf dip method for routine detection of pathogens such as viruses 5. Fletcher, J., and Eastman, C. E. 1984. identification of plant viruses using the latex and bacteria. We have shown that it is Translocation and multiplication of Spiroplasma agglutination test. Biol. Plant. 22:237-238. applicable for use in detecting the plantcitri in turnip (Brassica rapa). Curr. Microbiol. 15. Raju, B. C., and Nyland, G. 1981. Enzyme-linked 11:289-292. immunosorbent assay for the detection of corn pathogenic mollicute S. citri. Thus, LAT 6. Fletcher, J., Franklin, K. M., and Goodman, R. stunt spiroplasma in plant and insect tissues. might be applied in purification attempts M. 1982. Evidence for invalid results in the use of Curr. Microbiol. 5:101-104. or other laboratory procedures for which ELISA for detection of Spiroplasma citri in 16. Slack, S. A., Sanford, H. A., and Manzer, F. E. it would be helpful to know which plants and insects. (Abstr.) Phytopathology 1979. The latex agglutination test as a rapid 72:1005. serological assay for Corynebacterium fractions contained spiroplasma. Because 7. Fletcher, J., Leath, J. A., and Slack, S. A. 1983. sepedonicum. Am. Potato J. 56:441-446. of its specificity, it could also be used as a Serological detection of Spiroplasma citri by the 17. Whitcomb, R. F., Clark, T. B., Tully, J. G., very quick serological identification latex agglutination test. (Abstr.)Phytopathology Chen, T. A., and Bove, J. M. 1983. Serological method for cultured spiroplasmas. 73:807. classification of spiroplasmas: Current status. 8. Fletcher, J., Schultz, G. A., Davis, R. E., Yale J. Biol. Med. 56:453-459. Eastman, C. E., and Goodman, R. M. 1981. 18. Williamson, D. L., Whitcomb, R. F., and Tully, ACKNOWLEDGMENTS Brittle root disease of horseradish: Evidence for J. G. 1978. The spiroplasma deformation test, a We gratefully acknowledge the gifts of plant an etiological role of Spiroplasma citri. new serological method. Curr. Microbiol. material from C. E. Eastman, S. Haber, and L. R. Phytopathology 71:1073-1080. 1:203-207. 756 Plant Disease/Vol. 70 No. 8