The Use of Enzyme - Linked Immunosorbent Assay for Detection of Citrus Tristeza Virus

نویسندگان

  • D. Gonsalves
  • Mira Moscovitz
  • D. E. Purcifull
  • M. F. Clark
چکیده

BAR-JOSEPH, M., S. M. GARNSEY, D. GONSALVES, M. MOSCOVITZ, D. E. PURCIFULL, M. F. CLARK, and G. LOEBENSTEIN. 1979. The use of enzyme-linked immunosorbent assay for detection of citrus tristeza virus. Phytopathology 69:190-194. An enzyme-linked immunosorbent assay (ELISA) test was used to seedling yellows isolates of CTV from Israel and from Florida. Isolates that identify citrus tristeza virus (CTV) in extracts from citrus tissues. Alkaline produced only mild symptoms on lime (Citrus aurantifolia) 'Mexican' phosphatase conjugates were prepared with partially purified -y-globulin indicator seedlings could be detected by ELISA. The virus was detected in from antiserum to purified CTV. Citrus tristeza virus was detected quickly various phloem-containing tissues during warm and cold seasons, but was in extracts of experimentally inoculated plants kept in various indoor most readily detected from fruit pedicel bark. A test procedure that facilities, and in extracts of infected samples collected from the field. The incorporated composite sampling and mechanical homogenization was ELISA procedure was equally effective for detection of most common and developed to index large numbers of field trees. Additional key words: closterovirus. Citrus tristeza virus (CTV), an aphid-borne closterovirus, has immunodiffusion was practical. However, tissues must be collected been one of the most important citrus diseases for more than two at the proper time if the samples are to have a CTV titer high decades, especially in areas where extensive plantings still exist on enough for detection. Tsuchizaki et al (18) recently purified CTV the CTV-sensitive sour orange (Citrus aurantium L.) rootstock. from diseased fruits, and found that fluorescent-labeled antibodies Large-scale indexing of field trees currently is practiced in the San to the Hassaku strain of CTV could be used to detect the virus Joaquin Valley of California (16) and in Israel (4,15) to detect and within tissues of several citrus species. eradicate foci of infection and suppress further natural spread of Recently, the microplate enzyme-linked immunosorbent assay the virus. The CTV has not been transmitted mechanically to (ELISA) method, which is based on the double antibody sandwich herbaceous plants, and large-scale indexing is conducted by graft technique used by Voller et al (19) and Clark and Adams (5), has inoculation of lime (Citrus aurantifolia [Christm.] Swingle been shown to be an inexpensive, reliable, and sensitive assay 'Mexican') indicators, which are observed later for vein clearing method for several plant viruses (6,13,17). This paper reports and stem pitting (20). This procedure has several disadvantages: (i) detection of various CTV isolates from Israel and Florida by it is laborious and expensive, (ii) it requires 1-6 mo until symptoms ELISA, and application of this method for large-scale field can be read, and (iii) certain mild CTV isolates do not produce clear detection of CTV. symptoms. A method for rapid diagnosis, based on electron microscopy (EM) of partially purified CTV particles, has been MATERIALS AND METHODS developed (1) and was used until recently in Israel. The EM method enabled the rapid detection of new sources of CTV infection and, The experimental procedures for graft transmission of CTV and when combined with the lime test (4), it conserved indicator plants the isolates used extensively in these studies have been described without adding much to detection time. The main limitations of the previously (3). Table I summarizes the biological properties of five EM method are its high cost of operation and low sensitivity in of the Israeli CTV isolates used in this study. The Florida isolates diagnosing CTV in certain cultivars. included mild quick decline, seedling yellows, and uncharacterized Bar-Jospeh et al (2,3) reported the purification of CTV from the field isolates (9). leaves and bark of infected citrus seedlings, by polyethylene glycol Greenhouse conditions in Florida essentially were as described (PEG) precipitation and density-gradient centrifugation in CsCl by Garnsey et al (8). In Israel, Egyptian (=Mexican) lime seedlings after fixation with formaldehyde. By use of a similar purification were grown in a screenhouse in 5-L plastic pots that contained a procedure, antisera to CTV have been produced (9,11). Antisera to mixture of sand, loam, and peat (1:2:1). For some tests, unfixed purified CTV and to CTV coat protein degraded with commercially grown 2-yr-old sweet orange (C. sinensis[L.] Osb.), sodium dodecyl sulfate (SDS) were used successfully in an SDSgrapefruit (C. paradisi Macfad. 'Marsh Seedless'), mandarin (C. immunodiffusion procedure to detect CTV in infected citrus reticulata Blanco), and lemon (C. limon[L.] Burm. f. 'Eureka') (9-11). Garnsey et al (9) investigated many of the factors affecting budded on sour orange rootstock were grown in 10-L plastic application of serological procedures for field detection of CTV in bags in a cool (22±2 C) plastic chamber. Florida and showed that serological detection by SDS Tissues were collected also from field-grown trees in Israel and Florida, and processed fresh or after being stored frozen at -20 C. 00031-949X/79/000032$03.00/0 The CTV antiserum used in most tests was the antiserum * 1979 The American Phytopathological Society prepared to formaldehyde-fixed whole virus (CTV-FW) and

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تاریخ انتشار 2006