In Situ Immunofluorescence for the Detection of Citrus Tristeza Virus Inclusion Bodies

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Abstract
Transverse and longitudinal cryostat-cut sections, 30-40 .mu.m thick, were prepared from bark and petioles of five citrus species (Citrus aurantifolia, C. sinensis, C. aurantium, C. limon, C. medica) that were healthy or infected with various isolates of citrus tristeza virus (CTV). Sections were immersed in tetramethylrhodamine isothiocyanate (TRITC) labeled or fluorescein isothiocyanate (FITC) labeled immunoglobulin (IgG) for 1 hr at room temperature, rinsed in phosphate-buffered saline, mounted, and viewed with a fluorescence microscope. Orange-red fluorescing bodies were readily observed in phloem fibers, sieve tubes, and parenchyma cells of CTV-infected tissue sections incubated in TRITC-labeled IgG specific to CTV. Yellow-green fluorescence of the same cells was observed after incubation in FITC-labeled IgG. Autofluorescence was more evident with FITC-labeled IgG than with TRITC-labeled IgG. No fluorescing bodies were observed in sections incubated in normal IgG or in fluorescent labeled IgG specific for either tobacco etch virus or Pierce''s disease bacterium or in sections of healthy tissue incubated in TRITC-labeled CTV IgG. The same fluorescing bodies in CTV-infected tissue also were stained by Azure A, indicating that the nucleoprotein-containing inclusion bodies of CTV were the fluorescing structures.