Immunofluorescent Detection of Xylem-Limited Bacteria In Situ

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Abstract
Xylem-limited bacteria (XLB) were detected in situ in sectioned material using .gamma. globulins (IgG) specific to Pierce''s disease bacterium (PDB) or the ratoon stunting disease bacterium (RSDB) and conjugated to tetramethylrhodamine isothiocyanate (TRITC). Longitudinal and transverse cryostat-cut sections, 30-40 .mu.m thick, were prepared from plants affected by Pierce''s disease of grape, periwinkle wilt, plum leaf scald, a disease of ragweed, and sugarcane ratoon stunting disease. Sections were immersed in TRITC-labeled IgG for 1 h at room temperature, rinsed in phosphate-buffered saline, mounted, and viewed with a fluorescent microscope at 560-590 nm. Orange-red fluorescing bacteria were readily observed in and adhering to xylem vessels of diseased plants affected with Pierce''s disease, periwinkle wilt, plum leaf scald, and diseased ragweed when incubated in TRITC-labeled IgG to PDB. Fluorescing bacteria were observed in sugarcane with ratoon stunting disease when incubated in TRITC-labeled RSDB IgG. No fluorescing bacteria were observed in sections of infected plants immersed in normal serum or in IgG specific for Corynebacterium michiganense labeled with TRITC or in healthy plants incubated in PDB or RSDB specific IgG. Fluorescein isothiocyanate-labeled antiserum was not useful since xylem vessels autofluoresced at the same wavelength as fluorescein.