Detection of Xanthomonas campestris pv. citrumelo and X. citri from Citrus Using Membrane Entrapment Immunofluorescence

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Abstract
Xanthomonas campestris pv. citrumela (formerly X. campestris pv. citri strain E), the causal agent of citrus bacterial spot, and X. citri (formerly X. campestris pv. citri strain A), the causal agent of citrus bacterial canker, were easily detected in leaf extracts from symptomatic and asymptomatic citrus leaves and from cultures by immunofluorescence microscopy of bacteria that were trapped on 0.2-.mu.m black polycarbonate membranes. Leaf disks, 5 mm in diameter, were ground in phosphate buffered saline, and the extracts were successively passed through 5.0-.mu.m polycarbonate and 0.2 .mu.m black polycarbonate membranes. The 0.2-.mu.m membranes were washed and subsequently incubated in tetramethyl-rhodamine isothiocyanate (TRITC) labeled immunoglobulin (IgG) prepared against either X. capestris pv. citrulemo or X. citri, or in TRITC-labeled normal serum IgG. The membranes were rinsed, mounted on microscope slides, and viewed with an epifluorescence microscope using a 580-590 nm wavelength filter to detect the presence of fluorescing bacteria.