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Direct-Count Techniques for Enumerating Clavibacter xyli subsp. xyli Which Causes Ratoon Stunting Disease of Sugarcane. M. J. Davis, University of Florida, Institute of Food and Agricultural Sciences, Ft. Lauderdale Research and Education Center, 3205 College Avenue, Ft. Lauderdale 33314; Phytopathology 75:1226-1231. Accepted for publication 17 June 1985. Copyright 1985 The American Phytopathological Society. DOI: 10.1094/Phyto-75-1226.

A fluorescent-antibody-direct-count-on-filters (FADCF) technique was developed to enumerate Clavibacter xyli subsp. xyli from both pure culture and sugarcane. An acridine-orange-direct-count (AODC) technique, a fluorescent-antibody-direct-count-on-microscope-slides (FADCS) technique, and a counting-chamber technique were also developed for enumeration of the bacterium from pure culture but not from sugarcane. The FADCF and AODC techniques are similar, except that instead of an acridine orange stain a fluorescent-antibody is used to obtain increased specificity. In both techniques, stained bacteria are concentrated on the surface of polycarbonate membrane filters before enumeration with epifluorescence microscopy. With C. x. subsp. xyli from pure culture, similar cell counts were obtained with the FADCF and AODC techniques over a thousandfold range of concentrations. The lowest concentrations that were conveniently measured with the FADCF and AODC techniques were between 10⁴ and 10⁵ cells per milliliter. The FADCS and counting-chamber techniques were tenfold and one-thousandfold less sensitive, respectively. Since counts obtained with all four techniques generally followed a Poisson distribution, the accuracy of counts was directly related to the number of cells counted. When sap samples from sugarcane infected with C. x. subsp. xyli were examined, populations estimated with the FADCF technique were consistently higher than for plate-count estimates, but the correlation between the estimates was highly significant.