Isozyme Analysis of<i>Xanthomonas campestris</i>pv.<i>citri</i> (1989)

Kubicek, Q. B., Civerolo, E. L., Bonde, M. R., Hartung, J. S., and Peterson, G. L. 1989. Isozyme analysis of Xanthomonas campestris pv. citri. Phytopathology 79:297-300. Isozyme analysis of 14 putative isozymic loci by horizontal starch gel neopathotype strain. No isozymes were found in the citrus canker groups of electrophoresis was conducted on 36 strains of Xanthomonas campestris strains that distinguished any of the forms of citrus canker. As a subgroup, pv. citri representing four pathogenic variants associated with different the Asiatic citrus canker strains exhibited relatively little isozymic forms of citrus bacterial canker disease in eight countries. An additional 80 polymorphism despite their varied origins worldwide. In contrast, several strains of X. campestris associated with citrus bacterial spot disease, isozymic alleles were present only in the set of citrus bacterial spot strains primarily in Florida citrus nurseries, also were analyzed. Four enzymes isolated from Florida citrus nurseries. These strains also exhibited were monomorphic in all 116 strains. The number of isomorphs for the 10 extensive isozymic polymorphism. Isozyme analysis may be a useful remaining polymorphic loci ranged from two to five. Generally, all strains technique in epidemiological studies of phytopathogenic bacteria. of X. c. citri were isozymically similar, but not identical in all cases, to the Citrus bacterial canker disease (citrus canker) is caused by MATERIALS AND METHODS Xanthomonas campestris pv. citri (Hasse) Dye. Different forms of citrus canker are considered to be caused by pathogenic variants of Bacterial strains. The bacterial strains used in this study are the same pathogen distinguished by host range, geographical listed in Table 1. All strains were maintained in the U.S. distribution, pathogenicity, serology, phage typing, plasmid DNA Department of Agriculture collection of phytopathogenic bacteria analysis, and genomic fingerprinting (4-6,11). At least three in Beltsville, MD. A total of 116 strains from Argentina, Brazil, groups of strains of X. c. citri are recognized (4). Group A strains Florida, Guam, Japan, Mexico, North Yemen, and Pakistan were are associated with Asiatic citrus canker, group B strains with examined. They are maintained aseptically on Wakimoto semicancrosis B in Argentina and Uruguay, and group C strains with synthetic potato medium under sterile mineral oil at 3 C (5,6). The Mexican lime cancrosis in Brazil. Presumptive strains of X. c. citri strains represented a wide range of types based on a diversity of are associated with citrus bacteriosis in Mexico (4). Strains in each pathogenic variants, geographical origin, length of time in culture, of these groups produce raised lesions. A genomically and hosts from which they were isolated. The purity, pathoheterogeneous group of strains of X. campestris (11) is associated genicity, and phage sensitivity of each strain were verified before with a citrus bacterial spot (citrus spot) disease, primarily in isozyme analysis. The lysate from each strain was analyzed at least nurseries, in Florida. These strains generally produce flat, water- twice. soaked, or slightly raised lesions with associated necrosis and Culture conditions and preparation of bacterial enzyme lysates. chlorosis (18). The relationship(s) of these strains to variants of Each strain was grown from a single colony in 100 ml of M9 broth X. c. citri is not understood. (12) supplemented with 0.2% (w/v) Difco casamino acids and 0.1% Isozyme electrophoresis is widely used to assess genetic (w/v) Difco yeast extract at 30 C on an orbital shaker (120 rpm) for relatedness and variation within and among populations of 24 hr. The initial pH of the medium was 6.8. Cells were harvested bacteria (3,9,15,19,20, Bonde et al, unpublished). Isozyme by centrifugation at approximately 5,500 g for 10 min at 4 C, analysis is also a complementary tool for determining taxonomic resuspended once in 100 ml of ice-cold sterile saline (0.9% NaC1) relationships in eukaryotes (1,2,8,13,14). The applicability of plus 0.1% (v/v) Triton X-100, and washed in ice-cold sterile isozyme analysis is dependent on the extent of existing enzyme deionized water. After the final wash, the supernatant was quickly variation. Because the enzymes assayed are constitutively decanted and the inside of the tube was wiped dry. The cell pellet expressed, many genetic loci may be studied regardless of was resuspended in the remnant liquid and transferred to a sterile environmental conditions under which the strains were grown. 1.5-ml Eppendorf microcentrifuge tube. The tubes were stored at Comparisons of isozyme patterns are possible. These can be used -60 C until needed. Before electrophoresis, the frozen bacterial in conjunction with other genetic traits to determine taxonomic suspension was thawed on ice, frozen by submersion in liquid relationships among phytopathogenic bacterial strains. Differ- nitrogen, and macerated with a glass rod or a Teflon pestle ential isozyme patterns are related to distinct pathovars of previously dipped in liquid nitrogen. The pellet then was thawed in X. campestris (Bonde et al, unpublished), ice before refreezing in liquid nitrogen. These steps were repeated The purpose of this study was to examine the extent of isozyme once. After the final thawing, two or three drops of ice-cold 50 mM variation within different pathogenic variants and in widely Tris-HC1 buffer, pH 7.1, were added to the pellet. The tube was separated populations of X. c. citri, and the xanthomonads centrifuged at full speed in a microcentrifuge for 3 min. The lysate associated with citrus bacteriosis in Mexico and citrus spot in was drawn off in wicks of Whatman 3MM filter paper and Florida. subjected to electrophoresis (40 wicks/gel). The remnant lysate