Use of Monoclonal Antibodies to Monitor the Dissemination of Xanthomonas campestris pv . campestris

Yuen, G. Y., Alvarez, A. M., Benedict, A. A., and Trotter, K. J. 1987. Use of monoclonal antibodies'to monitor the dissemination of Xanthomonas campestris pv. campestris. Phytopathology 77:366-370. Monoclonal antibodies (MCA) were used to detect and differentiate on FS from 98% of samples (341 of 348) of lesions with definite black rot strains of Xanthomonas campestris pv. campestris isolated from cabbage symptoms; 88% of the 341 presumptive Xanthomonas cultures on FS were black rot lesions collected from the field. Extracts from leaf lesions were identified as one of the four test strains by serotyping cultures directly from spotted onto a semiselective medium (FS). Four-day-old cultures that the isolation medium. The method also was useful in testing 484 lesions appeared to contain Xanthomonas were tested without further purification with symptoms that did not resemble those of black rot; the inoculated with four MCA using indirect enzyme-linked immunosorbent assay. The strains of X. c. pv. campestris were recovered from 21%. Pathogenic and MCA reactivity patterns of four strains of X. c. pv. campestris were stable nonpathogenic xanthomonads with MCA patterns unlike those of the four through four sets of serial transfers onto culture media and serial passages introduced strains of X. c. pv. campestris also were found in the field trials through cabbage. The four strains were introduced to replicate field plots by this method. By monitoring the spread of the four inoculated strains of on inoculated transplants, and dissemination of each strain was tracked X. c. pv. campestris with MCA, disease progression and spatial patterns of independently with the MCA. Xanthomonas-like colonies were recovered infections were determined for each strain. Additional key words: Brassica oleracea, serology. Ecological and epidemiological studies of bacterial plant MATERIALS AND METHODS pathogens typically address population level phenomena. Techniques that allow accurate identification and monitoring of Bacterial strains and pathogenicity tests. Four strains of X. c. pv. individual strains within given populations have been limited to campestris were selected for field studies on the basis of reactivity those involving phage typing, serotyping, or mutant strains with patterns with four MCA that distinguished one strain from antibiotic resistance (5,8,9,11,14,17). Only a small number of another (Table 1). The pathogenicity of each strain was tested on strains can be investigated using antibiotic resistance markers, cabbage by spraying a susceptible cabbage (CG hybrid) with a whereas phage-typing and serotyping provide means to identify suspension of log-phase, washed bacteria (105 to 106 colony many strains. forming units [cfu] per milliliter) in a solution of 0.85% NaCl and In studies on black rot of crucifers caused by Xanthomonas 0.5% Tween 80. Plants were kept under high humidity for 24 hr campestris pv. campestris, phage typing and serotyping have after inoculation and maintained in the greenhouse at 23-37 C. provided varying degrees of success. Use of five bacteriophages Symptoms were recorded at 4-day intervals over a 21-day period. specific to X. c. pv. campestris allowed separation of 145 strains of Bacteria were reisolated from plant material on a semiselective X. c. pv. campestris from different geographic origins into two medium developed by D. Fieldhouse and M. Sasser (personal lysotypes and seven subtypes (12). Phage typing was used to communication). The medium (FS) contained 10 g of potato distinguish infections caused by three strains of X. c. pv. campestris starch, 500 mg of KNO 3, 800 mg of K2 H PO 4, 800 mg of K H 2 PO 4 , in a field study by introducing strains selected from different 100 mg of MgSO 4, 100 mg of yeast extract, 15 mg of methyl green, geographic locations for their distinct phage-sensitivity patterns and 15 g of agar per liter. Antibiotics (30 mg of trimethoprim, 50 (11). However, differentiation of strains in Hawaii by phage typing mg of cephalexin, and 150 mg of cycloheximide), I mg of was limited because 97% of the strains isolated from local cabbage pyridoxine-HCI and 3 mg of D-methionine were added aseptically farms were of a single lysotype (12). Monoclonal antibodies after autoclaving. In preliminary experiments, this medium was (MCA) subsequently were produced that differentiated Hawaiian strains of X. c. pv. campestris and separated them into six major TABLE 1. Reactivitya to four monoclonal antibodies of strains of groups and several subgroups (I). Xanthomonas campestris pv. campestris used in field experiments This study was initiated to test the feasibility of using MCA to identify the strain causing black rot in the field, to simultaneously Antibody follow the progress of black rot caused by two serologically distinct Strain X3 X9 XII X20 strains originating from different inoculum sources, and to Experiment I determine infection rates and spatial patterns for each strain. A249 + + +