Folia Microbiol. 46 (5), 371-375 (2001) http: //www. biomed, cas. cz/mbu/folia/ Multilocus Enzyme Electrophoresis Analysis of Pseudomonas syringae pv. phaseolicola K. GUVEN Department of Biology, Faculty of Science, Anadolu University, 264 70 EskiIehir, Turkey Received 9 May 2001 ABSTRACT. A multilocus enzyme electrophoresis tech- nique was developed to detect variation in seven enzyme loci among isolates of Pseudomonas syringae pv. phaseoli- cola, representing three races from different geographical locations, the causal agent of the halo blight disease of beans. Cellulose acetate gel electrophoresis of seven en- zymes revealed 19 electrotypes (ET) among 21 Pseudo- monas syringae pv. phaseolicola isolates. One of the patho- var syringae and one of the pathovar tomato isolates were represented by two different ET. The population of Turkish isolates and three races of the pathovar phaseolicola ap- peared to be genetically diverse. Halo blight is a seed-borne disease of Phaseolus beans caused by the bacterium Pseudornonas syringae pv. phaseolicola (Mohan and Schaad I987). Infected seeds give rise to infected seedlings (primary infectors) but these usually form a relatively small portion of the total number of seedlings present in the crop. Subsequent spread of the disease from these primary infectors is by rain splash, usually in the direction of the prevailing wind (Walker and Patel I964). The bacterium has three races differentiated by their pathogenicity on Phaseolus vulyaris cultivars (Ertu~rul and Giiven 1998). Many countries operate seed certification schemes but the standard cultural technique for detection of the organism is lengthy, taking at least three days (Taylor I97Oa). Serological assays, such as immuno- fluorescent cell staining and ELISA have overcome these problems but still have the disadvantage of the risk of a false positive serological reaction (Van Vuurde ~987; Wyatt et al. x989). Immunomagnetic separation technique combined the advantages of isolation and serological assays (Giiven and Mutlu 2000). In addition, inoculation to suitable host plants is often needed to discriminate between the many closely related pathovars of Pseudomonas syringae (Palleroni I984). Multilocus enzyme electrophoresis (MEE) detects the variation in an enzyme locus among isolates of a species, providing information on genetic structure, and elucidates the epidemiology of several bacteria (Selander et al. i986; Bibb et al. I99O). The method has been used as a typing or identification method for plant pathogenic bacteria (EI-Sharkawy and Huising I97Ia,b; Baptist et al. I97I, Giiven et al. I999) plant symbiotic bacteria (Barrera et al. I997; Silva et al. I999) and pathogenic microorganisms (Maiden et al. 1998) This study employs the MEE method to discriminate between the three races and different geographical origins of Pseudomonas syringae pv. phaseolicola on seven enzyme loci. MATERIALS AND METHODS Bacterial cultures. The bacteria used are shown in Table I. Nine strains of the pathovar phase- olicola were isolated from beans grown in different fields in Eski~ehir province in a previous work (Ertu[~rul and Giiven t998). Preparation of bacterial cultures. The method of Gargolla-Viola and Lopez (I99o) was used with some minor modifications to prepare protein extracts of the isolates. The isolate to be tested was grown on KB medium (Schaad i98o) at 28 ~ for 2 d and was examined visually for purity. Each isolate was then grown in 100 mL of Tryptie Soy Broth (Oxoid) at 28 ~ for 1 d. The bacteria were harvested by centrifugation (10000 g, 10 min) and washed twice with Tris-citrate buffer (100 mmol/L, pH 7.0) and stored at -20 ~ Frozen ceils were suspended in 4 mL of Tris-citrate buffer and sonicated (Sonics & Materials) for 4 min (30-s periods separated by a 30-s rest) with ice-bath cooling. The determination of breakage was carried out with a microscope (after a successful sonication treatment, the solution appeared transparent and only a few intact cells could be seen in the preparation by micro- scopy). A great deal of care was taken during sonication, not to overheat the sample. The crude sonicate was centrifuged (20 000 g, 20 rain, 4 *C) and then the supernatant was removed and stored at -40 ~ until used.