2.2.91
GENOTYPIC AND PHENOTYPIC VARIATION IN FIELD ISOLATES OF CLAVIBACTER MICHIGANENSIS SUBSP. MICHIGANENSIS

DW FULBRIGHT, J BELL, C MEDINA-MORA and MK HAUSBECK

Department of Botany and Plant Pathology, Michigan State University, East Lansing, MI 49924-1312, USA

Background and objectives
We have applied molecular fingerprinting using the polymerase chain reaction (PCR) and an array of primers which anneal to repetitive DNA elements in many prokaryotes (i.e. REP-PCR with REP, ERIC, BOX, and GTG) to the causative agent of bacterial canker of tomato, Clavibacter michiganensis subsp. michiganensis. The resulting genomic fingerprints divided a collection of 109 strains into four groups (A, B, C, and D) defined by banding pattern polymorphisms [1]. Historically, plant pathologists have been unable to determine major sources of initial inoculum or to predict the outcome of local or regional epidemics of bacterial canker. The discovery of the fingerprint groups and the fact that many strains in the collection were avirulent raised questions about the roles of the fingerprint groups in the epidemiology of C. michiganensis subsp. michiganensis. We therefore set out to characterize the original collection phenotypically and to characterize both genotypically and phenotypically a new collection formed by intensive sampling of three commercial tomato fields in 1995.

Materials and methods
A commercial processing tomato field in Ohio, a commercial processing tomato field in Michigan, and a commercial fresh market tomato field in Michigan were sampled in 1995. Up to 50 matched samples of spotted fruit and vegetative tissue were obtained from individual plants in each field; C. michiganensis subsp. michiganensis isolates were obtained from as many of these samples as possible. Multiple isolates were obtained from nine individual fruit lesions from the Michigan processing tomato field. REP-PCR was performed on the isolates as described by Versalovic et al. [2] with minor modifications [1]. Pathogenicity tests were performed by severing the first true leaf of tomato (Lysopersicon esculentum Mill. cv. Bonny Best) seedlings close to the stem with scissors dipped in a suspension of C. michiganensis subsp. michiganensis cells; symptoms were monitored over several weeks.

Results and conclusions In the original collection the strains examined fell into three virulence classes based on the symptoms produced in tomato seedlings. Virulent isolates were found in all fingerprint groups; they produced both stem cankers and leaf wilting. Strains of intermediate virulence were found in fingerprint groups A, C, and D; they produced cankers but no wilting. Avirulent strains were found in group A; they produced no symptoms. Thirty per cent of the group A isolates in the original collection were avirulent. In the 1995 collection, all fields sampled had mixed populations of C. michiganensis subsp. michiganensis fingerprint types; group A isolates predominated in the two commercial processing fields. Populations were mixed with respect to fingerprint group on some individual plants; different fingerprint groups were sometimes found in tissue and in fruit lesions. Populations within some individual fruit lesions were also mixed with respect to fingerprint group. All fields had substantial proportions of isolates that were not fully virulent, i.e., were of intermediate virulence or aviruient. Forty-eight per cent of the isolates from the Ohio processing tomato field and 38% of those from the Michigan processing tomato field were avirulent. Within each field, proportions in the three virulence classes varied between isolates from fruit and from vegetative tissue and within individual fruit lesions.

References
1. Louws FJ, Medina-Mora CM, Bell J, Smart CD, Opganorth D, Ishimaru CD, Hausbeck MK, de Bruijn FJ, Fulbright DW, 1998. Phytopathology, in press.
2. Versalovic J, Schneider M, de Bruijn FJ, Lupski JR, 1994. Methods in Cellular and Molecular Biology 5, 25-40.