GENETIC ANALYSIS OF INTRASPECIFIC VARIATION IN PATHOVARS OF Xanthomonas campestris (Pammel) Dowson BY HOST RANGE, PLASMIDS, AND RESTRICTION FRAGMENT-LENGTH POLYMORPHISM

GERARD RAYMOND LAZO

May, 1987

Xanthomonas campestris is always found in association with plants. Those X. campestris which are known to be pathogenic are differentiated into 123 pathovars (pathogenic variants), on the basis of limited pathogenicity tests. Some members of a pathovar may have a broader host range than others, since exhaustive pathogenicity testing is impractical. Other than these tests, there is no definitive means of classifying an unknown X. campestris isolate. Those X. campestris which are not pathogenic are unclassifiable by the pathovar system, yet they may exhibit similar host range specificity to those which are pathogenic. This work was conducted in efforts to better understand the variability of X. campestris. Over 115 strains of X. campestris were examined for plasmid content and restriction endonuclease profiles. Of the 26 pathovars examined, only 13 were found to contain plasmids. Restriction endonuclease digests of plasmid DNAs from strains within a given plasmid-containing pathovar gave surprisingly similar, but not always identical, digestion profiles by agarose gel electrophoresis. All 60 strains tested of X. campestris pvs. glycines, malvacearum, phaseoli, and vignicola could be accurately identified by pathovar from determination of the restriction fragment profile and/or by Southern hybridizations of that profile with a selected DNA probe. In no instance was the same plasmid profile seen in more than one pathovar. The apparent stability of the plasmids provides a natural genetic marker that can be strain specific and perhaps useful in epidemiological investigations. Cloned DNA fragments, derived from a cosmid library of a Florida isolate of X. campestris pv. citri strain 3041, were used to detect restriction fragment length polymorphism (RFLP) of total DNA from 87 strains of X. campestris, comprising 23 different pathovars. Autoradiographs of Southern transfers of genomic DNA probed with cosmid-sized clones revealed hybridization profiles which appeared to be highly conserved. The hybridization patterns observed between different pathovars suggested that some DNA fragments were conserved at the species level, others were conserved at the pathovar level, and still others were variable. The degree of variability appeared to depend on the DNA probe used. By using more than one DNA probe, or by digesting the genomic DNAs with different restriction endonucleases, all of the strains of X. campestris described as belonging to a given pathovar could be differentiated. All strains of X. campestris were readily grouped by RFLP phenotypes, and the classification based on RFLP patterns correlated well with the classification based on pathogenicity. Although certain pathovars may need to be redefined, this work supports and helps validate the natural taxonomic groupings provided by the pathovar naming system.

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
Chairman: Dean W. Gabriel, Major Department: Plant Pathology
INTRODUCTION

LITERATURE REVIEW

HOST RANGE OF PATHOVARS OF XANTHOMONAS CAMPESTRIS

CONSERVATION OF PLASMID DNA SEQUENCES AND PATHOVAR IDENTIFICATION OF STRAINS OF XANTHOMONAS CAMPESTRIS

* This chapter contains copyrighted material from the journal Phytopathology which was authorized for use in this dissertation.
ARE RACE-SPECIFIC AVIRULENCE GENES ENCODED BY PLASMIDS OF XANTHOMONAS CAMPESTRIS PV. MALVACEARUM?

PATHOVARS OF XANTHOMONAS CAMPESTRIS ARE DISTINGUISHABLE BY RESTRICTION FRAGMENT LENGTH POLYMORPHISM

* This chapter contains copyrighted material from International Journal of Systematic Bacteriology which was authorized for use in this dissertation.
SUMMARY

LITERATURE CITED

BIOGRAPHICAL SKETCH

ACKNOWLEDGMENTS

LIST OF TABLES

LIST OF FIGURES