Development of Molecular Tools for Genetic Analysis of Erwinia tracheiphila Pathogenesis
Erwinia tracheiphila (Et) is a gram negative bacterium in the family Enterobacteriaceae. It is the causal agent of bacterial wilt disease in cucurbit crops such as cucumber, melon, squash and pumpkin. The bacterium is vectored by striped cucumber beetles (Acalymma vittatum) and spotted cucumber beetles ( Diabrotica undecimpunctata howardi). The insects acquire E. tracheiphila from infected plants and spread the bacteria to young plants in the frass during feeding. Much is known about the insect vectors, but the pathogen has received little research attention. Understanding of the mechanisms of pathogenesis of this bacterium requires fundamental studies on traits such as drug resistance, growth characteristics and virulence. The goal of this study was to characterize strains of E. tracheiphila for virulence and cultural properties and develop molecular tools for investigations into their genetics. Strains of the bacterium isolated from melon, squash and cucumber were characterized for properties including, the presence of endogenous plasmids, antibiotic resistance profile, growth in different media, extracellular polysaccharide secretion (EPS), swarming motility, extracellular protease production, β-Galactosidase (LacZ) activity, and acyl-homoserine lactone (AHL) auto- inducer production. The strains BoCu 3-1, BoCu 4-1, HCAL 1-5, MCM 1-1, UNIS CU 1-1, ATCC 33245, E. Salisbury, LW4, and NJ11 possessed most of the virulent phenotypes. The minimal inhibitory concentrations of antibiotics were determined for these isolates. Spontaneous nalidixic acid-resistant mutants were isolated for strains HCAL 1-5, UNIS CU 1-1, and LW4, and these retained parental properties in growth and pathogenicity on muskmelon cultivar Athena hybrid plants. The nalidixic acid-resistant mutants also retained their physiological and pathogenic characteristics and could be used for electroporation, plasmid mating and transposon mutagenesis. The order of efficiency for transposon mutagenesis and electroporation of the isolates were UNIS CU 1-1 N3 > HCAL 1-5 N5 > LW4 N1, and for bi-parental mating HCAL 1-5 N5 > UNIS CU 1-1 N3 > LW4 N1. The molecular tools developed in this research will be useful for studies whose aim is to reveal the genetic basis for E. tracheiphila pathogenesis.
Sean Natacha Mason,
"Development of Molecular Tools for Genetic Analysis of Erwinia tracheiphila Pathogenesis"
ETD Collection for Tennessee State University.