ECOLOGICAL AND GENETIC CHARACTERISTICS ASSOCIATED WITH ALCALIGENES XYLOSOXIDANS DENITRIFICANS Project Director

A bacterium isolated from Homalodisca coagulata Say, found also to inhabit xylem of citrus and grape (Lauzon et al. unpubl.), shows potential for use in Symbiotic Control strategies against Xylella fastidiosa (Xf), the causal organism of Pierce’s disease (PD). The biology and “behavior” of the bacterium, identified as Alcaligenes xylosoxidans denitrificans (Axd), is under study to gather information that can be used to assess its efficacy and risk of use in the field. Real TimePolymerase Chain Reaction (RT-PCR) was used as a semi-quantitative means of monitoring Axd growth in lake water under semi-natural conditions. Axd grew better in autoclaved lake water than in lake water that contained indigenous microbial populations. Axd growth was also monitored in soil and on leaf surfaces under semi-natural conditions using microbiological and molecular techniques. Axd was not retrieved from soils containing indigenous microbial populations unless the soil was autoclaved. Axd was retrieved from leaf surfaces from citrus, strawberry, sage, and basil. We are currently examining the effect of introducing Axd to citrus leaf microbial communities using denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism. We have also monitored transfer and uptake of two plasmid vectors, DsRed (pIRES-DsRed Express, Invitrogen) and pTZ18r (Amersham Biotech). Transformation attempts included both chemical and electroporation protocols. E. coli was used as a control. In both cases, Axd resisted transformation while E. coli was successfully transformed. In addition, Axd was screened for the presence of endogenous plasmids. A strain of E. coli containing a single copy plasmid was used as a control. Our data suggest that Axd does not contain any endogenous plasmids from 5-150 kb. We are currently examining horizontal gene transfer potential between Axd and strains of E. coli and Shigella sp. that carry fluorescent and antibiotic-marked endogenous plasmids. Horizontal gene transfer is yet one factor used to assess harm and risk. It has been inferred that Axd is a potential new human pathogen based in part on its association with Pseudomonas aeruginosa (P. aeruginosa) infections in Cystic Fibrosis patients. We used RT-PCR to compare the growth of Axd and P. aeruginosa individually and in co-culture. We found that Axd and P. aeruginosa significantly affect each others’ growth. Our data suggest that Axd has the potential to mitigate harm associated with P. aeruginosa infections.