Disrup'on of Bacterial Endoparasi'sm of Plant Roots Using Butyric Acid James White and Kathryn Kingsley Dept. Plant Biology Rutgers University
[email protected]
Methodology • Poa ampla seeds sterilized with 30 min with 4% NaOCl. • Seeds placed on agarose media containing 0, 0.5, or 1.0 mM of butyric acid. • Seeds inoculated with endoparasi'c/endophy'c bacterium Pseudomonas fluorescens. • ASer several days seedlings were stained overnight with reac've oxygen stain diaminobenzidine (DAB), then counterstained with aniline blue to visualize bacteria within plant root hairs.
Root 'p showing long hairs (arrows) in a root that was not treated with butyric acid.
0.5 mM butyric acid treatment showing shorter root hairs due to fewer bacteria entering the root cells at the 'p meristem (blue arrow).
Note bacterial biofilm around meristem
1 mM butyric acid treatment showing total absence of root hairs due to cessa'on of intracellular invasion by bacteria. Without intracellular bacteria no root hairs form.
Root hair from treatment without butyric acid showing abundant intracellular bacteria. Arrows show intracellular bacteria. Bacteria s'mulate root hair development through produc'on of auxin. The bacteria exit root hairs at the 'p where the cell wall is thin. The slight reddish colora'on indicates presence of reac've oxygen around intracellular bacteria due to host defensive reac'on.
Root hair from treatment without butyric acid showing two bacteria (black arrow) exi'ng the hair at the 'p of the hair where the cell wall is thin. Bacteria are present in the root cells as L-‐forms (blue arrows). The exi'ng bacteria will reform cell walls to become rods.
Root hair from 0.5 mM butyric acid treatment. Intracellular bacteria are sparse in this treatment. Most of the bacteria have already exited the hair. No L-‐forms are visible in this hair. Due to presence of butyric acid in the medium few bacteria entered the root cells at the growing root meristem. The few bacteria that did enter cells s'mulated root hair forma'on but because their number was few, root hair length growth was restricted.
Another root hair from the 0.5 mM butyric acid treatment. All intracellular bacteria have already exited the hair.
Another hair from the 0.5 mM butyric acid treatment with few if any L-‐forms in the hair.
Hair from the 0.5 mM butyric acid treatment showing an exi'ng bacterium at the 'p (arrow). Three bacteria are present within the hair below the exi'ng bacterium.
Conclusions • Butyric acid reduced (0.5 mM level) and completely eliminated (1 mM level) conversion of Pseudomonas fluorescens from the free-‐living state to the endoparasi'c. • The quan'ty of bacteria that enter into root 'p meristem cells directly correlates with the length of root hairs. A greater number of bacteria that enter at the root meristem results in longer root hair growth.