Paenibacillus polymyxa invades plant roots and forms biofilms

Abstract

Paenibacillus polymyxa is a plant growth-promoting rhizobacterium with a broad host range, but so far the use of this organism as a biocontrol agent has not been very efficient. In previous work we showed that this bacterium protects Arabidopsis thaliana against pathogens and abiotic stress (S. Timmusk and E. G. H. Wagner, Mol. Plant-Microbe Interact. 12:951-959, 1999; S. Timmusk, P. van West, N. A. R. Gow, and E. G. H. Wagner, p. 1-28, in Mechanism of action of the plant growth promoting bacterium Paenibacillus polymyxa, 2003). Here, we studied colonization of plant roots by a natural isolate of P. polymyxa which had been tagged with a plasmid-borne gfp gene. Fluorescence microscopy and electron scanning microscopy indicated that the bacteria colonized predominantly the root tip, where they formed biofilms. Accumulation of bacteria was observed in the intercellular spaces outside the vascular cylinder. Systemic spreading did not occur, as indicated by the absence of bacteria in aerial tissues. Studies were performed in both a gnotobiotic system and a soil system. The fact that similar observations were made in both systems suggests that colonization by this bacterium can be studied in a more defined system. Problems associated with green fluorescent protein tagging of natural isolates and deleterious effects of the plant growth-promoting bacteria are discussed.

FIG. 1.

Micrographs of GFP-tagged P. polymyxa isolate B1 cells: fluorescence micrographs (GFP) (A and D) and phase-contrast micrographs (B and E) of bacterial cells containing plasmid pBSVG101 (A and B) or pCM20 (D and E) after 24 h of incubation in LB. Panel C is an overlay of panels A and B, and panel F is an overlay of panels B and D. The arrows indicate cells with low levels of fluorescence.

FIG. 2.

Distribution of fluorescence signal intensities of single P. polymyxa B1 cells. Fluorescence intensities of individual cells were measured. For each strain-plasmid combination, 100 cells were measured. The approximate detection limit was 100 U.

FIG. 3.

Fluorescence (GFP) micrographs of plant roots colonized by P. polymyxa B1::pCM20. P. polymyxa colonization and biofilm formation on plant roots in the gnotobiotic experiments (A and B) and in the soil experiments (D, E, and F) are compared, and bacterial localization in intercellular spaces is shown in panel C. Roots were prepared and analyzed as described in Materials and Methods. The roots in panels D, E, and F were visualized directly from soil. (A) Bacterial biofilm formation on an A. thaliana root in the gnotobiotic system after 2 h of incubation. (B) Bacterial biofilm formation in the gnotobiotic system after 5 h of incubation. Note that P. polymyxa B1::pCM20 is localized between A. thaliana root cells (arrows) (see panel C and Fig. 5) and that there is an indication of damage to the epidermal root cap. (C) Deconvoluted optical section of 20-μm slices of surface-sterilized A. thaliana roots, 5 h after inoculation. (D) Bacterial biofilm formation on an A. thaliana tap root after 1 week of colonization in the soil assay. (E) Bacterial biofilm formation on a barley (H. vulgare) root colonized by P. polymyxa B1::pCM20 in a soil assay after 1 week of incubation. (F) Control roots treated with a mock solution. Note that the bacterial accumulation (indicated by brackets) in the gnotobiotic system (A) and in soil (D and E) occurred mainly around the root tip.

FIG. 4.

SEM micrographs of plant roots colonized by P. polymyxa B1::pCM20. P. polymyxa B1::pCM20 colonization and biofilm formation on A. thaliana roots in the gnotobiotic system after 2 h of colonization (A, C, and E) and in nonsterile soil assays after 1 week of colonization (B, D, and F) are compared. Roots were prepared and analyzed as described in Materials and Methods. Images were taken at the root tips (A, B, C, and D) and at tip-distal regions (E and F). Note the biofilm formation in the gnotobiotic system (A and C) and the soil system (B and D) around the root tip. Many fewer bacteria than in the biofilm region colonize the regions behind the root tip in the gnotobiotic system (E) and in the soil system (F). In the nonsterile system only P. polymyxa was present in the biofilm-covered regions (D), whereas P. polymyxa cells mixed with indigenous bacteria were found in the distant regions of the plant root (F). Note the degradation pattern on the root tip indicated by arrows in panel B.

FIG. 5.

Sizes of the P. polymyxa B1::pCM20 populations in the rhizoplane and endorhizosphere of A. thaliana in the gnotobiotic system and in soil. The data are expressed as log₁₀ CFU per gram (fresh weight) of plant material. The error bars indicate standard deviations for four samples. Note the different time scales in the gnotobiotic system and the soil experiments.