Transparent soil microcosms allow 3D spatial quantification of soil microbiological processes in vivo

Abstract

The recently developed transparent soil consists of particles of Nafion, a polymer with a low refractive index (RI), which is prepared by milling and chemical treatment for use as a soil analog. After the addition of a RI-matched solution, confocal imaging can be carried out in vivo and without destructive sampling. In a previous study, we showed that the new substrate provides a good approximation of plant growth conditions found in natural soils. In this paper, we present further development of the techniques for detailed quantitative analysis of images of root-microbe interactions in situ. Using this system it was possible for the first time to analyze bacterial distribution along the roots and in the bulk substrate in vivo. These findings indicate that the coupling of transparent soil with light microscopy is an important advance toward the discovery of the mechanisms of microbial colonisation of the rhizosphere.

Keywords: CFU, colony forming units; FISH, fluorescent in situ hybridization; GFP; GFP, green fluorescent protein; Nafion; PGPR; PGPR, plant growth promoting rhizobacteria; Pseudomonas fluorescens; RI, refractive index; Rhizobia; T-RFLP, terminal restriction fragment length polymorphism; confocal; lettuce, image analysis; plant roots; refractive index matching.

Figure 1.

Maximum projection confocal images of GFP-labeled Pseudomonas fluorescens colonies (green) on the surface of lettuce root tissues (gray) in situ in transparent soil with Nafion particles from the substrate labeled with sulphorhodamine B fluorescent dye also visible (red). (A) The majority of the bacterial fluorescence is associated with the root tissue. Scale bar = 150 μm. (B) Bacteria are present on the root tip and in this case also the surfaces of Nafion particles in close proximity to the root have bacterial fluorescence associated with them. Scale bar = 150 μm. (C) At higher resolution, bacterial colonisation was predominantly observed in the intercellular junctions of root epithelial cells. Scale bar = 45 μm.

Figure 2.

Quantification of Pseudomonas fluorescens in the rhizosphere. (A) Bacteria, Nafion particles and roots were processed sequentially to allow quantification. (i-ii) Bacterial fluorescence before and after processing with a median filter and thresholding facilitated measuring the bacterial abundance. Scale bar = 40 μm. (iii-iv) Original images of particle surfaces were processed and skeletonised. Gray lines in (iv) represent skeleton of particle surfaces in (iii). It was then possible to select the volumes inside particles (shown here in blue) to measure them to correct for available area (pore space). Scale bar = 200 μm. (v-vi) Example image of a section of lettuce root before and after the application of a median filter and subsequent thresholding were applied. This allowed the selection of the internal volume of the root for measurement (shown in blue). Scale bar = 200 μm. (B) Quantification of bacterial distribution in transparent soil with small (500–850 μm) and large particles (850–1200 μm). The positions R1 to B3 represent a 3 × 3 grid of points on and around the roots, where R is on the root and A and B are at intervals perpendicular to the root. 1 is the root tip and 2 and 3 are closer to the shoot. See Figure S1 for schematic. (i) There was higher bacterial abundance in images that include a section of plant root. At all other positions, there was a consistent area of bacterial fluorescence as a proportion of the area of backgound in images without plant roots. These values were corrected for available area. (ii) Number and (iii) average size of bacterial aggregates at the 3 horizontal (X) positions (R, A & B) and at the 3 vertical (Y) positions (1, 2 and 3) in samples with or without plants. Letters above the bars indicate the results of Fisher's protected LSD tests.