Rhizosphere visualization
The Rhizosphere is the zone surrounding the roots of plants
in which complex relations exist among the plant, the soil microorganisms
and the soil itself. The plant roots and the biofilm associated with them
can profoundly, influence the chemistry of the soil including pH and nitrogen
transformations.
The fluorescence microscopic image below shows red
fluorescing fungal filaments across the rhizosphere of spring wheat together
with mainly red fluorescent bacteria. The original image was at 100X and
is a part of the ASM Biofilm Collection.
This exercise developed by J.R. Heckman (Plant Science
Department, Rutgers University, New Brunswick, NJ.) and J.E. Strick (Program
in History of Science, Princeton University, Princeton, NJ. illustrates
a technique by which the rhizosphere of plants can be visualized by its
effect on soil pH.
Background
As explained by Heckman and Strick (1996), nutrients are
absorbed by the roots of plants as cations and anions. The differential
uptake of these ions can affect the pH of the rhizosphere. Among the most
important of these ions are nitrate NO3- and ammonium NH4+. The absorption
of ammonium ions, for example, promotes the efflux of H+ ions and reduces
the pH of the rhizosphere, while the absorption of NO3- ion promotes the
efflux of OH- and raises the rhizosphere pH. This change in pH is localized
to the region adjacent to the root and does not affect the bulk soil pH.
Instructions
In this exercise, 5-10 seeds (Ryegrass was used orignally,
although other seeds should also work) are planted in soil contained in
a box with a glass window which permits observation of the roots growing
below the level of the soil. During germination and growth of the plants,
the window is covered with an opaque sheet of paper or plastic in order
to exclude light. The progress of root growth can be observed by occasionally
removing the covering. When the roots have grown so that they cover most
of the surface of the glass plate visualization of the rhizosphere pH can
be attempted.
A water agar suspension is made by adding 7.5 g of purified
agar to 1 liter of distilled water. To this is added 0.06 g of bromcresol
purple and 0.136 g of CaSO4. This solution is brought to a boil to dissolve
the agar and is then cooled to just above the point of solidification (about
38 C).
The glass plate covering the plant roots is carefully removed so as to
not disturb the soil or roots. The agar solution is slowly poured over
the surface of the soil to a depth of a few mm. As the first layer hardens,
another can be added until a layer of about 5 mm thickness is reached.
Within ten minutes, color changes begin to occur in the agar which outline
the rhizosphere. These changes are complete within 20 to 30 minutes.
Preparation of the soil
A sandy loam admixed with organic matter and with a pH of
about 6.2 and low in available nitrogen was used in the investigation.
The soil is sieved to pass through a 2-mm sieve. To the soil was added
0.44 g of CaHPO4, 0.19 g of KCl and 1.0 g of CaSO4 per kg of soil. Several
different soil mixtures were used in the original study, those that seemed
to work best were the following.
To the soil mixture described above were added one of the
following:
-
Ca(NO3)2 at 0.59 kg-1 soil. Or
-
(NH4)2SO4 at 0.47 g kg-1 and N-Serve 24, Nitrapyrin [2-chloro-6-(trichloromethyl)pyridine]
and 0.01 g kg-1 soil. The N-Serve is prepared as a stock solution 200 mg
N-Serve in distilled water. Fifty ml of the stock solution is dribbled
over the soil which is then thoroughly mixed.
Note: N-Serve is hazardous and inhalation and
contact with skin must be avoided. The stock solution should be prepared
in a hood. The function of the N-Serve is to prevent Nitrification by resident
soil organisms which might obscure the pH reaction. N-Serve is available
from DowElanco, Indianapolis, IN 46268.
The growth chambers
The original study employed Root-Vue Farms available from
Edmund Scientific Co., Barrington NJ. These have the advantage of uniformity
and convenience although they are rather costly.
A home made alternative can be made from half gallon paper
milk containers and acetate sheets. This alternative is modeled on an design
described in the Microcosmos Curriculum Guide to Exploring Microbial Space
(Kendall Hunt Pub. Co.) an exercise called Subterranean Space Station.
A window is cut into a milk container as described in the accompanying
diagram. A piece of acetate sheet is inserted into opening on the inside
of the milk container. This sheet will be held in place by the soil as
it is added. The acetate sheet must be long enough so that it may subsequently
removed. Soil preparation and seed germination and growth are as described
above. In order to add the agar solution containing the bromocresol purple,
a frame of the dimensions of the window will be needed to contain the agar
while it hardens.
References
Heckman, J.R. and J.E. Strick, 1996, Teaching Plant-Soil
Relationships with Color Images of Rhizosphere pH. Journal of Natural Resources
and Life Sciences Education.
Zook, Douglas, 1992, The Microcosmos Curriculum
Guide to Exploring Microbial Space, Kendall Hunt Publishing Company, Dubuque,
Iowa. ISBN 0-8403-8386-X