EXERCISE 2
TO OBSERVE THE INFECTION PROCESS
Clover rhizobia enter their host's roots through the root hairs.
Infection is preceded by a deformation of root hairs and the
forming of an infection thread which can be observed directly
under the microscope.
Root hair deformations may also be caused
by non-nodulating strains of Rhizobium.
Non-nodulating strains
used in this chapter cause no infection threads to form.
Key steps/objectives
l)
Culture strains of Rhizobium in YM broth
2)
Sterilize and germinate clover seeds
3)
Mount seedling on microscope slide
4)
Incubate the seedlings in inoculated mineral medium
5)
Observe root hair deformation and infection threads
6)
Compare root hair deformations caused by different kinds of
rhizobia strains
(a)
Culturing strains of rhizobia in YM broth
(Key step 1)
Inoculate 50 ml flasks or test tubes containing 20 ml of YM broth
in duplicate with the strains listed below:
1)
Rhizobium leguminosarum bv. trifolii (TAL 382) isolated from
nodules of Trifolium semipilosum
2)
R.l. bv. trifolii non-infective isolated from nodules of
Trifolium sp.
3)
R.l. bv. trifolii (TAL 1185) isolated from nodules of
Trifolium repens
4)
R.l. bv. phaseoli (TAL 182) isolated from nodules of
Phaseolus vulgaris
5)
R. meliloti (TAL 380) isolated from nodules of Medicago
sativa
6)
Bradyrhizobium sp. (TAL 764) isolated from nodules of
Lupinus angustifolius
Other strains of the same species of rhizobia may be substituted.
Incubate at 25-30C for 5-7 days on a rotary shaker.
(b)
Germinating seeds
(Key step 2)
Choose a small-seeded legume.
Clover, especially Trifolium
repens or T. glomeratum, is most suitable for this exercise.
Surface sterilize seeds according to the procedure outlined in
Appendix 10.
Some clover species may need scarification with
sulfuric acid.
Others, like Nolan's white clover and strawberry
clover (Trifolium fragiferum) germinate easily without
scarification.
Wash seeds with at least eight changes of sterile
distilled water.
Aseptically place the seeds onto water-agar
plates for germination.
Incubate the plates inverted for 48 h or
more until roots are 6-8 mm long.
(c)
Preparing a Fahraeus slide
(Key step 3)
Prepare 10 ml of Fahraeus carbon and nitrogen free medium
(Appendix 3) containing 0.6% agar in a 15 ml tube.
Cool the
liquid agar medium to 48C in a water bath.
For each strain of Rhizobium used, prepare two sterile 50 ml
boiling-tubes containing 25 ml Fahraeus carbon and nitrogen-free
medium without agar.
uninoculated controls.
Set up two additional tubes for
Cover with 50 ml beakers.
Transfer approximately 0.2 ml of agar medium to a sterile
microscope slide using a Pasteur pipette fitted with a rubber
bulb.
Leave one-half of the slide empty.
This is best done by
lining up the slide and a long coverslip side by side in a
sterile Petri dish (Figure 2.1).
Place the agar in five or six
drops onto the bottom half of the slide.
Immediately, transfer a
well formed seedling to the slide with a sterile inoculation
loop.
Place the seedling onto the slide in such a way that the
root tip is immersed in the agar and the cotyledons are in the
empty half of the slide.
With sterile forceps, carefully place
the long coverglass over the agar and the root tips.
If the seed
coat adheres to the cotyledons on the seedling, carefully remove
it with sterile fine tipped forceps.
Figure 2.1. Petri dish with
Figure 2.2.
Placement of
Components of Fahraeus slide
seedlings on Fahraeus slide
Transfer the slide mounted seedlings to the tubes containing the
Fahraeus mineral medium.
(d)
Inoculating the seedlings
(Key step 4)
Using the broth cultures which have been set up for this
experiment in (a), inoculate two seedlings with each of the six
strains of Rhizobium by adding five drops of the cell suspensions
to individual tubes containing the mineral medium and the
Fahraeus slides.
Alternatively, the seedlings may be inoculated by incorporating a
cell suspension into the Fahraeus agar medium before the seedling
is placed onto the slide.
This speeds up the infection process.
Add five drops of sterile broth medium to the controls.
Incubate at 25-30C in a well-lighted environment.
(e)
Observing the root hairs under the microscope
(Key step 5)
After 24 h remove Fahraeus slide from the nutrient tube and
examine it under the microscope.
absorbent filter paper.
Remove the excess solution with
Observe with phase contrastor ordinary
bright field microscope under low and high power magnifications.
Search for root hair deformations and/or curling and infection
threads.
Mark the position of your slide on the microscope stage
so that the same spot may be found in later observations of the
same root hair infections.
12-24 h.
Make observations in intervals of
Periodic observation may be made at shorter intervals
if inoculation was done by including the cell suspension into the
agar medium. Return the slide to its tube between observations.
Take precautions against undue contamination when returning the
slide to the mineral medium.
Aseptic conditions cannot be
maintained beyond the first observation.
However, contamination
usually does not interfere provided the root hairs chosen for
observations are not located at the edges of the microscope
slide.
(f)
Comparing root hair deformations
(Key step 6)
Photograph or draw the root hair deformations or curling caused
by each strain.
Distinguish full curling from slight curling and
root hair branching.
the root hairs.
Note the effects of noninvasive strains on
Compare the deformations caused by the various
strains used.
Typical root hair deformations, like the shepherd's crook, are
shown in Figure 2.3.
Figure 2.3. Deformed white clover root hair infected with R.
trifolii 0403.
thread.
Note the sheperd’s crook and the infection
(Photo courtesy of F. Dazzo)
Figure 2.4. Selective proliferation and colonization of
Rhizobium trifolii on a root hair of its host legume clover
in a Fahraeus slide system. (Photo contributed by B.B.
Bohlool)
Figure 2.5. Rhizobium trifolii inside infection thread in
the root hair of its host clover (Trifolium repens). (Photo
contributed by B.B. Bohlool)
Requirements
(a)
Culturing R. leguminosarum bv. trifolii strains in YM broth
Rotary shaker
Twelve 50 ml flasks (or tubes) containing 20 ml culture
broth each
Inoculation loop, flame
Slant cultures of clover rhizobia strains TAL 382, TAL 1185,
TAL 182, TAL 380, TAL 386, noninfective strain of clover
rhizobia
(b)
Germinating seeds
Incubator
Materials and tools for sterilizing seeds (Appendix 10)
Plates of water agar (7.5 g agar per liter distilled water)
Seeds of clover (Trifolium repens, T. glomoratum or other)
(c)
Preparing a Fahraeus slide
Water bath
Sterile microscope slides (1 mm x 24 mm x 40 mm)
Coverslips (kept in sterile Petri dishes)
Pasteur pipettes (sterile); rubber bulbs
Inoculation loop, forceps, flame
Fahraeus C and N free medium
Fahraeus medium plus 0.6% agar in 15 ml tube
Seedlings of clover
Fahraeus medium (25 ml) in tubes (39 mm x 150 mm) with
covering 50 mm beakers
(d)
Inoculating the seedlings
Growth chamber (or well lighted environment) at 25-30C
Pasteur pipettes (sterile); rubber nipples
Tubes with seedlings from (c)
(e)
Observing the root-hairs under the microscope
Microscope with phase or bright field condenser
Forceps
Filter paper (sterile and absorbent)
Seedlings in inoculated Fahraeus solution from (d)
(f)
Comparing root hair deformations
Microscope as in (e) with camera attachment