EXERCISE 16
TO SCREEN RHIZOBIA FOR NITROGEN FIXATION POTENTIAL
The nitrogen fixation potential of a number of strains of pure
cultures of Bradyrhizobium japonicum in symbiotic association
with soybean is compared.
The most effective strains in this
exercise will be compared later in potted field soil.
Key steps/objectives
1)
Prepare Leonard jars
2)
Culture rhizobia
3)
Prepare water-agar plates
4)
Sterilize and plate seeds for germination
5)
Plant and inoculate seedlings in Leonard jars
6)
Observe progress of experiment
7)
Harvest experiment
8)
Analyze data
(a)
Experimental design and treatments
The experiment is set up as a Randomized Complete Block Design
(RCBD) with three blocks or replications (Figure 16.1).
There
are 14 inoculation treatments, a plus-nitrogen control with no
inoculation, and a non-inoculated control with no nitrogen. The
plus-nitrogen control will contain 70 ppm N applied as a 0.05%
KNO3 (w/v) solution.
The nitrogen is added to the nutrient
solution in the reservoir of the Leonard jar assembly.
(b)
Preparing Leonard jars
(Key step 1)
A total of 48 Leonard jar assemblies will be required.
Prepare
the jars as explained in Appendix 11.
(c)
Culturing the rhizobia
(Key step 2)
Each of the 14 strains of B. japonicum to be evaluated is
cultured for 5-7 days prior to planting.
Grow the rhizobia in
100 ml Erlenmeyer flasks containing 20 ml of yeast-mannitol
broth.
Incubate these at room temperature (25-30C) on a rotary
shaker for 5-7 days.
(d)
Surface-sterilizing the seeds
(Key step 3 and 4)
Check the germination (percentage viability) of the soybean seeds
and surface sterilize a sufficient number of uniform, undamaged
seeds to give about 200 germinated seeds.
Sterilize by immersing
seeds in 3% sodium hypochlorite solution for 3-5 minutes as
described in Appendix 10.
Germinate the seeds by plating on
sterile water- agar (0.75% [w/v]) and incubate at room
Figure 16.1.
An example of a randomized complete block design
experiment.
temperature (25-30C) until the radicles are 0.5 - 1.0 cm long.
Avoid overcrowding agar plates with the seeds.
(Contact between
seeds in an overcrowded plate increases the risk of
cross-contamination from a partially sterilized seed to
neighboring seeds.
Uncrowded plates [approximately 25-30 seeds]
produce more uniform and better germination due to better
availability of moisture.)
(e)
Planting and inoculating of seeds
(Key step 5 and 6)
Follow the method for planting and inoculating the seeds
described in Exercise 15.
each jar.
Plant three, well-germinated seeds in
Plant three jars per treatment.
indicate block (replicate) assignment.
Label the jars and
Group the treatments
according to block assignment and keep them separated.
Remove all Leonard jars of Block I to the growth room (or
glasshouse) bench.
Randomize the placement of the jars within
Block I.
Similarly randomize the placement of the Leonard jars of Block II
and Block III.
Make daily observation of the experiment.
Five to ten days after
planting, thin to two uniform plants per jar.
down the controls first.
Begin by thinning
Excise the shoot of the unwanted plant
with sterilized scissors.
Bear in mind that growing conditions
such as temperature and light intensity during this experiment
must be in the range to which the species are adapted.
Excessive
temperatures are particularly damaging and can severely impair
the infection process, nodule development, and nodule function.
Plants may "green-up" gradually at the time that nodules begin to
function, delivering fixed nitrogen for plant metabolism.
Plants
inoculated with ineffective strains of rhizobia, and also the
uninoculated controls, will remain yellow (chlorotic) and
stunted.
(f)
Harvesting the plants
(Key step 7 and 8)
Harvest the plants after 30 days.
To minimize errors during
harvest, the stem should be cut at the point of cotyledon
attachment.
This point is marked by a scar on the stem.
These
scars are not visible in some species.
The stem should then be
cut at the level of the growth medium.
Place the plant shoots in
labeled paper bags.
Dry to constant weight at 70C for 2 days.
Each bag should contain the plant shoots from only one jar.
(Paper envelopes may be substituted for smaller plants, e.g.,
Centrosema, Trifolium, Desmodium, etc.)
Roots and adhering rooting medium are dislodged into a coarse
sieve.
Wash the rooting medium from the roots using a gentle
stream of water.
Describe the nodule distribution mentioned in
Appendix 1 (e.g., prolific tap-root nodulation; occasional
nodules on lateral roots and distant from the tap-root; large
numbers of small nodules; small number of large nodules).
Detach
the nodules, count them, determine their total fresh weight, and
place them in vials or aluminum foil weighing-boats for drying.
Dry the nodules to constant weight at 70C for 2 days.
(Nodule
harvest from each Leonard jar must be treated individually as in
the case of the shoots.)
Do not pool nodules of the three
replicates of any one treatment into a single vial.
Determine dry weight of shoots and of nodules for all treatments.
Perform an analysis of variance on the dry weight data (shoots
and nodules) using the method as described in Appendix 17.
Plot the mean shoot weight (Y-axis) against the mean nodule dry
weight (X-axis).
Determine the correlation coefficient (r) of
the plot and test the significance of r at the 5% and 1% levels
of confidence.
Draw the "best" regression line on your plot after determining
the regression equation for the regression line.
Shoot weight and nodule weight are usually highly correlated,
thus shoot weight is used routinely as an indicator of relative
strain effectiveness.
Other parameters that are highly correlated with shoot weight are
total nitrogen of shoot and nodule dry weight.
Nitrogenase
activity (acetylene reduction) may not easily correlate unless
done under very controlled conditions.
Requirements
(a)
Experimental design and treatments
No special requirements
(b)
Preparing Leonard jars
48 Leonard jars
(c)
Culturing the rhizobia for testing
Agar-slant cultures of B. japonicum
Yeast-mannitol broth
Shaker
(d)
Surface-sterilizing the seeds
Soybean seeds
Sodium hypochlorite solution (3%) or commercial bleach
(Chlorox)
Water agar plates
Incubator
(e)
Planting and inoculating of seeds
Broth cultures of B. japonicum from (c)
Pregerminated seeds
Sterile pipettes (1 ml) or Pasteur pipettes
Alcohol lamp and matches
Forceps, glass rods, and alcohol spray bottle
0.05% KNO3 (w/v) solution
Bench space in greenhouse
(f)
Harvesting the plants
Scissors, paper envelopes or bags
Coarse sieve, vials or aluminum foil weighing boats
Drying oven (70C)
Weighing balance