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Soil Science 113

(Soil Microbiology)

Exercise 4
Rhizobium-Arbuscular Mycorrhizal- Legume Symbiosis

Objective: Demonstrate that rhizobial inoculation and arbuscular mycorrhizal inoculation can
improve growth of legumes.

Plastic pots
Legume and AMF inoculants


Pot Experiment

1. Label each pot with the treatment, date, and your team number. The treatments are the

Pot# Treatments

1 uninoculated

2 plus mycorrhizal fungi

3 plus Rhizobium

4 plus mycorrhizal fungi and Rhizobium

2. Fill the pots with unsterilized 1 kg soil.

3. Apply 0.1 g complete fertilizer (14-14-14) to all pots.
4. With spatula, dig hole approximately 5 cm at the center of the pot; to pots #2 and #4,
place 5 g of mykovam to previously made hole while to pots #1 and #3, put sterilized
sand into the hole.
5. Sow three surface sterilized peanut seeds in the hole. To pots #3 and #4, sow peanut
seeds pre-inoculated with Rhizobium.
6. Add water to each bag to bring the soil to field capacity.
7. After one week, thin the plants to one plant per pot. The experiment will end in 6 weeks.
8. The plants will be harvested at 6 weeks after sowing. With a razor blade, cut the plant
top off at the soil surface and get the wet weight. Subsequently place the plant top in
labeled paper bag and dry in an oven set at 80 0C.
9. Carefully invert the pot and remove the root boll. As much as possible, save the roots
and nodules intact by gently removing the soil from the roots. Collect all detach roots
and nodules and add them with the intact root system. As soon as most of the soil is off
the roots, wash the roots gently under running water. Continue washing the roots until all
the soil is removed.
10. Pat the roots dry with paper towel and detach the nodules from the roots. Record the
number and wet weight of nodules. Place the nodules in small vials with silica gel for
future isolation of rhizobia.
11. With a razor blade, cut approximately 1 g roots from each treatment pot. Place these
root samples in 50 % ethyl alcohol and set aside for laboratory exercise on mycorrhizal
infection determination.


1. What is leghemoglobin and what is its significance

2. Discuss the course of events in the formation of root nodules
3. Why did you use unsterilized soil? Will the result be different if you had used sterilized
4. Why is there a need to surface sterilize the seeds prior to planting?
5. Describe the steps you followed in inoculating the seeds with Rhizobium?