1. Killer Plant?
The Effect of Allelochemicals on the Formation of Root Nodules in Leguminous Plants
Amrita Sukhavasi

2. Problem:
Does alfalfa- leaf extract, an allelochemical, affect the formation of root nodules of leguminous plants?

3. Introduction:
Allelopaths are plants that release chemicals into their surroundings that inhibit the growth/development of the plants around them
Allelochemicals are chemicals that are released from allelopaths’ roots, passed through the soil, and absorbed by other plants. They harmful because they can restrain other plants’ nutrient intake or impede their root growth.
Leguminous plants are plants such as peas, beans, lentils, and peanuts that have a symbiotic relationship with nitrogen fixing bacteria in the soil, rhizobia.
These rhizobia attach onto these plants’ roots and form nodules to fix nitrogen for the plant.
Nitrogen Fixation is a process by which nitrogen (N₂) in the atmosphere is converted to inorganic nitrogen compounds such as ammonium (NH₄⁺). This can then be turned into amino acids and used by the plant.

4. Hypothesis:
The alfalfa-leaf extract will restrict the formation of root nodules by nitrogen-fixing bacteria.

5. Materials: 10 cm. diameter plastic pots
2 lbs. sterile sandy soil (4 parts sand to 1 part soil)
Autoclave
2 lbs. topsoil
50 g. alfalfa seeds
Plastic wrap
40 white clover seeds
Microscope
Beaker
Water
Growth Light

6. Procedure:
Prepare alfalfa-leaf extract: Grow alfalfa seeds for 2 weeks and soak sprouts in 50 ml of water for two days in a cool place.
Fill pots with sterilized, sandy soil (4 parts sand to 1 part topsoil) 1 cm. from the top.
Label pots with either “distilled water” or “alfalfa-leaf extract
Plant 10 clover seeds in each pot. Water each pot with either distilled water or alfalfa-leaf extract depending on the pot’s label.
Grow clover for about 6 weeks.
Gently remove plants from pots with the roots and rinse them to remove extra soil.
Using a microscope, count the number of nodules on each plant’s roots.
Record results and find the mean number of root nodules per plant.

7. three weeks after planting seeds
Experiment Pictures:
three weeks after planting seeds
six weeks after planting seeds
Plant Roots
Root Nodules

8. Experiment:
Independent Variable: the substance that the plant is watered with (either distilled water or alfalfa-leaf extract)
Dependent Variable: the amount of root nodules that form on the plant roots as a result of what it was watered with
Control: the plants watered with distilled water
Constants: The type of soil, amount of light, amount of water, and growing time for each pot

9. Data: Number of Root Nodules Substance Used to Water Plant Water
Number of Root Nodules
Substance Used to Water Plant
Water
Alfalfa-Leaf Extract 17 9 14 7 18 10 8 16 11 13
Average
15.33
9.25
Standard Deviation
1.97
2.05
T Test

10. Graph:
This graph shows the average number of root nodules for the plants watered with alfalfa- leaf extract and the plants watered with distilled water. Notice that the clover plants watered with the alfalfa- leaf extract formed fewer root nodules than the plants watered with water. This suggests that alfalfa- leaf extract impedes the formation of root nodules.

11. Possible Errors:
The plant’s roots could have choked each other out since they were planted so close to each other in the pots
Different percentages of plants grew in both groups (60% of the seeds planted in the control group grew and 80% in the experimental group grew)
Some nodules may not have fully formed yet and thus may not have been accounted for in the data

12. Conclusion:
Hypothesis supported: The alfalfa-leaf extract will restrict the formation of root nodules by nitrogen-fixing bacteria.
This experiment contributes to the agricultural society by showing not to plant leguminous plants such as peas, beans, lentils, and peanuts near allelopathic plants. This ensures that the leguminous plant is able to healthily grow root nodules and fix nitrogen from the atmosphere.
This can lead to other useful studies, such as determining whether allelopathic plants can be used as natural pesticides instead of artificial fertilizers which are harmful to the environment.

13. Resources:
Achenbach, J., & . (n.d.). Plants on the Warpath. National Geographic Magazine,
Ferguson, J. J., & Rathinasabapathi, B. (n.d.). Allelopathy: How Plants Suppress Other Plants [Data file]. Retrieved from edis.ifas.ufl.edu/‌pdffiles/‌HS/‌HS18600.pdf
A Study of Allelopathy in Plants. (2000) (M. DeLacy & L. Selger, Eds.). In Encouraging Student Biological Research (pp , 46-51). National Association of Biology Teachers.
Tebo, M. (2010). Chemical Warfare in the Plant Kingdom. In Killer Plants. Retrieved from
Westra, E. P. (n.d.). Can Allelopathy be Incorporated into Agriculture for Weed Suppression? [Data file]. Retrieved from
Wojciechowski, M. F., & Mahn, J. (2006). Nitrogen Fixation and the Nitrogen Cycle. Retrieved from