"Battle of Space Invaders-Episode III-Allelopathic Suicide"

The purpose of this experiment is to test the autotoxic properties of alfalfa stem and leaf leachates. It is hypothesized that autotoxic allelochemicals exist in alfalfa leaves and stems that will inhibit germination of alfalfa seeds. Furthermore, it is hypothesized that these allelochemicals are water soluble and will be found in the water and methanol fractions rather than the heptane fractions.

Four leachates are produced by first adding 20 grams of alfalfa leaves to each of two 960mL. jars and 20 grams of chopped alfalfa stems to two more 960mL. jars. To one jar of leaves and one jar of stems, 700mL. of methanol is added, and an equal volume of distilled water is added to the other two jars. After soaking for 24 hours later, the lids are removed, and the water and methanol are allowed to evaporate until approximately 200mL. of liquid in each jar is left. The leachates are strained to remove the solid debris. Then, 70mL. of each of the leachates are mixed in a separating flask with an equal volume of heptane. Once two layers form in the flask, the layers are drained into separate beakers, creating eight fractions. Alfalfa seed germination trials are used to confirm the presence of allelochemicals in each fraction. Germination trials using distilled water, methanol, and heptane are performed to use as controls.

The average percent germination for each distilled water and methanol fraction was compared to the average percent germination for the corresponding control. These fractions inhibited germination at least 20% more than the control. Therefore, an autotoxic allelochemical was present in the leaves and stems. With one exception caused by a technical error, the heptane fractions had germination percentages within 5% of the controls, indicating that only small amounts of the allelochemicals were found in the heptane fractions. The data supported both hypotheses.

It has been known by botanists for hundreds of years that plants are constantly at war with one another using a weapon called allelopathy. They produce and release allelochemicals into the environment that are toxic to specific plants. However, no one imagined that plants could also produce chemicals that are harmful to themselves. That was until the 1980’s, when horticulturalists discovered that the re-establishment of Medicago sativa, commonly known as alfalfa, was often unsuccessful due to allelopathic suicide created by the “autotoxic effects of the crop on seedlings” (1).

According to well known botanist Dr. Alan Putnam, “Autotoxicity is an intraspecific form of allelopathy that occurs when a plant species releases chemical substances that inhibit or delay germination and growth of the same plant species” (1). The process that produces many potentially autotoxic compounds is called secondary metabolism, “the production of any compound not essential to the growth and basic needs of the plant,” (3). The environment is an important factor that affects the type and amount of secondary metabolites produced (3). Phenolic compounds, terpenoids, non-protein amino acids, and alkaloids are just a few examples of allelochemicals (3). These chemicals have the ability to do such things as damage membranes, inhibit plant growth, and poison essential proteins (3).

Alfalfa’s autotoxicity is well documented in field studies conducted in Alberta, Canada and in Nebraska (6). Signs of autotoxicity in the perennial legume are inhibited germination, poor establishment, and low productivity when grown after itself (2). Inhibition of root and shoot growth is the result of the aqueous extract from alfalfa shoots (2). Roots can become swollen, curled, discolored, and lack root hairs (1). In the 1980s, it was discovered that medicarpin is the autotoxic allelochemicals in alfalfa (2). When tested in a germination trial using alfalfa seeds, this chemical reduced germination by fifty-nine percent (2).

Producers of this forage crop must know how to reduce the occurrence of allelopathic suicide, and there are a number of ways to do so. Some methods include seeding at least two weeks after plowing, maintaining adequate soil fertility and pH, and removing top growth by grazing or burning before plowing (2). Another way of controlling autotoxicity is to use crop rotation (5).

Since alfalfa is a legume, it has a taproot with symbiotic bacteria capable of fixing free nitrogen from the atmosphere (4). This fixed nitrogen is available to the alfalfa plant and about 25% of the nitrogen in the plant stubble becomes available for other crops following tilling of the alfalfa field (5). Cereal grain crops and annual forage grasses are the best rotational crops to use with alfalfa (5). To plant alfalfa in September, it is best to harvest a small grain crop in June thus allowing time for proper seedbed preparation (5). Soybeans, peanuts, and other legumes do not make good rotational crops for alfalfa as they are not resistant to the allelochemicals produced by alfalfa (5).

No-till agriculture is becoming a popular method of soil conservation. Producers leave what is left of the harvested crop in the field and reseed with the same plant or rotate with another one. Some horticulturalists believe that this method can work with alfalfa as long there is adequate soil pH and fertility, good surface and internal drainage, proper weed and insect control, and enough moisture for germination and growth of alfalfa seedlings (5). This method would work if alfalfa seed was planted in small cereal grain stubble. However, because of autotoxicity, alfalfa stubble would have to be burned or grazed smooth in order for this no-till method to work. Otherwise, the chemical in the remaining stems would poison the newly planted alfalfa.

Some scientists are researching ways to genetically modify alfalfa to decrease the autotoxic effects. One such modification involves the formation of specialized cells or tissues to segregate the toxic compounds in organelles (3). Another idea involves a backup detoxification system within the plant that protects the plant when sensitive tissues come in contact with toxins (3). Autotoxicity may be a severe problem; however, it is one that is preventable if the producer is well informed about allelopathic suicide.

The purpose of this experiment is to test the autotoxic properties of alfalfa stem and leaf leachates. Methanol and water are used to produce the leachates. In turn, these leachates are extracted with heptane to test the solubility of the allelochemical(s) and to produce eight fractions. Germination trials are used to detect the presence of autotoxic allelochemicals in each fraction.

It is hypothesized that autotoxic allelochemicals exist in alfalfa leaves and stems that will inhibit the germination of alfalfa seeds. In nature, rain and ground water is responsible for leaching allelochemicals from the plant. Therefore, it is hypothesized that these allelochemicals are water soluble compounds and will be found in the water and methanol fractions and not in the heptane fractions.

Materials and Procedures

4 – 960mL. jars with lids

Methanol

Heptane

distilled water

alfalfa seed

round foil trays 70mm in diameter

8 – 120mL. amber plastic bottles

filter paper

wire mesh strainer

separating flask

100mL. graduated cylinder

10mL. graduated cylinder

gloves

protective goggles

re-sealable plastic bags

marker

paper towels

I. Preparation of Leachates

1. Weigh out 20 grams of alfalfa leaves and put into each of two 960mL. jars.
2. Repeat using chopped alfalfa stems.
3. Pour 700mL. of distilled water into one jar of leaves and one jar of stems.
4. In the two remaining jars, add 700mL. of methanol.
5. Label the jars accordingly and with the lids on, shake well and allow to soak overnight.
6. Remove the lids and place under a heat lamp to evaporate for approximately three days so the chemicals that leached from the leaves and stems are more concentrated.
7. Observe and record changes in color.

II. Heptane Extractions

1. Filter out the stems and leaves using a wire mesh strainer..
2. Rinse and dry the four 960mL. jars and return the remaining liquids back into the corresponding jars.
3. Using the 100mL. graduated cylinders, measure out 30mL. of one sample.
4. Then, add an equal amount of heptane and shake well.
5. Pour mixture into a separating flask and allow the two layers to once again settle out.
6. Carefully remove the bottom layer into a labeled 120mL. bottle.
7. Next, drain the heptane layer into a separate bottle.
8. Repeat steps 3 through 7 except use 40mL. of sample and heptane in step 3 to attain a total of 70mL..
9. Repeat steps 3 through 8 for each of the four jars.

III. Germination Trials

1. Label the foil trays, allowing two for each of the eight fractions and three controls: distilled water, methane, and heptane.
2. Place the filter paper in the trays and moisten with 2cc of sample from each bottle.
3. Allow the solvent to evaporate overnight.
4. After evaporation, place 50 alfalfa seeds in each tray and re-moisten with 1cc of distilled water.
5. Place in re-sealable plastic bag on top of a paper towel to absorb excess moisture.
6. Allow to germinate in a dark place for three days.
7. Next, count and record the number of germinated seeds.
8. Repeat steps 2 through 7 for each sample and the three control samples.

Alfalfa Seed Germination Trials
Controls
	Percent Germination
	Trial 1	Trial 2	Average
Distilled Water	28%	27%	27.5%
Heptane	27%	24%	25.5%
Methanol	29%	32%	30.5%
Leaves
	Percent Germination
	Trial 1	Trial 2	Average
Distilled Water Fraction	0%	13%	6.5%
Distilled Water Heptane Fraction	32%	25%	28.5%
Methanol Fraction	0%	8%	4.0%
Methanol Heptane Fraction	27%	21%	24.0%
Stems
	Percent Germination
	Trial 1	Trial 2	Average
Distilled Water Fraction	0%	0%	0.0%
Distilled Water Heptane Fraction	32%	16%	24.0%
Methanol Fraction	0%	0%	0.0%
Methanol Heptane Fraction	18%	7%	12.5%

In the separation flask, the heptane fractions formed the top layers. The distilled water and methanol fractions formed the bottom layers. Two germination trials are performed and an average percent germination is calculated for each fraction and control.

This graph shows that the average percent germination for the leaves and stems fractions is at least 20% less than the distilled water control, indicating that autotoxic allelochemicals are present in these fractions.

This graph of the methanol leaf and stem fractions shows that the average percent germination of these two fractions is at least 20 % less than the methanol control sample. Autotoxic allelochemicals in the methanol fractions are responsible for the inhibition of germination of the alfalfa seeds.

This graph compares all four heptane extracted fractions with the heptane control sample. Except for the methanol stem fraction, the average percent germination values are within 5% of the control, indicating that the autotoxic allelochemicals are not heptane soluble. The methanol stem fraction has an average percent germination value of 12.5%, which is 13% less than the control. One explanation for this result could be that an autotoxic allelochemical is present in the stems that is slightly soluble in heptane. Another possible explanation is that this fraction was contaminated with the lower methanol soluble fraction containing autotoxic allelochemicals during the separation process.

Conclusions

The germination trials on the eight fractions did confirm both hypotheses. An autotoxic allelochemical was present in the alfalfa leaves and stems that inhibited the germination of alfalfa seeds. Also, the allelochemical(s) were more soluble in methanol and distilled water fractions than the heptane fractions.

Recommendations for Future Studies

To aid alfalfa producers in their search for suitable rotational crops, germination trials could be performed using alfalfa leachates. Also, a bioassay could be performed to test alfalfa’s allelopathic effects on other crops.

Acknowledgements

I would to thank the following people for all of their help and useful advice during this project:

§ Mr. Steven Kirby, Vinita High School Chemistry Teacher, for his help with the chromatography and supplying the chemicals for the germination trial.

§ Mr. & Mrs. Darrel Skidgel for providing me with the hard to find alfalfa seeds.

§ Dr. Mike Smith, Oklahoma State University Professor of Horticulture, for providing resources for this paper and advising me on the procedure.

§ and last but not least, my mom, Dr. Debbie Fimple, for sponsoring my project and providing me with helpful procrastination prevention tips.

Bibliography

1. Alfalfa Autotoxicity. 18 Feb. 2002. <http://www.gov.on.ca/OMAFRA/english/crops/fi

eld/autotox.htm>.

2. Autotoxicity and Alfalfa Establishment. 18 Feb. 2002. <http://www.forages.css.orst.ed

u/Oregon/Publications/Species/Alfalfa/Alfalfa_Autotoxicity.html>.

3. Autotoxicity in Plants. 18 Feb. 2002. <http://www.colostate.edu/Depts/Entomology/c

ourses/en570/papers_1994/marko.html>.

4. Beard, James B. Turfgrass: Science and Culture. New Jersey: Prentice Hall, 1973.

5. Crop Rotation. 23 Feb. 2002. <http://www.agr.okstate.edu/alfalfa/pub/stand-949/crop.htm>.

6. Miller, Darrell A. “Allelopathic Effects of Alfalfa.” Journal of Chemical Ecology 9 (1983): 1059 – 1072.