“Battle of the Space Invaders”

Episode II

A Study of the Allelopathic Properties of Garden Mulches

Allelochemicals are chemicals found in many plant tissues that inhibit the growth of competing plant species in different ways, such as prohibiting photosynthesis, stunting root growth, or inhibiting seed germination. In trees, these chemicals are found in the leaves and in the resins in the limbs and trunk. Allelochemicals can be released from the wood or leaves as the tree decays, or they can be released via the roots. Therefore, the study of the allelopathic properties of garden mulches made from shredded tree material should be of great importance to gardeners, landscapers, and horticulturalists.

This experiment is designed to test for the presence of allelochemicals in six mulches in an attempt to find a mulch that inhibits the growth of such “space invaders” as bermuda and fescue grasses without adversely affecting the growth of radish and tomato plants. The mulches are evaluated for allelopathic properties in several different trials that include a seed germination inhibition trial; measurements of soil pH; measurements of fescue root and blade length to look for stunting of root growth post-mulching; spectrophotometer analysis of chlorophyll content of bermuda grass before and after grass is sprayed with mulch teas; and measurements of plant height and fruit or vegetable production post-mulching. Visual observations of plant health are also recorded.

The data confirmed the hypothesis for the Juniper and Blue Spruce mulches as they leached allelochemicals that adversely affected bermuda and fescue without affecting radish and tomato production. Allelochemicals in the Pine mulch also stunted bermuda and fescue grass production. The soil pH data shows that the Pine and Cypress mulches leached allelochemicals that reduced soil pH and adversely affected plant growth.

Mulch is an attractive covering used in gardening and landscaping and is usually made from shredded trees. Mulches help the soil retain moisture, protect the plants from extreme temperatures, and add nutrients to the soil as the mulch decays. Weed control is another important property of some mulches. (7)

The leaching of allelochemicals from the mulch material may play a role in controlling those unwanted space invaders, like weeds and grasses. Allelochemicals are compounds released by one plant species that affects other nearby plants to prevent overcrowding. (3) However, those allelochemicals released from the mulches may also affect the growth of flowers, trees, shrubs, and fruit and vegetable producing plants.

Allelopathy can be described as chemical warfare between plants. Allelochemicals can be released from the roots, leaves, fruits, or decaying plant materials. Chemicals with allelopathic properties have also been isolated from microorganisms associated with plants and soils. (3) Allelochemicals are specifically targeted and dissipate quickly which is an advantage over many synthetic herbicides.

Allelochemicals use several methods to kill the targeted plant. They can stunt the growth of the plant by either interfering with the activation of plant growth hormones or by blocking protein synthesis. Some allelochemicals can inhibit cell division in the meristematic zone of the roots, which decreases root growth. Allelochemicals can also starve a plant to death by interrupting photosynthesis. In a more complex method of killing a plant, allelochemicals uncouple the oxidative phosphorylation biochemical process within the plant cells. This in turn reduces energy production required for cell metabolism and plant life. (3)

The study of allelopathy is becoming more important in agriculture as the need for new herbicides is inevitable. In the United States, crop losses are estimated at $9-$10 billion annually. Some of the synthetic herbicides available today are causing undesirable environmental contamination and are becoming less effective and less selective. (1)

Research investigating the use of allelochemicals as biological herbicides involves testing crop rotation as a method of releasing allelopathic residues into the soil as the allelopathic cover plant dies. The most successful weed suppressors were sorghum, wheat, rye, oats, and barley. This approach to weed control may be timely, since there is now enthusiasm among farmers for reducing tillage and for using surface plant residues to conserve soil and moisture. (3)

The largest selling synthetic herbicide in the world is Round-Up. (4) The active ingredient, glyphosate, prohibits an enzyme involved in the formation of certain amino acids, and therefore disrupts protein synthesis in the plant. (5) This herbicide is very effective and kills all kinds of plants. Another advantage is that it does not spread in the soil and kills only the plants it comes in contact with. However, this property can also be a major disadvantage. It is necessary to apply Round-Up very carefully to ensure only the undesirable weeds are killed. This herbicide is also expensive. A 9.5 liters (2.5 gallons) container of Round-Up, which will treat about five acres, cost approximately $125. This herbicide is so widely used that genetically altered crops are being developed that are resistant to the chemical. These genetically modified plants are creating quite a stir in the world of organic agriculture as well as global politics. (4)

It is doubtful that plant products will be active enough to be packaged and sold as herbicides unless more unique and active compounds are discovered. (2) But, this does not mean that isolation and identification of allelochemicals is not important. Natural plant products can provide clues to the synthesis of safe, selective herbicides. In the future, plants may be genetically manipulated to increase their yields of useful allelochemicals. (2)

The purpose of this experiment is to study the allelopathic properties of garden mulches. The Blue Spruce mulch is used in this study because in last year’s experiment it proved to be a very effective seed germination inhibitor. Two other native Colorado trees, Pinyon and Juniper, are used to make mulches for this year’s experiment based on information provided by the Rio Grande National Forest Service. Botanist Dean Erhard suggested that the release of allelochemicals from Pinyon or Juniper trees in the Rocky Mountain foothills might be responsible for reduced vegetation in those areas, which is also affecting the wildlife population. Paper mulch made from shredded plain white paper is selected to determine if allelochemicals present in the other mulches are responsible for damage to the test plants or if the physical blockage of sunlight by a thick layer of mulch is responsible for plant growth inhibition. Two other mulches commonly marketed in Oklahoma, Cypress and Pine, are also included in this study.

A series of tests are used to evaluate the six different mulches. The Seed Germination Inhibition Trial and the Bermuda Grass Trial are designed to test for the presence of allelochemicals in mulch teas. Mulch teas are made by soaking the mulches in water for seven days. The Fescue Seed Trial, Radish Production Trial, and Tomato Production Trial are designed to test for allelochemicals that leach from the mulch as the planters are watered. Soil pH is measured during the Tomato and Radish Production Trials to see how the mulches affect this parameter.

If a mulch with the allelopathic properties that successfully inhibits fescue and bermuda grass growth without affecting radish or tomato production can be identified in this experiment, then, this mulch could be used in future studies to isolate allelochemicals that could be used to develop a new herbicide for vegetable gardens. Plus, after further testing on other types of plants and flowers, it might be possible to market this mulch as an “all natural product” that eliminates the need for expensive, synthetic herbicides.

It is hypothesized that allelochemicals with herbicidal properties are present in the garden mulches and will leach from the mulches causing adverse affects on invading bermuda and fescue grass without affecting production of the radish and tomato plants.

MATERIALS AND PROCEDURE

Test mulches

Tomato Plants

Radish Seeds

Bermuda Grass Seeds

Fescue Grass Seeds

Potting Soil

Fourteen plastic pots

(25cm. diameter)

Fourteen planters (60cm. x 17cm.)

Meter stick

Science Workshop 500 Interface

pH and temperature probes

Spectrophotometer

Graduated cylinder

250mL. beakers

70% Isopropyl Alcohol

Hot plate

Digital gram scale

Safety goggles

Six 960mL. sealable plastic containers

Large resealable plastic bags

Plain paper towels

Preparing Test Materials

1. Mulch teas are prepared by soaking g 720cc. of each mulch with 240cc. of distilled water in labeled plastic containers for seven days. Measure and record the pH of each tea.
2. Prepare planters and pots by spreading gravel in the bottom of the containers and adding equal amounts of an enriched potting soil leaving enough space for mulch.
3. Plant a tomato plant in each of the seven pots and secure them with a pipe cleaner to a dowel rod for support.
4. 15cc of Bermuda grass seed is planted in the other seven plastic pots.
5. Plant 5cc of radish seeds in seven more planters and thin out the sprouts leaving about 5cm. between plants before mulching.
6. Plant 15cc of Fescue seed in another seven planters.
7. Water all plant groups as needed. Apply fertilizer and insecticide as needed to protect the plants.

Seed Germination Inhibition Trial

1. Place twenty seeds onto a plain paper towel.
2. Saturate the towel with 15cc of the mulch tea and 15cc of the mulch.
3. Place them into labeled plastic bags.
4. Allow seeds to germinate for one week in a dark place at room temperature.
5. Repeat steps one through four for each mulch type and seed type to be tested.
6. Count and record the number of germinated seeds to determine the percent of inhibition rate.

Bermuda Grass Trial

1. Collect grass clippings from each pot before and seven days after spraying with the corresponding mulch tea.
2. Spray the Control pot with distilled water.
3. Observe and record observations on growth and color of grass blades daily.
4. Boil two grams of each grass sample individually with 50cc. of alcohol and pour them through a strainer into a graduated cylinder. Add alcohol to increase all samples to 100cc.
5. Determine the transmission percentage with a spectrophotometer set at a blue-green wavelength about 508nm.
6. Do three measurements on each sample to obtain an average percent transmission.

Fescue Grass Seed Trial

1. Mulch planters with a layer of mulch 10cm. deep as soon as the seeds are planted. Add no mulch to the Control planter.
2. Record visual observations daily on the amount of growth.
3. Count the number of sprouts in each planter two weeks post-mulching.
4. Pull ten sprouts at random from each planter.
5. Measure the blade and root length for each sprout.
6. Calculate the average blade and root lengths for each planter.

Tomato Production Trial

1. Apply a layer of mulch 10cm. in depth and label pots according to mulch used.
2. Measure plant height from top of much layer to highest leaf on the plant.
3. Add no mulch to the Control.
4. Wait one week and begin recording weekly pH readings and measuring plant heights.
5. Make and record visual observations on plant health, insect damage, and flower and tomato production every 7-14 days.
6. At eight weeks post planting, count all of the tomatoes and measure their circumferences with a ruler and a string or pipe cleaner.

Radish Production Trial

1. Mulch the radish planters with a layer of mulch 8cm. deep. Add no mulch to the Control planter.
2. Record observations on plant height, color, uniformity and insect damage and measurements of soil pH weekly.
3. Four weeks after mulching, count all of the radishes and measure and record the circumference of the ten radishes selected at random from each planter.

DATA

Seed Germination Inhibition Trial

	Percent Inhibition
	Radish Seeds	Tomato Seeds	Fescue Seeds	Bermuda Seed
Control	5%	5%	15%	100%
Blue Spruce	5%	45%	25%	100%
Cypress	0%	0%	20%	100%
Juniper	10%	60%	35%	100%
Paper	0%	0%	0%	100%
Pine	0%	10%	25%	100%
Pinyon	5%	5%	10%	100%

This trial shows that allelochemicals leached by the Blue Spruce, Pine, and Juniper mulches are the most effective in preventing the germination of unwanted fescue grass in a vegetable garden. However, the Blue Spruce and Juniper mulches should not be applied to the garden before the tomato and radish seeds have germinated. The Cypress mulch could be applied before germination of the radish and tomato seeds, but is not as effective as the Juniper mulch in keeping the fescue out of the garden. This procedure is not adequate for evaluating bermuda seed germination. Bermuda is a warm season, drought resistant grass and it is suspected that the moisture suppressed germination rather than the allelochemicals.

Bermuda Grass Trial

The visual observations made on the Bermuda grass pots did indicate that the Blue Spruce, Pine, and Juniper mulch teas did affect the growth of the grass as these three pots had more brown at the base of the grass blades and spots of retarded growth. Allelochemicals in these three mulch teas may have affected root growth, protein synthesis, or chlorophyll production.

The pots are rated seven days post-spraying in order of greatest amount of brown and areas of retarded growth to the greenest and most uniform in growth. The results are as follows:

1. Blue Spruce

2. Juniper

3. Cypress

4. Pinyon

5. Paper, Control.

Results of Chlorophyll Analysis (% transmission on spectrophotometer)
	Control	Blue Spruce	Cypress	Juniper	Paper	Pine	Pinyon
Pre-Spray Samples	47.2	49.3	52.6	48.3	40.1	45.4	46.2
Post-Spray Samples	67.5	67.6	64.6	61.0	66.0	65.1	69.9

Quantitative analysis with a spectrophotometer is used to evaluate chlorophyll content in grass clippings obtained before and after spraying the pots with the mulch teas. These results do not indicate that allelochemicals are responsible for the decrease in chlorophyll. All seven pots, including the Control sprayed with tap water, have an increase of percent transmission in the post-spray samples. This test procedure may have been more accurate if stolons had been clipped at soil level rather than at the top of the pot.

The amount of chlorophyll affects the percent transmission of light. A decrease in chlorophyll results in an increased percent transmission value.

Fescue Grass Trial

The planters are mulched as soon as the fescue grass seed is planted. Two weeks later, the blades of grass are counted and the test planters are ranked from most amount of growth to the least. The four planters with the least amount of growth, Juniper, Blue Spruce, Pine, and Cypress, also have the highest fescue seed germination inhibition rates.

Visual observations indicate that many of the Juniper and Cypress blades are yellow. This suggests that allelochemicals in these two mulches may also affect chlorophyll production.

Mulch	Number of Blades	Number of Inhibited Seeds	Seed Germination Inhibition Rate
Control	224	3 of 20	15%
Pinyon	146	2 of 20	10%
Paper	116	0 of 20	0%
Blue Spruce	100	5 of 20	25%
Pine	98	5 of 20	25%
Cypress	77	4 of 20	20%
Juniper	74	7 of 20	35%

Ten shoots are pulled from each planter two weeks post-mulching, and the blades and roots are measured. The average blade and root lengths are calculated. The following chart presents the average root and blade lengths for each mulch compared to the Control. A comparison of seed germination inhibition is also included. The values for the six test mulches are expressed as percent of deviation from the Control value.

	Average Blade Length (cm.)	Percent Deviation	Average Root Length (cm.)	Percent Deviation	Seed Germination Inhibition Percent Deviation
Control	5.93	0%	4.22	0%	3 seeds out of 20
Pinyon	10.83	+ 82.60%	2.86	- 32.20%	- 33.00%
Paper	6.49	+ 9.40%	4.07	- 3.30%	- 100.00%
Blue Spruce	9.50	+ 60.20%	3.38	- 19.9%	+ 66.00%
Pine	10.78	+81.00%	4.41	+ 4.50%	+ 66.00%
Cypress	9.62	+ 62.20%	2.82	- 33.10%	+ 33.00%
Juniper	10.92	+84.10%	3.97	- 5.10%	+ 133.00%

No correlation is found between root lengths and blade lengths. Therefore, inhibition of seed germination by allelochemicals rather than stunting of root growth appears to have the greatest effect on the amount of fescue grass growth in the planters.

One reason for using mulch is to hold moisture in the soil. All planters were mulched to a depth of 10 cm. However, even with a protective screen on top, the paper mulch was easily blown out of the planter. Without the protective layer of mulch, the Control and Paper planters would lose moisture more rapidly that other five planters. Also, the Pine mulch consisted of chunks of pine bark rather than shredded mulch like the Cypress, Juniper, Pinyon, and Blue Spruce. The water retention in the Pine planter could have been compromised by the lack of density of that mulch. The Pine, Control, and Paper planters had the longest average root lengths. The reason for this could be because the moisture evaporated more rapidly from these planters so the roots in those three planters had to reach deeper into the soil for moisture.

Radish Production Trial

The data for this trial showed that all of the mulches performed better than the Control planter with no mulch. Radish production was extremely poor for all seven planters. The type of radish seed used was recommended for early summer harvest. The hot July and August temperatures could have adversely affected radish production.

The Cypress mulch performed only slightly better than the Control in plant height and had 0% radish production. As mentioned in the Soil pH Trial, an allelochemical leached from the Cypress mulch could be responsible for the drastic drop in soil pH and poor plant performance. Therefore, the Cypress mulch should not be used in a radish garden.

The following chart presents the radish plant production data with the values for the six mulches expressed as percent of deviation from the Control value. A radish is counted as a developed radish if it has a bulb formation on its root equal to or greater than two centimeters in circumference. The circumference of five radishes pulled at random from each planter is used to calculate the average circumference.

	Average Total Height (cm.)	Percent Deviation	Developed Radishes	Percent Deviation	Average Circumference (cm.)	Percent Deviation
Control	9.5	0%	0 out of 18	0%	1.25	0%
Pinyon	14.0	+ 47.3%	1 out of 46	+ 100.0%	1.87	+ 50.0%
Paper	16.8	+ 76.8%	2 out of 45	+ 200.0%	4.87	+ 290.0%
Blue Spruce	15.0	+ 57.9%	1 out of 59	+ 100.0%	1.75	+ 40.0%
Pine	17.5	+ 84.2%	2 out of 38	+ 200.0%	3.75	+ 200.0%
Cypress	10.0	+ 5.2%	0 out of 28	0%	1.5	+ 20.0%
Juniper	16.5	+ 73.6%	1 out of 36	+ 100.0%	1.75	+ 40.0%

Tomato Production Trial

Visual observations on the overall health of each plant based on color of the stems and leaves, number of blooms, and leaf condition as well as insect damage are made bi-weekly. During the second week, all seven plants are viscously attacked by spider mites. An insecticide containing Cyfluthrin and Imidacloprid is used.

The following chart indicates the bi-weekly ranking of the plants in order from most to least healthy looking:

	Week 2	Week 4	Week 6	Week 8
1^st	Blue Spruce	Pine	Pinyon	Paper
2^nd	Pine	Blue Spruce	Blue Spruce	Blue Spruce
3^rd	Cypress	Paper	Pine	Cypress
4^th	Juniper	Pinyon	Juniper	Juniper
5^th	Pinyon	Cypress	Paper	Pinyon
6^th	Paper	Juniper	Cypress	Pine
7^th	Control	Control	Control	Control

The colored cells indicate that those plants are tied for the bi-weekly rating.

The hot Oklahoma August temperatures adversely affected tomato production on all seven plants. Also, none of the tomatoes ripened on the vine as the red color of the tomato fruit does not develop when temperatures are above 30°C (86°F).

Tomato plant heights are measured weekly and at the end of eight weeks, all tomatoes are harvested and their circumferences are measured. Based on tomato production, plant, growth, and condition ratings, the Blue Spruce and Pinyon mulches are the best mulches to use in a tomato garden.

The Cypress and Juniper mulches perform better than the Control on plant height, health, and production and appear to be safe to use on established tomato plants. The Paper mulch needs a longer testing period to be ruled in or out as an effective mulch as plant height and health improves at the end of the eight weeks.

The Pine mulch plant has the worst tomato production and the lowest plant health rating at the end of the trial. One reason for this poor performance could be that the soil pH for this mulch dropped from 7.1 to 5.0. The Pine mulch could have leached an allelochemical that over time dropped the pH of the soil below the desired pH level for tomato production (6.5). Therefore, the Pine bark mulch is not recommended for a tomato garden.

Mesonet data shows that the average high temperature for August is 35°C (95°F) and 31°C (88°F) in September. There are twelve days during the tomato trial where temperatures exceeded 37.7°C (100°F).

Do Mulches Affect Soil pH?

The starting soil pH is 7.1. The mulch teas range in pH from 6.9 to 5.0. The soil pH’s for all of the test planters dropped significantly including the Control planter with no mulch during the first two weeks. This drop in pH corresponds to the time period when the radish and tomato plants are growing rapidly. The removal of nutrients such as nitrogen, phosphorus, and potassium could have an effect on soil pH.

However, the pH and production data show that the Cypress mulch may leach an allelochemical that adversely affects the growth of the radish plants by dropping the soil pH below the tolerance range for the radishes (6.8 to 5.5). The same phenomenon occurs in the tomato trial for the Pine mulch.

CONCLUSIONS

Evaluation of the Test Mulches' Allelopathic Properties
	Control	Blue Spruce	Cypress	Juniper	Paper	Pine	Pinyon
Inhibited seed germination*		TF	F	RTF		F
Inhibited bermuda growth		X		X		X
Inhibited fescue growth		X	X	X		X
Adversely affected soil pH			X			X
Adversely affected Tomato Plant	X					X
Adversely affected Radish Plant	X		X

*Seed Type: R=Radish, T=Tomato, F=Fescue. The conclusions for the bermuda seed inhibtion trial are not included, as the procedure did not work for this seed type.

The data for the Juniper and Blue Spruce mulches confirm the hypothesis that allelochemicals with herbicidal properties are present in these garden mulches and do leach from the mulches causing adverse affects on bermuda and fescue grass without affecting the production of the radish and tomato plants. However, these mulches should not be applied until after the radish and tomato seeds have germinated.

The Pine mulch also leaches allelochemicals that adversely affected fescue and bermuda growth. However, the Pine mulch should not be used in a tomato garden due to its adverse affects on soil pH and tomato plant health and production.

The Cypress mulch inhibits fescue growth, but has adverse affects on radish plant growth and production due to an allelochemical that caused the soil pH to drop.

The Paper and Pinyon mulches prove to have no allelopathic affects on the plants tested in this experiment. However, these mulches help to demonstrate that the blockage of sunlight alone is not responsible for inhibition of the bermuda and fescue grass growth.

RECOMMENDATIONS FOR FUTURE STUDIES

• Try the Blue Spruce and Juniper mulches on flowerbeds this spring.

§ Test allelopathic properties of the mulch of the Junepirus virginiana tree that invades pastureland in central Oklahoma.

§ Attempt to isolate allelochemicals from the Blue Spruce and Juniper.

§ Test mulches again in late spring and early summer to improve production data.

AKNOWLEDGEMENTS

There are several people that deserve thanks for helping me with the project and giving me advice:

• Mrs. Sue Gray-Melaugh, OSU Extension Horticulturists, for her advice and expertise
• Mr. Roy Ball, OSU Extension Agriculture Agent, for providing the Mesonet Data
• Mrs. Kathy Davis, Vinita High School Biology Instructor, and Mr. Steve Kirby, Vinita High School Chemistry Instructor, for helping with my procedure, providing the Science Workshop 500 Interface Unit, and the spectrophotometer
• Mr. Brian Stecklein, for his help with the statistical analysis
• My dad, Stan Fimple, for helping me collect the Colorado tree samples and for getting them mulched
• And last but not least, my mom, Dr. Debbie Fimple, for her enthusiasm and constant encouragement (i.e. nagging) throughout my project.

BIBLIOGRAPHY

1. Kebede, Zewdu. “Allelopathic Chemicals: Their Potential Uses for Weed Control in Agroecosystems.” <http://www.colostate.edu/Depts/Entomology/courses/en570/papers_1994/kebede.html>. (1 Jan. 01).

2. Putnam, Alan R. “Allelochemicals from Plants as Herbicides.” Weed Technology Oct. 1988.

3. Putnam, Alan R. “Allelopathic Chemicals.” Chemical and Engineering News 4 Apr. 1983: 34-45.

4. “Round-Up Ready Corn Under Attack in California.” 1999. <http://www.inforshop.org/new4/ca_corn.html>. (1 Jan. 01)

5. Shepherd, Hurley. “Where Did the Genes Come From for the Round-Up Immune Soybeans?” 1997. <http://www.madsci.org/posts/archives/dec97/873488056.Ag.r.html>. (1 Jan. 01)

6. “Tree Allelochemicals . . .: Ways and Means.” The University of Georgia Extension Forest Resources. <http://www.forestry.uga.edu./efr/docs/pfor99%2D005.html>. (4 Jan. 01)

7. “Why Should I Use Mulch?” <http://www.acergarden.com/html/mulch.html>. (1 Jan. 01)