AP Environmental ScienceMrs. DoumaAllelopathy Lab

The Effects of Allelopathy

INTRODUCTION

From childhood most of us have known that animals are territorial and that they aggressively defend resources needed for survival. Vivid memories of carnivores attacking territorial challengers have been embedded into our minds from numerous nature shows and classroom discussions.

What many of us do not realize is that plants can be just as aggressive in defending their resources. Many plant species are capable of poisoning the soil and air around them. They do this by exuding toxic chemicals called allelotoxins from their leaves and roots. Allelotoxins prevent the germination of seeds and the encroachment of vegetative growth from other plants. By keeping competitors at bay, the defending plant can reserve precious resources such as soil nutrients, water, and sunlight for itself. Those plants which have evolved the strongest defenses will be the most likely to survive.

For instance, have you ever noticed the ground under a pine tree? Generally, if you examine a pine forest, you will discover that no plants are growing beneath the trees. This paucity of growth is due to more than just a lack of sunlight. This happens because fallen pine needles contain acid which leaches down into the soil as they decompose, thus, lowering the pH beyond the range of tolerance for most plant species. The acidic soil which does not affect the pine tree essentially eliminates other contenders for precious water and mineral resources beneath the forest floor.

In all ecosystems, species, both plant and animal, are competing for limited resources. Numerous strategies are applied in the ongoing struggle for survival. Most of us are familiar with camouflage, mimicry, ambush predation, and thorns, spines, and needles. As stated above, plants, because they cannot move from place to place, have evolved a heavy reliance on allelotoxins – poisonous chemicals. Some of these allelotoxins are transferred to other plants through volatilization which is when allelochemicals are exuded into the air, and, in turn, are absorbed through the leaves of competing plants killing them. Other protective strategies by plants include the release of defensive chemicals into the soil through their roots, which assures that intruding roots from other species cannot grow, and are thus rendered incapable of monopolizing valuable water and mineral supplies. Other defenders poison the soil around them as their leaves drop to the ground and transfer allelochemicals onto the topsoil and thus prevent the germination of seeds from their own species or other species.

Competition, which can be defined as one organism having a negative or restrictive effect on another, may be intraspecific, occurring between individuals of the same species, or interspecific, occurring among different species. In addition, it should be noted that there are two general ways an organism can limit the resources available to another organism: exploitation competition or interference competition.

Allelopathy is a type of chemical interference competition utilized by plants. The word allelopathy is derived from two basic root words: allelon (of or from each other) and pathos (to suffer). Allelopathy involves a chemical inhibition of one species by another. Molecules produced by one plant, mostly secondary metabolites, are released into the environment and then influence the growth and development of neighboring plants. For example, in the Mojave Desert of the Southwestern United States, where water is a limiting factor for growth, creosote shrubs exude allelochemicals from both their leaves and roots that prevent other desert plants from encroaching upon their immediate territory. When viewed from a distance, the creosote bushes are spaced out, like pegs in a cribbage board, for as far as one can see with a life-sustaining three to five meters between them.

OBJECTIVE

The purpose of this lab is to analyze the effect of allelopathic chemicals on the germination and growth of seeds and to understand the role of chemical defense in plant ecology.

VOCABULARY

allelopathyexudation

intraspecific competitionherbicide

interspecific competitionvolatilization

inhibitory thresholdleaching

exploitation competitionallelochemicals

interference competitionallelotoxins

phytotoxicitymetabolite

range of tolerancelimiting factor

MATERIALS

  • 150ml to 200ml small plastic pots or cups
  • Small trays for holding each groups pots
  • Soil for filling pots (regular soil works much better than prepackaged potting mix)
  • Markers for labeling pots; if desired, small marker flags can be constructed out of toothpicks and sticky labels and then inserted into the edge of the pots.
  • Balances for massing soil and leaf
  • Soil sieves or 1/8” to ¼” metal screen for grinding up dried leaf material
  • Spring water or distilled water for watering seeds
  • Graduated cylinders
  • Metric rulers
  • 250ml beakers or large plastic cups for holding water
  • Plastic/paper bags for storing dried leaves
  • Indoor plant growing lights if natural lighting is not available
  • Test seeds: radish (if small size seed is desired) or mung bean (if larger size seed is desired)
  • Dried leaves from one or more allelopathic plants. The following is a list of some of the more notable allelopathic plants. Other local plants can be tested as well.

Black walnutBarleyEucalyptus

Pine needlesApricotSage

MintRagweed (allergy caution)Sunflower

Tree of heavenWormwood Chrysanthemum

Crown vetchKnapweedTobacco

SorghumCreeping buttercupPea

GarlicFragrant sumacAlfalfa

PROCEDURE

  1. Obtain the crushed dried leaves of one or more known allelopathic plants. If your experiment is going to be more research oriented, you may want to try plants with unknown allelopathic effects. Be careful about the plants you chose. Some species may be extremely toxic, for example, poison ivy and poison oak can cause severe skin problems, while plants like castor bean and oleander can be fatal. Check with your instructor. You will need to develop a hypothesis for your research. There are some factors below under data analysis if you need ideas.
  1. You have to decide how many allelopathic plants you will be testing. In addition, you will have to figure out the crushed leaf to soil ratio for each of the pots you use. This ratio should be based on mass not volume because crushed leaf volume can vary greatly. Below there is a data chart that includes four different test conditions (1.25, 2.5, 5.0, and 10.0 grams of crushed leaf respectively) and a control. You may use these suggested amounts or you can decide on the ratio of crushed leaf to soil yourself.
  1. It is recommended that you use small 150ml plastic pots or cups which hold a 100 gram mixture of crushed leaf and soil with room at the top for watering. At this time you will need to figure out how many test conditions (pots) are going to be incorporated into your experiment. If your group test five different species and follow the data chart, you will need 25 plastic cups.
  1. For each of your test conditions, mass out the amount of crushed leaf needed and dump it into a plastic, sealable baggie. Then mass out enough soil to bring the total mixture of leaves and soil up to 100 grams. For example, if you are putting 5 grams of crushed leaves into the plastic baggie, then you will need to mass out 95 grams of soil. Dump the soil into the baggie with the crushed leaves. Seal the baggie and shake thoroughly to mix the soil and crushed leaves together.
  1. Obtain one of the small plastic pots or cups and carefully pour the 100 gram mixture into the container. Tap the pot or cup on the table a couple of times to settle the contents and smooth out the top surface with your fingers. Identify the amount of crushed leaf in the pot or cup by either marking on the side of the container or making a small identification flag with a toothpick and sticky label and inserting it into the soil mixture.
  1. In order to allow the allelochemicals to thoroughly leach into the soil mixture, water your containers 48 hours before you plant your test seeds. Each pot or cup should be watered in an identical manner. Fill each 150 ml container with the various 100 gram soil and crushed leaf mixtures. Pour 40 ml of water into each container. Place the watered containers in a location where they will not dry out quickly. As stated above, remember to wait two full days before planting any seeds.
  1. Once the allelochemicals have had a chance to penetrate into the soil mixture, it is time to plant your seeds. Two of the most reliable and easy to use seeds are radish and mung bean. Both seeds are small (but not excessively tiny) with radish being about one-tenth the size of mung bean.
  1. Twenty radish seeds can be planted in a 150 ml container while only nine mung bean seeds can be placed in the same sized container without severe overcrowding. You will have to decide how many seeds to plant, but it should be remembered that better statistical results are obtained with larger numbers.
  1. The easiest and most uniform way to plant your seeds is through the use of a stiff probe such as a nail. Mark the probe one-quarter inch from the tip (seed planting depth) so you will know how far to push it in when you are making holes for the seeds. Decide how many seeds you are going to plant in each pot and the planting pattern you will use for all the pots. After making the holes, use a pair of tweezers to put each seed in its hole. Use the probe to shove it down to the proper depth if necessary.

  1. Once planted, the pots will need to be placed in a warm area to stimulate germination. Natural or artificial lighting will provide the necessary warmth. Make sure the containers do not dry out. If watering is necessary, all pots or cups should be watered uniformly giving each container the same amount of water. Make sure to record the day and time that each container was planted. Make sure all the containers are clearly marked.

DATA ANALYSIS

There are several factors that can be analyzed as you observe the results of this experiment:

  • How did allelochemicals affect the length of germination (time)?
  • Did the same allelochemicals affect different species of seeds differently?
  • What percent of the seeds germinated in the control and the various test conditions?
  • How did the germination rate vary with increasing concentrations of allelochemicals?
  • How did different types of allelochemicals affect a single species of seed?
  • How did the allelochemicals affect the post-germination growth of the various plants?
  • What are the confounding variables and sources of error?

QUESTIONS

  1. Why is allelopathy important to the long-term stability of an ecosystem?
  1. Did your experimental results indicate that there is a wide range in the effectiveness of various allelopathic chemicals used by plants?
  1. Did your experimental results indicate that different types of seeds vary in their ability to resist allelopathic chemicals?
  1. What is the danger of using seeds such as radish and mung bean which may not be found in the ecosystems of the allelopathic plants being tested?
  1. What were some of the possible sources of error in your experimental design?
  1. How could the control container with 100% soil and no leaves be a possible source of error in the experiment?
  1. How might knowledge about allelopathic chemicals be used to create natural herbicides and help to promote sustainable agriculture?
  1. Do you think that scientists should use their bioengineering skills to remove the genes from allelopathic plants that are responsible for producing allelotoxins and splice those genes into non allelopathic food crop plants in an effort to reduce the need for herbicides and insecticides?
  1. Why might knowledge of allelopathic effects be important to farmers who are concerned about crop to crop and weed to crop interactions?
  1. Many ecosystems are dependent on low intensity ground fires as opposed to catastrophic wildfires which can destroy important soil decomposers such as fungus and nitrogen fixing bacteria. How do allelochemicals help to reduce the possibility of catastrophic wildfires occurring in forest and other ecosystems?