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Factors affecting Nodule Formation in Legumes

Factors affecting Nodule Formation in Legumes

Introduction: (Initial Observation)

The growth of all organisms depends on the availability of mineral nutrients, and none is more important than nitrogen, which is required in large amounts as an essential component of proteins, nucleic acids and other cellular constituents. All legumes have the capacity to fix their own nitrogen. Rhizobia bacteria combine with legume roots to form nodules that, in turn, carry on nitrogen fixation – the process by which legume plants obtain (fix) the nitrogen they need from the gaseous nitrogen in the air.

In this project, you will make use of soil bacterium of the genus Rhizobium and a host legume to demonstrate the effects that soil microbes can have on soil fertility and nutrient availability.

By completing this project, you will be able to:

  • Collect, record and interpret your data.
  • Identify the control and the variables in the experiment.
  • Describe the growth of plants with and without nitrogen fixing bacteria.
  • Name the bacterium responsible for making nitrogen available to the plant.


This project guide contains information that you need in order to start your project. If you have any questions or need more support about this project, click on the “Ask Question” button on the top of this page to send me a message.

If you are new in doing science project, click on “How to Start” in the main page. There you will find helpful links that describe different types of science projects, scientific method, variables, hypothesis, graph, abstract and all other general basics that you need to know.

Project advisor

You will need 9 to 10 weeks time for your experiments.

Care should be used in handling the fertilizers and the bacterial inoculums.

Information Gathering:

Although the project is about nodule formation in legumes, you actually need to gather information and research on bacteria that form such nodules on the plant’s root. Study about conditions for bacteria growth and practice avoiding cross contamination when experimenting with bacteria. Read books, magazines or ask professionals who might know in order to learn about the factors that may affect bacteria growth and nodule formation in legumes. Keep track of where you got your information from.

Make yourself familiar with the following terms before you continue.

aerial parts of the plants – Those parts of a plant that appear above ground.

nitrifying bacteria – Any of various soil bacteria that take part in the nitrogen cycle, oxidizing ammonium compounds into nitrites or nitrites into nitrates.

nitrogen fixing – The conversion by certain soil microorganisms, such as rhizobia, of atmospheric nitrogen into compounds that plants and other organisms can assimilate.

legume – A plant of the pea family. Capable of forming a symbiotic relationship with Rhizobium bacteria on the nodules of the roots. The bacteria fix nitrogen for the plant and the plant provides a place for the bacteria to live.

inoculated – To implant microorganisms in a substance.

roots – The underground portions of a plant. Nodules form on the roots of legumes that contain nitrifying bacteria.

fertilizer – Any of a large number of natural and synthetic materials, including manure and nitrogen, phosphorus, and potassium compounds, spread on or worked into soil to increase its capacity to support plant growth.

culture plate – A specialized covered dish with a growth medium, usually agar, in which bacteria are introduced for growth.

Rhizobium – Any of various nitrogen-fixing bacteria of the genus Rhizobium that form nodules on the roots of leguminous plants, such as clover and beans.

symbiotic – A close prolonged association between two or more different organisms of different species that may, but does not necessarily, benefit each member.

Boron, molybdenum, chloride

Exist in the soil as anions or uncharged molecules, because they are anions their chemistry is quite different than the metal cations

A. Boron
1. Functions in plants

  • new cell development in meristematic tissue
  • proper pollination and fruit or seed set
  • translocation of sugars, starch, N and P
  • protein synthesis
  • nodule formation in legumes
  • regulation of carbohydrate metabolism

B. Molybdenum
Plant function

  • Absorbed as molybdate (MoO4-2)
  • Essential component of nitrate reductase enzyme
  • Structural component of nitrogenase enzyme, involve in N2 fixation by bacteria in root-nodules
  • < 1ppm in plants

What are the numbers used to define fertilizers?

The correct type of fertilizer to use on your orchids is very important!
All fertilizers consist if three main ingredients:

nitrogen–(N)–which promotes general plant growth
phosphorus–(P)–which promotes flowering
potassium–(K)–which promotes strong roots.

The ingredients are mixed in various combinations because plants have different needs. The combinations are indicated by a three number code:

  • The first number is the percent of nitrogen (N)
  • The second number is the percent of phosphorus (P)
  • The third number is the percent of potassium (K)

So for example 10-5-10 means 10% Nitrogen, 5% phosphate and 10% potassium.

Question/ Purpose:

What do you want to find out? Write a statement that describes what you want to do. Use your observations and questions to write the statement.

What factors affect the formation of nodules in legumes? What is the effect of a nitrifying bacteria such as Rhizobium on formation of nodules and plant growth?

Identify Variables:

When you think you know what variables may be involved, think about ways to change one at a time. If you change more than one at a time, you will not know what variable is causing your observation. Sometimes variables are linked and work together to cause something. At first, try to choose variables that you think act independently of each other.

Variables that may affect the formation of nodules on legumes are:

1. Presence of bacteria in the soil (inoculum)

2. Presence of nutrients, but shortage of nitrogen fertilizers


Based on your gathered information, make an educated guess about what types of things affect nodule formation in legumes. Identifying variables is necessary before you can make a hypothesis.

Hypothesis Sample 1:

Bacteria in soil have no effect on formation of nodules. Formation of nodules is a property of legumes and some other plants. My hypothesis is based on my observation of other plant roots that have no nodules. If bacteria in soil were causing nodules, they would do the same for other plants too.

Hypothesis sample 2:

I think the shortage of nitrogen in soil promotes the growth of nitrogen fixing bacteria and formation of nodules. The fact that nodules does not form on the roots of other plants may have something to do with the resistance of those plants against bacteria attack.

Experiment Design:

Design an experiment to test each hypothesis. Make a step-by-step list of what you will do to answer each question. This list is called an experimental procedure. For an experiment to give answers you can trust, it must have a “control.” A control is an additional experimental trial or run. It is a separate experiment, done exactly like the others. The only difference is that no experimental variables are changed. A control is a neutral “reference point” for comparison that allows you to see what changing a variable does by comparing it to not changing anything. Dependable controls are sometimes very hard to develop. They can be the hardest part of a project. Without a control you cannot be sure that changing the variable causes your observations. A series of experiments that includes a control is called a “controlled experiment.”


In this experiment you grow a leguminous plant, such as garden pea or green bean in various condition in order to see how different factors may affect plant growth and nodule formation. All along during this experiment you will monitor the plant growth (plant height) and after completing your experiments you will also observe the plant roots and look of nodules.


  1. Label each container with a different label:
    no additions (sand and plants)
    inoculated — no fertilizer (sand, bacteria and plants)
    not inoculated — fertilized (sand, fertilizer and plants)
    inoculated — fertilized (sand, fertilizer, bacteria and plants)
  2. Put half of the sand (1 qt ) into a mixing bowl or container and add 1 teaspoon of the 0-20-20 fertilizer to the sand and mix thoroughly. If using a 0-10-10 grade fertilizer, or a 0-10-5 fertilizer, use 2 tsps. For other grades, adjust the amount proportionately.
  3. Place 1 pint of the fertilized soil in each of pots #3 & #4. Place 1 pint of the unfertilized soil in each of pots #1 & #2.
  4. Plant 5 seeds in each of the pots.
  5. Add Rhizobium inoculums to the surface layer of the sand in pots #2 and #4.
    Note: Avoid Rhizobium contamination in the not inoculated pots.
  6. Moisten the soils of each pot until water just starts to run out of the holes in the bottom. Use tap water or distilled water. If your water is chlorinated, it may be best to let it stand in a container for a day to give the chlorine a chance to dissipate before use.
  7. Place each of the pots in separate shallow dishes or trays. Put them together (to eliminate environmental variables) in a warm, well-illuminated location, and water as needed to maintain moisture.
  8. If one or more of the pots has less than 5 plants after the seeds have germinated and developed into shoots several inches tall, remove the smallest plants so that each pot contains the same number of plants.
  9. Have the students predict the results (based on the introductory remarks for this lab) and record those predictions.
  10. Make and record measurements/observations at least 2x per week. It will take about six weeks for the plants to develop root nodules, so the experiment should run for at least 9-10 weeks to be able to observe any differences between the treatments with and without Rhizobium.
  11. Measure and record the height of the aerial parts of the plants in each pot (cm). Determine the mean height (cm) for the plants in each pot. Record in the data table.
  12. Observe the appearance of the plants, including color, leaf size, leaf spacing, and any other observations and record them.
  13. At the end of the nine-week growth period harvest the plants. Harvest by immersing the container in a tub of water and carefully washing the soil off the roots. Be careful while harvesting to avoid loss of plant material.
  14. Record the wet mass of the plants for each separate pot.
  15. Sketch and/or describe the comparative differences between root growth, nodule formation and overall vigor of the plants in each pot.
  16. Air dry the plants for several days in a sunny location until a constant dry weight is obtained. Record the dry weight of the entire group of plants for each separate pot.
  17. Interpret the measured / observed results and reconcile them with your predictions as a written conclusion.

Materials and Equipment:

  • 20 seeds of a leguminous plant, such as garden pea, green bean, or clover (Red clover seeds are supplied with the Nitrogen-Fixation Study from Carolina supply.) These seeds should not be pre-inoculated. (A colored coating usually indicates inoculation.)
  • an inoculums of Rhizobium bacteria that is host-specific to the chosen host plant. If the Nitrogen-Fixation Study Kit from Carolina Biological (see reference) is not used, then appropriate host seed and bacterial inoculum can be obtained from a seed retailer.
  • 4 identical or closely similar pots (or other suitable containers with drainage holes) that are large enough to hold 1 pint of soil
  • 4 pints of fine mason’s sand (sandbox sand)
  • 1 teaspoon of a 0-20-20 fertilizer or &#0;2 teaspoon of 0-10-10 or 0-10-5. Different ratios, such as 0-12-6 can be used as long as they contain no (or almost no) nitrogen.
  • balance
  • tubs to wash soil from plants

Where to get Rhizobium bacteria?

Check the links at the end of this project and send a few emails to those who may sell Rhizobium. You may also have to make a few phone calls.

If you can’t find Rhizobium, try to find an existing legume plant and take out some nodules from the root. grind them, mix them with water and use them as inoculum.

If none of the above works for you, get some regular soil that usually contains Rhizobium and many other bacteria. Dissolve it in water and use the polluted water as inoculum.

Results of Experiment (Observation):

Experiments are often done in series. A series of experiments can be done by changing one variable a different amount each time. A series of experiments is made up of separate experimental “runs.” During each run you make a measurement of how much the variable affected the system under study. For each run, a different amount of change in the variable is used. This produces a different amount of response in the system. You measure this response, or record data, in a table for this purpose. This is considered “raw data” since it has not been processed or interpreted yet. When raw data gets processed mathematically, for example, it becomes results.

Data Table


Pot 1

No Treatment

Pot 2


No Fertilizer

Pot 3

Not Inoculated


Pot 4




Find individual plant height, then the average for each pot.

Record the appearance, including color, leaf size, leaf spacing, and other significant observations.


Using the data collected in the data table, create a line graph with time on the horizontal axis and plant height in cm on the vertical axis. Include all four pots on one graph for comparison. Plot the average for each pot only.

Using the data collected from the harvesting of the plants, create a bar graph with mass on the vertical axis and each pot’s label on the horizontal axis. Include the wet mass and the dry mass as two separate bars for each pot.

Write a paragraph that summarizes the differences in the four groups of plants using the data in the chart and graphs.

Report presence, size and the number of nodules.


Pot 1

No Treatment

Pot 2


No Fertilizer

Pot 3

Not Inoculated


Pot 4



Number of nodules
Average size of nodules


No calculations is required for this experiment.

Summary of Results:

Summarize what happened. This can be in the form of a table of processed numerical data, or graphs. It could also be a written statement of what occurred during experiments.

It is from calculations using recorded data that tables and graphs are made. Studying tables and graphs, we can see trends that tell us how different variables cause our observations. Based on these trends, we can draw conclusions about the system under study. These conclusions help us confirm or deny our original hypothesis. Often, mathematical equations can be made from graphs. These equations allow us to predict how a change will affect the system without the need to do additional experiments. Advanced levels of experimental science rely heavily on graphical and mathematical analysis of data. At this level, science becomes even more interesting and powerful.


Using the trends in your experimental data and your experimental observations, try to answer your original questions. Is your hypothesis correct? Now is the time to pull together what happened, and assess the experiments you did.

Was your hypothesis supported? Why or why not?

What questions came up during the lab that might be answered with further investigations?

What effect do you think it would have on a crop of peas to use the Rhizobium inoculum?

How could the hypothesis that “Rhizobium Bacteria adds nitrogen to the soil” be tested by further experiments with the current set up?

Related Questions & Answers:

What you have learned may allow you to answer other questions. Many questions are related. Several new questions may have occurred to you while doing experiments. You may now be able to understand or verify things that you discovered when gathering information for the project. Questions lead to more questions, which lead to additional hypothesis that need to be tested.

Possible Errors:

If you did not observe anything different than what happened with your control, the variable you changed may not affect the system you are investigating. If you did not observe a consistent, reproducible trend in your series of experimental runs there may be experimental errors affecting your results. The first thing to check is how you are making your measurements. Is the measurement method questionable or unreliable? Maybe you are reading a scale incorrectly, or maybe the measuring instrument is working erratically.

If you determine that experimental errors are influencing your results, carefully rethink the design of your experiments. Review each step of the procedure to find sources of potential errors. If possible, have a scientist review the procedure with you. Sometimes the designer of an experiment can miss the obvious.