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Project Summary

Difficulty	6
Time required	Long (a couple of weeks)
Prerequisites	You will need a plant with an existing crown gall infection as a source of bacteria for this project.
Material Availability	Readily available
Cost	Low ($20 - $50)
Safety	Use caution when cutting with sharp knife or razor blade.

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Abstract

Objective

The objective of this project is to determine whether or not a garlic powder solution can prevent crown gall in tomato plants.

Introduction

Crown gall is a plant disease caused by the bacterium Agrobacterium tumefaciens. Crown gall affects a large variety of broad-leaved (dicotyledonous) plants, including tomato, apple, pear, cherry, almond, raspberry, and rose plants. It is characterized by the development of galls—large, tumor-like swellings—on the plant. Typically (but not always), the galls appear on the crown of the plant, just about the soil level. (Deacon, Roberson and Isbister, date unknown) Figure 1, below, shows photographs of crown galls on a raspberry root and an apple branch.

Figure 1. Examples of crown galls: crown gall on a raspberry root (left) and crown galls on an apple branch (right) (Ellis, date unknown).

Crown gall is especially interesting to biologists because the bacteria introduces some of its DNA into the plant cells, and this bacterial DNA is incorporated into the plant cell's own DNA. The bacterial DNA causes the plant cells to produce proteins that alter the cells' metabolism. The end result is that the plant makes galls, which are a favorable environment in which the bacteria can grow. You can see that nature has been making transgenic plants long before scientists did! Scientists have used genetically-engineered A. tumefaciens to introduce new genes (including even non-plant genes) into plant genomes. (Deacon, Roberson and Isbister, date unknown)

A. tumefaciens infects plants through wound sites, e.g., from pruning or transplantation. Cellular components such as sugars leaking from the wound site can serve as chemoattractants for the bacteria. In fact, specific strains of A. tumefaciens can detect a particular compound released from plant wounds, acetosyringone, at 1 part in 10,000,000! The bacteria can direct their movement towards higher concentrations of the compound (this is called chemotaxis) to find the wound site.

Crown gall is usually not much of a problem for mature plants, but seedlings and young plants that are infected can be severely stunted. The A. tumefaciens bacteria can remain viable in the soil for long periods of time (up to two years). How can young garden and crop plants be protected from infection in soil that harbors A. tumefaciens? Antibiotics can not be used for agricultural purposes, because resistant bacterial strains will develop with widespread use, resulting eventually in loss of effectiveness of the antibiotic against human disease.

How about testing a natural product as an anti-bacterial agent to protect plants? Garlic powder has been shown to have anti-bacterial properties under some conditions (Jonkers et al., 1999). Can it protect tomato plants from A. tumefaciens? In this project you will grind up garlic tablets in order to make solutions with a known amount of garlic powder.

You will need a plant infected with crown gall as a source of A. tumefaciens bacteria for this experiment. To infect tomato seedlings, you will use a "stab innoculation" technique, using a pin. Half of the plants will be stabbed with sterile pins that have been scraped on the inside surface of a cut-open crown gall (to innoculate the pin with A. tumefaciens bacteria). The remaining plants will be stabbed with sterile pins (as a negative control).

Terms, Concepts and Questions to Start Background Research

To do this project, you should do research that enables you to understand the following terms and concepts:

• crown gall,
• Agrobacterium tumefaciens.

Bibliography

• Here are several excellent references on crown gall disease and the bacteria that causes it:
  • Gleason, M., 1995. "All About Crown Gall," Department of Plant Pathology Iowa State University, [accessed October 4, 2006] http://www.ipm.iastate.edu/ipm/hortnews/1995/5-19-1995/cgall.html.
  • Ellis, M.A., date unknown. "Bacterial Crown Gall of Fruit Crops," Ohio State University Extension, Fact Sheet, Plant Pathology [accessed October 4, 2006] http://ohioline.osu.edu/hyg-fact/3000/3301.html.
  • Nameth, S. and J. Chatfield, date unknown. "Bacterial Crown Gall on Ornamentals in the Landscape," Ohio State University Extension, Fact Sheet, Plant Pathology [accessed October 4, 2006] http://ohioline.osu.edu/hyg-fact/3000/3054.html.
  • Moore, L.W., 2006. "Crown Gall Disease of Nursery Crops," An Online Guide to Plant Disease Control, Oregon State University [accessed October 4, 2006] http://plant-disease.ippc.orst.edu/articles.cfm?article_id=8.
  • Deacon, J., A. Robertson, and A. Isbister, date unknown. "The Microbial World: Biology and Control of Crown Gall (Agrobacterium tumefaciens)," Institute of Cell and Molecular Biology, The University of Edinburgh [accessed October 4, 2006] http://helios.bto.ed.ac.uk/bto/microbes/crown.htm.
• Jonkers, D. et al., 1999. "Antibacterial effect of garlic and omeprazole on Helicobacter pylori," Journal of Antimicrobial Chemotherapy 43(6): 837‐839, available online [accessed October 4, 2006]: http://jac.oxfordjournals.org/cgi/content/full/43/6/837.

Materials and Equipment

To do this experiment you will need the following materials and equipment:

• plant infected with crown gall as a source of A. tumefaciens bacteria:
  • a county agricultural extension agent may be able to help with identifying crown gall infection;
  • you can also check with experts at local nurseries.
• sharp knife or razor blade for cutting gall open,
• at least 24 tomato plant seedlings,
  • Note: tomato plant seedlings are usually available for purchase in spring to early summer. If you are doing your project at another time of year, you will have to grow your own plants from seed, which will add several weeks to the project, so be sure to plan ahead!
• potting soil,
• 4 large plant pots (big enough to hold 6 tomato seedlings each),
• garlic tablets,
• clean tools for crushing garlic tablets,
• water,
• graduated cylinder (100 mL),
• 1 ml disposable pipettes (or eyedropper),
• pins.

Experimental Procedure

1. In this project you will have four different sets of tomato plants, with 5 plants each. The table below shows the experimental conditions for the four sets.

   #	description	innoculation	treatment
   1	control #1	stab with sterile pin	plain water
   2	control #2	stab with sterile pin	garlic solution
   3	control #3	stab with crown gall pin	plain water
   4	test	stab with crown gall pin	garlic solution

2. Pots 1 and 2 are negative controls. These tomato seedlings will be "sham innoculated" (stabbed with sterile pins). None of these plants should develop galls (this is why these are called "negative" controls). The stabs on the seedlings in pot #1 will be treated with plain water. The stabs on the seedlings in pot #2 will be treated with garlic solution. Comparing these two pots will show you if the garlic solution has any effects (positive or negative) on uninfected tomato plants.
3. Pot #3 is a positive control. These tomato seedlings will be stab innoculated with bacteria, and the stabs will be treated with plain water. You should expect all of these seedlings to develop galls (that's why it is called a "positive" control.) It is important to include this control condition. These plants must develop galls. If they do not, your experiment cannot show that garlic has a preventive effect.
4. Pot #4 has the test seedlings. These will be stab innoculated with bacteria, and the stabs will be treated with garlic solution. Comparing these seedlings to those in pot #3 will tell you if the garlic treatment has a preventive effect.
5. For plants treated with garlic solution (pots 2 and 4), pipette 1 mL of the garlic solution over the stab wound immediately after innoculation, and then once a day for the next four days (5 treatments total).
6. For plants treated with water (pots 1 and 3), pipette 1 mL of water over the stab wound immediately after innoculation, and then once a day for the next four days (5 treatments total).
7. Allow the plants to grow for 2–3 weeks. Observe the plants daily. Make notes of your observations in your lab notebook.
8. Figure 2, below, shows an example of a tomato seedling that has developed a crown gall three weeks after stab innoculation with A. tumefaciens.

   Figure 2. Examples of crown galls developing on tomato seedlings three weeks after stab innoculation (arrows) with A. tumefaciens. (Deacon, Roberson and Isbister, date unknown)

9. Compare the number of seedlings with with galls between pots 3 and 4. Is garlic effective at preventing crown gall at the concentration you tested?
10. Compare the growth of the seedlings between pots 1 and 2. Does the garlic solution have any effect (positive or negative) on sham innoculated seedlings (pots 1 and 2)?

Making Garlic Powder Solution

1. In this experiment, you will treat each plant once a day with 1 mL of garlic powder solution for a total of 5 days. How much solution will you need?
   1. amount per plant per day × number of plants × number of days = amount of solution needed
   2. 1 mL/day/plant × 12 plants × 5 days = 60 mL solution needed
2. Prepare garlic solution by grinding up tablets into very fine powder and dissolving in warm water.
   1. Choose a single garlic concentration between 1 and 20 g/L. For example, let's suppose your desired final concentration is 5 g/L, and that each garlic tablet contains 40 mg (0.04 g) of garlic powder.
   2. To make 60 mL (0.05 L) of 5 g/L solution, you'll need:
      5 g/L / 0.04 g/tablet × 0.06 L = 7.5 tablets.
   3. It's good to have a little extra solution, so round up to 8 tablets.
3. How much garlic powder is that? 8 tablets  ×  0.04 g/tablet = 0.32 g garlic powder.
4. So how much water do I dissolve that in? 0.32 g / 5 g/L = 0.064 L = 64 mL.

Stab Innoculating Tomato Seedlings

1. Sterilize pins by wrapping them in aluminum foil, and then heating them in the oven at 300°F for 30 min.
2. Use a sharp knife or razor blade to cut open the crown gall you will use to innoculate your tomato seedlings.
3. Scrape the point of a sterile pin inside the gall to innoculate it with A. tumefaciens bacteria. You only need a small amount of material. Use the same amount of material for each innoculation.
4. Stab plant near base of stem with tip of the pin. The pin should penetrate to the center of the stem, but no further.
5. For sham innoculations (seedlings in pots 1 and 2), do not innoculate the pin with crown gall material. Instead, stab the seedlings near the base of the stem with the tip of a sterile pin. Again, the pin should penetrate to the center of the stem, but no further.
6. It's a good idea to mark the location of the stab innoculation by drawing a circle around it with permanent marker. You'll be able to tell where you innoculated each plant, even if the wound site heals. Mark the sham innoculated plants first, then mark the plants innoculated with crown gall scrapings (or use separate markers). This way, you won't risk contaminating the sham innoculated plants.

Variations

• The procedure above uses only a single concentration of garlic solution. If the garlic treatment is not effective against crown gall, it might be that garlic just isn't effective against A. tumefaciens. On the other hand, it might be the wrong concentration of garlic solution, or perhaps 5 days of treatment is not enough.
  • If your negative controls show that the garlic solution has a negative effect on the tomato seedlings, try again with a lower concentration of garlic than the one suggested above.
  • If your negative controls show that the garlic solution has no effect, or a positive effect on the tomato seedlings, try again with a higher concentration of garlic than the one suggested above, or extend the treatment for a longer time period, or both.
• If garlic is effective against crown gall, you could do additional experiments with different concentrations of garlic to find the most effective concentration. Also, as you dilute the garlic solution, the preventive effect should go away, which will add support for the hypothesis that something in the garlic solution is acting as an anti-microbial agent.
• Advanced. There are commercially available preventive treatments for crown gall disease that use a biological control agent, a closely-related bacterial strain called Agrobacterium radiobacter which competes with A. tumefaciens and prevents it from infecting plants. Galltroll is one of the commercial names for this project. Galltroll is not effective against every strain of A. tumefaciens (see Deacon, Roberson and Isbister, date unknown). Design an experiment using the stab innoculation technique to test the effectiveness of Galltroll against the A. tumefaciens strain(s) from galls in your local area.

• For more science project ideas in this area of science, see Microbiology Project Ideas.

Credits

Andrew Olson, Ph.D., Science Buddies

Sources

This project is based on:

• Cabrera, C.J., 2003. "Can Garlic Prevent Crown Gall?" California State Science Fair Abstract [accessed October 4, 2006] http://www.usc.edu/CSSF/History/2003/Projects/J1405.pdf.

Last edit date: 2006-10-13 22:00:00

Career Focus

If you like this project, you might enjoy exploring careers in Microbiology.

Microbiologist Microorganisms (bacteria, viruses, algae, and fungi) are the most common life-forms on Earth. They help us digest nutrients; make foods like yogurt, bread, and olives; and create antibiotics. Some microbes also cause diseases. Microbiologists study the growth, structure, development, and general characteristics of microorganisms to promote health, industry, and a basic understanding of cellular functions.

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