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Utilizing FGE to Improve Agricultural Health

Jessica Rhee and Camille Hall

Rockdale Magnet School

for Science and Technology

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TABLE OF CONTENTS

INTRODUCTION – Page 3-4

MATERIALS, METHODS, AND PROCEDURES – Page 5-6

RESULTS (DATA OR FINDINGS) – Page 7-8

DISCUSSION, CONCLUSIONS, DATA ANALYSIS – Page 9-10

LITERATURE CITED – Page 11-12

APPENDICIES – Page 13-15

ACKNOWLEDGEMENT OF MAJOR ASSISTANCE – Page 16

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INTRODUCTION

Crown gall (a disease that consists of tumor-like galls found in roots and trunks) has been

found across 93 families and 750 different species of plants, decreasing the yield per tree for

many commercially grown fruit tree crops: pears, apples, peaches, and cherries (Khan, n.d.). This

is an economic problem, especially for farmers who grow trees and rely on those plants for

produce also causes management-related labor costs to increase. It is estimated that damage

caused by crown gall in fruit and nut trees in California reached up to US $23 million dollars of

loss in 1976 (Kennedy, 1980). This project is focused on using simple, easily obtainable

ingredients such as garlic and cloves, to kill crown gall bacteria found in plants. The Kirby Bauer

test would be used to determine whether the growth of the bacteria, bacteria Agrobacterium

tumefaciens, can be slowed or stopped. Garlic will also be planted along with beans that are

coated with the bacteria to determine whether it can be utilized as a growing/support agent.

Crown gall is easily identifiable: the bacteria Agrobacterium tumefaciens causes large

lump-like growths that are found on stems and roots of the infected plant. Agrobacterium

tumefaciens can be spread through movement of contaminated soil, water, or and infected plant

material, and enters plants through wounds or natural plant openings. Plant cells are then

stimulated to have unregulated growth, producing a gall (Joy, 2005). This is an issue because as

the gall grows, the plant tissues start to become disorganized and normal transport processes are

disrupted (The Royal Horticultural Society, n.d.).

After breaking down over time, the gall bacteria can survive for many years after by

colonizing the roots of different plants in the area after being deposited in the soil (Khan, n.d.).

Plants that are young and have multiple galls tend to be stunted as well as being more susceptible

to damage caused by drought or winter injury (The Morton Arboretum, n.d.). Losses in nursery
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stock of fruit and ornamental plants were around $112 million in Canada in 1986, which is equal

to approximately 10% of the nursery plant material (Carter, 1989).

Garlic has been found to help prevent many diseases, as it is a natural antibiotic. Multiple

modern studies, such as FGE on Candida albicans Biofilms and FGE enhancing the antimicrobial

activities of antibiotics on resistant strains confirm that garlic can be effective against a wide

spectrum of bacteria, fungi and viruses (Shuford, 2005). The antimicrobial activities of garlic are

linked to the presence of some bioactive compounds as well (Li, 2015). Using garlic for this

project may be beneficial, as it may have an effect on the bacteria Agrobacterium tumefaciens.

If garlic and other easily obtainable ingredients can be used to stop the growth of crown

gall, this allows for the opportunity for other detrimental plant diseases to be prevented as well.

In turn, this would increase crop yields and support tree farmers. The use of garlic would help

prevent the spread of antibiotic-resistant bacteria for more common medicines. The increased

abuse of antibiotics enhances the need for alternative agents, such as garlic, that would relieve

that strain (Li, 2015). This project hopes to benefit people, the environment, and the economy.

Hypothesis

If fresh garlic extract is used on the plant disease agrobacterium tumefaciens, it will improve

agricultural health by preventing further growth of the bacteria. This may allow for the

opportunity for other detrimental plant diseases to be prevented as well.

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MATERIALS AND PROCEDURES

Set up:

All materials were gathered, and gloves were put on. Materials included forceps, Chinese

white garlic, crown gall bacteria, petri dishes, antibiotic sensi discs, blank sensi discs which

would be used as a control, a stirring rod, hotplate, centrifuge, agar nutrient mix, a pipette,

sharpie, and a ruler. The work area was also made clean.

Fresh Garlic Extract:

100 grams of Fresh Chinese white garlic bulbs were blended in 50 mL sterile distilled

water. Then, the mixture was crushed finely using a mortar and pestle. The resulting paste was

centrifuged at 3000 rmp for 30 minutes and the liquid was strained from the solid using a pipette.

The FGE was then stored in 1.5 mL micro test tubes at -20°C until use. The FGE was then put

onto blank sensi discs using a pipette and stored at -20°C.

Testing:

First, a sharpie was used to mark on lid of the agar plate (trial number, date, name of

bacterium). The surface of the agar plate was inoculated with the different bacterium using

separate, sterile swabs for each bacterium. The swab was then immersed in the culture tube and

the excess culture was squeezed on the inner side of the test tube. Next, the swab was streaked on

the surface of the agar plate three times and the plate was rotated 60 degrees after each streaking.

The swab was run around the edge of the agar to ensure the whole surface had been seeded. The

culture was allowed to dry on the plate for ten minutes at room temperature with the lid in place.

Next, the FGE discs (or antibiotics, depending on the trial) were dispensed on the plates using

forceps. Contact with the disc and culture was ensured by gently pressing the disc with the

sterilized forceps. The plates were then incubated for eighteen hours at 37°C.
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Retrieving Results:

To retrieve results, the metric ruler was placed across the zone of inhibition, at the widest

diameter, and measured from one edge of the zone to the other edge. Difficult-to-see zones were

held up to the light. Millimeter measurements were used—the disc diameter was part of that

number. If there was no zone at all, it was reported as 0 even though the disc itself was around

7mm.

DATA COLLECTION

The zone of inhibition’s diameter was recorded in millimeters and the result was reported

on the table. All four trials with each bacterium were averaged to create a bar graph that pictured

the average (mean) zone diameters for each bacterium and a graph with each individual

bacterium’s trial.

DATA ANALYSIS

The data was analyzed through an ANOVA test, as there was more than one group being

compared. An average was used (from multiple trials) using mean data. Individual T-tests for

each trial were also used, to test for p-value when compared to the control.
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RESULTS

Average Zone of Inhibition for FGE Concentration and

Antibiotic Trials

Streptomycin

Erythromycin
FGE Concentrations and Antibiotic Trials

4.0 mL/4g

3.5 mL/4g

3.0 mL/4g

2.5 mL/4g

2.0 mL/4g

1.5 mL/4g

1.0 mL/4g

0.5 mL/4g

0.0 mL/4g

0 5 10 15 20 25 30 35
Zone of Inhibition (mm)

Figure 1- Average zone of inhibition

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Figure 2- p-values of all trails completed with T-tests

As shown in Figure 1, the average zone of inhibitions for all FGE and antibiotic trials

were calculated using mean. The 1.0 grams of garlic and 4 mL of water had the widest zone of

inhibition. The 4.0 grams of garlic and 4 mL of water had the shortest zone of inhibition. Many

of the FGE concentration trials resulted in wider zones of inhibitions than the antibiotic trials.

Penicillin was not included, as penicillin is effective for positive gram bacteria and

agrobacterium tumefaciens is a negative gram bacterium, resulting in ineffectiveness.

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DISCUSSION & CONCLUSION

The hypothesis was: If fresh garlic extract is used on the plant disease agrobacterium

tumefaciens, it will improve agricultural health by preventing further growth of the bacteria. This

may allow for the opportunity for other detrimental plant diseases to be prevented as well.

In this project, the investigation was whether or not fresh garlic extract could be used to

prevent the growth of crown gall, which would help reduce financial loss for tree farmers.

Multiple tests were run to interpret the data: first an ANOVA, which yielded a p-value of

0.45964 (a confidence of 54%). Based on this value, overall, the data was not significant.

However, the control was not calculated into this test—this merely compares each trial to itself.

Meaning, that no one concentration (for garlic and including antibiotics) performed any better or

worse than the other. To determine if the trials were significant and effective when compared to

the control, twelve individual T-tests were performed. Apart from penicillin, all of the trials had

p-values that were less than 2%, as seen in figure 2. It can be said that with 98% confidence, that

all trials (not including penicillin) were statistically significant. This is because a p-value of 0.02

(2%) means that there is a 2% chance that nothing occurred or that what occurred happened by

chance. Penicillin, an antibiotic that best works against gram-positive bacteria, was not effective

against agrobacterium tumefaciens, a gram-negative soil bacterium. Looking at the results from

the T-tests performed, the evidence was sufficient to reject a null hypothesis.

Limitations for this project included time. One source of error could have been due to

differences in the streaking of the plates on each day. A way to solve this problem in the future

would be to have the same person streak the plate so that the technique remains the same. To

expand on this project in the future, different potential antibiotics could be used in place of

garlic, such as cloves. Testing garlic extract on plant diseases other than crown gall and more
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variation in concentrations as well as more trials to evaluate if a particular concentration

performed better in comparison to the others is a consideration to make in the future.

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LITERATURE CITED

Cab International. (2021). Invasive Species Compendium. Xanthomonas campestris (black rot

of crucifers). https://www.cabi.org/isc/datasheet/56918

Carter. (1989). Crown Gall of Stone Fruits and Nuts, Economic Significance and Diversity of

its Causal Agents: Tumorigenic Agrobacterium spp. Journal of Plant Pathology. 92(1).

87 https://www.jstor.org/stable/41998760

Joy, Ann and Hudelson, Brian. (2005). Crown Gall. Extension. University of Wisconsin-

Madison. https://hort.extension.wisc.edu/files/2014/11/Crown-Gall.pdf

Kennedy and Alcorn. (1980). Bio-Care Technology. Providing BioLOGICAL Solutions for

Crown Gall Control. http://bio-caretechnology.com/economic-loss-crown-gall/

Khan, Awais. Fact Sheet: Crown Gall. KhanLab at Cornell University.

https://blogs.cornell.edu/applevarietydatabase/fact-sheet-crown-gall/

Li, Guoliang, Ma, Xudong, Deng, Lisha, Zhao, Xixi, Wei, Yuejiao, Gao, Zhongyang, Jia, Jing,

Xu, Jiru, Sun, Chaofeng. (May, 2015). Fresh Garlic Extract Enhances the Antimicrobial

Activities of Antibiotics on Resistant Strains in Vitro. Jundishapur Journal of

Microbiology 8(5). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4458355/

Pique, Nuria, Minana-Galbis, David, Merino, Susana, Tomas, Juan. (2015). Virulence Factors

of Erwinia amylovora: A Review. International Journal of Molecular Sciences 16(6).

https://pubmed.ncbi.nlm.nih.gov/26057748/

Reynolds, Jackie. (2021). Biology LibreTexts. Kirby-Bauer (Antibiotic Sensitivity).

https://bio.libretexts.org/Learning_Objects/Laboratory_Experiments/Microbiology_Lab

s/Microbiology_Labs_I/09%3A_Kirby-Bauer_(Antibiotic_Sensitivity)
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Shuford, Jennifer, Steckelberg, James, Patel, Robin. (2005). Effects of fresh garlic extract on

Candida albicans biofilms. American Society for Microbiology ASM Journals 49(1).

https://pubmed.ncbi.nlm.nih.gov/15616341/

The Morton Arboretum. Plant Galls. https://mortonarb.org/plant-and-protect/tree-plant-

care/plant-care-resources/plant-galls/

The Royal Horticultural Society. Crown Gall. https://www.rhs.org.uk/disease/crown-gall

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APPENDICIES

Appendix A: Experimental Design Diagram (EDD)

INDEPENDENT VARIABLE: Different concentrations of garlic (g) to water (mL) & antibiotic trials

units of measurement: millimeters (diffusion)

Control Independent Variable change 1: Independent Variable change 2: Independent Variable change 3:

-Blank, sterile disks used on all 4 g of garlic/ 0mL of water 4 g of garlic/ 0.5mL of water 4 g of garlic/ 1mL of water

strains of bacteria

Independent Variable change 4: Independent Variable change 5: Independent Variable change 6:

4 g of garlic/ 1.5mL of water 4 g of garlic/ 2mL of water 4 g of garlic/ 2.5mL of water

Independent Variable change 7: Independent Variable change 8: Independent Variable change 9:

4 g of garlic/ 3mL of water 4 g of garlic/ 3.5mL of water 4 g of garlic/ 4mL of water

Independent Variable change 10: Independent Variable change 11: Independent Variable change 12:

Penicillin used Erythromycin used Streptomycin used

Trials: 5 for control Trials: 4 for each concentration Trials: 4 for each antibiotic TOTAL TRIALS:

53

DEPENDENT VARIABLE: diffusion of the bacteria across the plate (Kirby Bauer Test)

units of measurement: millimeters (diffusion)

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Appendix B

Figure 2- Kirby-Bauer test results for antibiotic (Erythromycin, Streptomycin, and Penicillin)

Appendix C

Figure 3- Kirby-Bauer test results for Fresh Garlic Extract trials with control trial

Appendix D

Figure 4- Fresh Garlic Extract in centrifuge to separate liquid from solid

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Appendix E

Figure 5- creating FGE sensi discs

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ACKNOWLEDGEMENT OF MAJOR ASSISTANCE

Thank you to the Rockdale Magnet School for Science and Technology for providing lab

space and funds for this project.