The purpose of this experiment is to analyze the effects of the fungal disease, Pythium aphanidermatum on Bibb lettuce in a microgravity environment. This project is significant because in order to live long term in space, it is necessary to have an understanding of how crops will respond to diseases in microgravity as well as how to effectively treat them.
It was hypothesized that plants infected with P. aphanidermatum would suffer a decrease in health and number in both environments, treating plants infected with P. aphanidermatum would allow the plants to recover faster than non-treated plants in both environments, treating plants not infected with P. aphanidermatum would cause the plants to suffer a decrease in health and number in both environments, and that introducing plants to microgravity would cause a decrease in health and number in comparison to their hydroponics counterparts.
For this project, two separate environments, hydroponics and microgravity, were set up with 80 plants in each environment. A clinostat, a machine that slowly rotates the plants, was used to simulate microgravity. Both environments contained four subgroups: a control (no Pythium or treatment), plants treated with tea tree oil (no Pythium), plants exposed to Pythium (no treatment), and plants exposed to both Pythium and the tea tree oil. Each subgroup contained 20 plants. Leaf color, number of leaves, leaf width, and stem height were measured weekly.
During experimentation, two drops of pure tea tree extract were applied to each plant in the appropriate groups. It is believed the treatment was too strong as the plants began to die. In future trials, the treatment dosage will be reduced to one drop of a 50% concentration.
Trial one of this experiment has ended. The clinostat control, hydroponics Pythium, and clinostat Pythium were the only experimental groups to reach completion. After performing a z-test, the researcher rejected the first hypothesis as the hydroponics Pythium group actually performed better than the hydroponics control group. She also accepted the fourth hypothesis as the clinostat control showed less overall growth and was in worse health. A second trial of this experiment is currently underway.
In order to live long term in a space environment, it is necessary for one to have an understanding of how crop plants will grow in space for the purposes of food production. This project is also meaningful in determining the effects a disease will have on a hydroponics system either in or out of space. Hydroponics systems are used frequently in areas with poor soil or limited growing space and it’s important to be able to maintain healthy crop plants.
The purpose of this experiment is to determine what effect the introduction of a disease to the environments will have on the plants. Tea tree oil will also be applied to a group of the plants to test its viability as a treatment.
The disease I choose to work with was Pythium aphanidermatum, which is a fungus that attacks the roots of seedlings, causing them to rot from the bottom up and topple over. This disease is said to thrive in cool, wet environments, such as a hydroponics system.
I chose to use tea tree oil as a treatment since it is used primarily in the treatment of various fungal diseases in humans such as Athlete’s foot. Because of its usefulness in one species, it may be transferrable to another.
Photosynthesis
Photosynthesis is the process of using light energy to convert carbon dioxide and water into glucose and oxygen. The formula for photosynthesis is 6CO2 + 12H2O + Light Energy C6H12O6 + 6O2 + 6H2O, which means that six carbon dioxide molecules plus twelve water molecules combined with light energy produce a molecule of sugar, six molecules of oxygen, and six molecules of water. Photosynthesis occurs in the chloroplasts of the plant cell. During the process of photosynthesis, the plant releases oxygen, a vital element in life processes. As long as a plant receives its required amount of light, it should produce plenty of oxygen. (Farabee)
Microgravity
Microgravity is not the absence of gravity; it is simply any environment with gravity less than that of Earth’s. However, microgravity doesn’t just exist outside of Earth. On Earth, it is possible to create brief instances of microgravity. When roller coasters move in a steady up and down movement (parabolas), they give you a few seconds of microgravity. This is the same concept used to create the “Vomit Comet.” (NASA)
Microgravity Simulation and the Clinostat
A common instrument used to simulate microgravity is the clinostat. The clinostat is essentially a rotating wheel that contains compartments for plants. By rotating, it disrupts the plant’s sense of gravity (gravitropism), a effect similar to that of an actual microgravity environment. (O’Donnell, Personal)
Terraformation
Terraformation on Mars is something that may have a chance of becoming a reality. The current annual temperatures of Mars is -55oC, much too cold for human habitation (Horack). At one NASA conference, several ideas were thrown around. One possible solution came from Margarita Marinova, an MIT undergraduate at the time. Marinova suggested using perfluorocarbons (PFCs) to help initiate the planet’s warming process. Marinova has studied the effects of PFCs in partnership with Chris McKay of NASA’s Astrobiology Institute. There are several advantages to using PFCs. They are considered super greenhouse gases, so a small amount would warm the planet significantly. PFCs also have a long lifespan and they don’t have any negative effects on living organisms. In addition, PFCs don’t destroy ozone (Horack).
Plant Hormones
Two common plant hormones are auxins and cytokinins. Both are vital to a plant’s growth and development. Auxin’s primary functions are to stimulate cell elongation, cell division, differentiation, and root initiation; mediate phototropism and gravitropism; and delay leaf senescence. A common natural auxin is indole-3-acetic acid (IAA). Cytokinins, such as kinetin, affect root growth and differentiation; stimulate cell division and growth, germination, leaf expansion, shoot initiation, and bud formation; delay senescence; and release of apical dominance. (Auxins, Cytokinins)
Lighting and Plants
Plants require light in order to grow. They use light energy to convert carbon dioxide and water into glucose and oxygen (6CO2 + 12H2O + Light Energy C6H12O6 + 6O2 + 6H2O). White light contains all wavelengths of light. Plants use very little of the yellow and green wavelengths of light, reflecting them instead, which is why they appear green. They mostly use red and blue wavelengths, requiring more red than blue. Incandescent bulbs are discouraged because while they release a lot of red light, they are a poor source of blue light (Trinklein). “Cool-white fluorescent tubes produce a small amount of red rays in addition to orange, yellow-green, and blue rays. However, the red light produced usually is not enough for plants unless windows or other artificial lights produced additional red rays…. Special fluorescent tubes have also been developed for growing plants. These have a higher output in the red range to balance out the blue output (Trinklein).” The plants should receive 16-18 hours of light if that is the only light they are receiving. If they are receiving light from another source, 12-14 hours is sufficient. If plants do not receive adequate lighting, they may have longer stems and lengths between leaves. The leaves may be smaller than those that receive adequate lighting. The coloring may be a pale green and some leaves, typically lower ones, may yellow and drop (Trinklein).
Hydroponics
Hydroponics is the study of growing plants in materials other than soil. There are a plethora of different medias available besides soil, including expanded clay pebbles, rockwool, and perlite. There are several techniques useful for a hydroponics environment, the most common being ebb and flow, drip method, nutrient film technique, and the passive system. Since this project only uses rockwool and the ebb and flow technique, that is all that will be described here. (Hydroponics Online School)
Rockwool
Rockwool is basalt rock, heated at high temperatures and spun into different shapes, usually cubes. It is important to avoid inhalation as it may cause irritation. (Hydroponics Online School)
Ebb and Flow
The ebb and flow technique generally consists of using a reservoir filled with nutrient solution which will pump solution up for a set time then let it drain and repeat. (Hydroponics Online School)
Setting Up the Environments
This experiment involves the use of both a hydroponics environment as the control and a simulated microgravity environment as the experimental environment (Fig. 1). The simulated microgravity environment is created through the use of a clinostat. The clinostat rotates slowly, disrupting the plant’s usual sense of gravity. The plants are contained in black trays, which revolve along with the wheel. The plants receive nutrients as the wheel rotates through a reservoir located at the bottom of the clinostat. The hydroponics group was placed into a large black tray. Nutrient solution continuously flowed past the plants and drained into the tub below. Both environments contained plants grown in rockwool, spun rock shaped into cubes. The nutrient solution used was Flora-Nova in the concentration of a teaspoon for every two gallons.
Data Collection
Over a period of 7 weeks, leaf color, leaf count, leaf width, and stem height were monitored in an effort to determine the health of the plants during their development.
Leaf Color
Leaf color was measured using a specialized chart created by the researcher (Fig. 2). Plants are assigned a number between one and seven, where one is the lowest value on the chart – brown, and seven is the highest value – bright green. The color of the leaves is a useful qualitative measurement of health. The lower the number/value on the scale, the worse the plant is faring.
Leaf Count
Leaf count was measured as a quantitative indicator of health and growth. The healthier a plant is, the more leaves are present. Leaf count also increases as the plant develops. It becomes obvious that the plant is not faring well if in the fourth or fifth week of development, the plant has only developed two or three leaves.
Leaf Width
Leaf width was measured using an electronic digital caliper at the widest part of the leaf. Only one leaf per plant was selected and was chosen from the midsection of the plant in order for the sample to be an adequate representation of the plant. Leaf width was chosen to be measured because it is an indicator of health and growth. As the plant matures, its leaves grow wider and longer. If a plant is in its fourth or fifth week of development and only has a width of 2 mm, it is apparent that the plant is not faring well.
Stem Height
Stem height was measured using an electronic digital caliper from the base of the plant to where the first nodes begin to appear. Stem height typically follows a set pattern. Lengthening of the stem is usual in the first few weeks and then will begin to taper off and possibly even decrease as the plant begins to develop adventitious roots and additional nodes. Stem height is used primarily as an indicator of growth and maturity rather than an indicator of health.
Data Analysis
Averages for each week were calculated. At the conclusion of the experiment, each group was compared back to the control (Hydroponics – non-Pythium, non-treatment). All clinostat groups were also compared back to the clinostat control (non-Pythium, non-treatment). A z-test was conducted in order to test whether or not there was a difference between each experimental group and the control.
Z Test
The z-test is a statistical test used to determine if there is a difference between the mean of an experimental group and the mean of the control group. This experiment allows for a 5% margin of error and since values can decrease or increase, a critical value of 1.96 is used. If the z-value calculated by the formula shown in Fig. 3 falls between -1.96 and 1.96, the null hypothesis is accepted.
Hydroponics Control
The hydroponics control group performed outstandingly well. There were no fatalities. All of the plant were in good health and maintained that status until the end of experimentation. At the end of experimentation, the averages for the group were as follows:
Leaf Color: 7.0
Leaf Count: 24.20
Leaf Width: 103.58
Stem Height: 25.22
Clinostat Control
The clinostat control group fared exceedingly well in comparison to previous years. Only four plants were lost during experimentation, which is normal for this group. At the end of experimentation, the averages for the group were as follows:
Leaf Color: 6.0
Leaf Count: 12.63
Leaf Width: 52.22
Stem Height: 20.15
Hydroponics Pythium
The hydroponics Pythium group is still undergoing experimentation. As of week 4, the averages are as follows:
Leaf Color: 7.00
Leaf Count: 24.95
Leaf Width: 116.37
Stem Height: 19.90
Clinostat Pythium
The clinostat Pythium group is still undergoing experimentation. As of week 4, the averages are as follows:
Leaf Color: 5.80
Leaf Count: 6.70
Leaf Width: 18.35
Stem Height: 24.52
Treatment groups
Shortly after the introduction of the treatment, the plants exposed perished. As a result, there is no data for any of the treatment groups.
Z Values
Clinostat Control to Hydroponics Control
Leaf color: undefined
Leaf count: -15.22
Leaf width: -16.05
Stem height: -7.40
Hydroponics Pythium to Hydroponics Control
Leaf color: undefined
Leaf count: 1.08
Leaf width: 4.36
Stem height: -8.46
Clinostat Pythium to Clinostat Control
Leaf color: undefined
Leaf count: -4.30
Leaf width: -4.48
Stem height: 2.49
Hypothesis 1
Plants infected with Pythium will suffer a decrease in health and number in both environments.
Group Comparison
Hydroponics Pythium to Hydroponics Contol
Clinostat Pythium to Clinostat Control
Based on the z values for these groups, we can see that overall, the hydroponics Pythium group appeared to have performed better on average that the hydroponics control group. The only exception to this was the leaf count value, which fell into the null range.
The clinostat Pythium group, however, appears to have been in worse health than the clinostat control group, as expected.
As of this trial, the first hypothesis has failed to be accepted, because of this discrepancies between the two experimental groups.
Hypothesis 2
Treating plants infected with Pythium will allow the plants to recover faster than non-treated plants in both environments
Group Comparison
Hydroponics Pythium with Treatment to Hydroponics Pythium
Clinostat Pythium with Treatment to Clinostat Pythium
Hypothesis two can neither be accepted or rejected at the time since the experimental groups involved have perished.
Hypothesis 3
Treating plants not infected with Pythium will suffer a decrease in health and number in both environments
Group Comparison
Hydroponics Treatment to Hydroponics Control
Clinostat Treatment to Clinostat Control
Hypothesis three can neither be accepted or rejected at the time since the experimental groups involved have perished.
Hypothesis 4
Introducing plants to microgravity will cause a decrease in health and number in both environments.
Group Comparison
Clinostat Control to Hydroponics Control
Based on the z values for these groups, we can see that, for the most part, the clinostat control group did not perform as well as the hydroponics control group, as was predicted.
However, in this experiment, the stem height is shown to be shorter than that of the hydroponics control. In prior research, the clinostat control stem height is generally longer than that of the hydroponics control.
As with most projects, things have a tendency to not work as planned. This year’s project involved the use of tea tree oil to combat the fungal disease, Pythium aphanidermatum. This oil, as far as I have been able to discover, has not been considered as a way to try to combat Pythium infections. However, tea tree oil has been proven to be an effective topical treatment for ridding humans of their fungal diseases. The solution used was a pure extract from Melaleuca alternifolia and two drops were applied to each plant. Because tea tree oil has not been used on plants, it’s impossible to know what effect the oil will have on the plant. Since the two drops killed the plants, I have decided to run a 1 week side experiment in which I test varying strengths of the tea tree oil against the plant’s ability to survive. After discovering under which concentration the plant can survive, I will rerun the experiments.
In addition, the Pythium culture used does not appear to have infected the plants. Following the completion of the current trial, a second trial will be conducted with the remaining plate, which appears to have more growth than the previous plate.
In the second trial, although the disease showed plentiful growth in the beaker, once administered to the plant, it doesn’t appear to be having any effect on the plant. I contribute this primarily to the discrepancies between my sources and their information on what temperature this disease will thrive at. Lettuce plants enjoy a cooler temperature, in the late 50s to mid-60s while apparently, P. aphanidermatum enjoys warmer climate. With that in mind, P. aphanidermatum may not be a primary pathogen to lettuce.
Aeroponics International. Antifungal properties discovered for growing plants without pesticides. Retrieved August 11, 2010 from: http://www.biocontrols.com/ aero120.htm.
Andrew, Scott. Pythium infection on root rot. Retrieved on August 30, 2010 from: http://hydrocentre.com.au/blog/2006/06/ pythium-infection-on-lettuce-root.html
Bagnall, Roger. Control of Pythium wilt and root rot of hydroponically grown lettuce by means of chemical treatment of the nutrient solution. April 2007. Retrieved on August 17, 2010 from: http://upetd.up.ac. za/thesis/submitted/etd-04242008-115346/unrestricted/dissertation.pdf
Barry, Patrick L. Leafy green astronauts. April 5, 2010. Retrieved January 19, 2011 from: http://science.nasa.gov/science-news/science-at-nasa/2001/ast09apr_1/
Barry, Patrick L. and Tony Phillips. Sowing seeds in a magnetic field. Retrieved January 24, 2011 from: http://weboflife. nasa.gov/currentResearch/currentResearchFlight/sowingSeeds.htm
Biggs, Alton. Et al. Biology: The Dynamics of Life. McGraw-Hill: New York, 2006. Pages 622-623
Bohn, Guy Weston. The important diseases of lettuce. Retrieved on September 8, 2010 from: http://science-in-farming. library4farming.org/PlantDiseases_2/ Vegetable-Crops/Diseases-of-Lettuce.html
Buell, C., et al. Pythium genome database. Retrieved on August 30, 2010 from: http://pythium.plantbiology.msu.edu/
Campbell, Neil. A, Jane B. Reece, and Lawrence Mitchell. Biology: Concepts and Connections. 2nd edition. Benjamin Cummings Publishing: Menlo Park, CA, 1997. Chapter 13.3
Carr, Anitra. Tea trees and their therapeutic properties. Retrieved October 6, 2010 from: http://lpi.oregonstate.edu/f-w98/teatrees.html
Cornell University Plant Diagnostic Clinic. Pythium blight and root rot on turfgrass. Retrieved on September 14, 2010 from: http://plantclinic.cornell.edu/factsheets/ pythium/pythium.htm
Duble, Richard. Pythium blight. Retrieved on October 4, 2010 from: http://aggie-horticulture.tamu.edu/archives/parsons/turf/publication/PythBlight.html
Farabee, Michael. Plant hormones, nutrition, and transport. May 18, 2010. Retrieved on November 20, 2010 from: http://www.emc.maricopa.edu/faculty/farabee/biobk/biobookplanthorm.html
Latin, Richard. Pythium blight. Retrieved on October 4, 2010 from: http://www.extension.purdue.edu/extmedia/BP/BP-109-W.pdf
McGraw Hill. Biology: The Dynamics of Life. Columbus, 2006. Pages 622-623.
National Aeronautics and Space Administration. What is microgravity? Retrieved on January 19, 2011 from: http://www.nasa.gov/centers/glenn/shuttlestation/station/microgex.html
Olsen, Cynthia. The benefits of tea tree. 1998. Retrieved October 6, 2010 from: http://aztec.asu.edu/iaha/articles/Tea_Tree_Article/tea_tree_article.html
Olsen, Mary. Diseases of lettuce in Arizona. February 12, 2010. Retrieved on August 11, 2010 from: http://ag.arizona.edu/plp/ plpext/diseases/vegetables/lettuce/ lettuce.html
Farabee, Michael. Plants and their structure. June 6, 2007. Retrieved on November 18, 2010 from: http://www.emc.maricopa.edu/ faculty/farabee/biobk/ biobookplantanat.html
Farabee, Michael. Plants and their structure II. June 6, 2007. Retrieved January 19, 2011 from: http://www.emc.maricopa.edu/ faculty/farabee/biobk/ biobookplantanatII.html
Fauth, Peter. Plant hormones. Retrieved November 20, 2010 from: http://users.hartwick.edu/fauthp/ horthormone.ppt
Flinn Scientific. Hydroponics nutrient solution MSDS. Retrieved on September 21, 2010 from: http://www.flinnsci.com/ Documents/MSDS/H/HyNuSo.pdf
Fuller, Michael. Plant structure and function. Retrieved on November 18, 2010 from: http://www.uic.edu/classes/bios/ bios100/labs/plantanatomy.htm
Ginsberg, Elizabeth. Pythium wilt. Retrieved on August 30, 2010 from: http://www.backyardgardener.com/alive/ ga58.html
Globus, Ruth. Hydroponics. March 30, 2010. Retrieved November 28, 2010 from: http://settlement.arc.nasa.gov/teacher/lessons/contributed/thomas/hydroponics/ hydroponics.html
Gonsalves, Andrew K. and Stephen A. Ferreira. Pythium primer. June 1994. Retrieved October 26, 2010 from: http://www.extento.hawaii.edu/kbase/crop/type/pyt_prim.htm
Gruener, Raphael. Are cells capable of sensing gravity? Retrieved on January 19, 2011 from: http://www3.physiology.arizona.edu/ people/gruener/clinostat/cansense.html
Gruener, Raphael. The clinostat’s history. Retrieved on January 19, 2011 from: http://www3.physiology.arizona.edu/ people/gruener/clinostat/history.html
Gruener, Raphael. How does the clinostat work? Retrieved on January 19, 2011 from: http://www3.physiology.arizona.edu/ people/gruener/clinostat/howworks.html
Gruener, Raphael. Why simulate icrogravity? Retrieved on January 19, 2011 from: http://www3.physiology. arizona.edu/people/gruener/clinostat/ whysim.html
Henderson, Donna. Plant Pathology. Retrieved on October 11, 2010 from: http://ceimperial.ucdavis.edu/ Plant_Pathology/
Inglis, Debra Ann and Erik Vestey. Crop profile for lettuce in Washington. Retrieved on August 30, 2010 from: http://users.tricity.wsu.edu/~cdaniels/profiles/Lettuce.pdf
James, Donald. What is microgravity? Retrieved on January 19, 2011 from: http://quest.nasa.gov/smore/background/ microgravity/MGintro1.html
Kimball, John. Auxin. January 2, 2011. Retrieved January 19, 2011 from: http://users.rcn.com/jkimball.ma.ultranet/ BiologyPages/A/Auxin.html
Kimball, John. Cytokinins. January 18, 2011. Retrieved January 19, 2011 from: http://users.rcn.com/jkimball.ma.ultranet/ BiologyPages/C/Cytokinins.html
Kimball, John. Tropisms. March 30, 2009. Retrieved on November 21, 2010 from: http://users.rcn.com/jkimball.ma.ultranet/ BiologyPages/T/Tropisms.html
Knee, Michael. Plant hormones. Retrieved on January 20, 2011 from: http://www.hcs.ohio-state.edu/ hcs300/hormone.htm
Koike, S.T. Pythium root rot. March 10, 2009. Retrieved on August 20, 2010 from: http://www.ipm.ucdavis.edu/PMG/r280100211.html
Koning, Ross. Apical dominance. Retrieved on August 11, 2010 from: http://plantphys.info/apical/apical.html
Parker, Kala. Pythium aphanidermatum. Retrieved December 1, 2010 from: http://www.cals.ncsu.edu/course/pp728/ Pythium/Pythium_aphanidermatum.html
Parsons, Jerry. Hydroponics as a hobby. Retrieved on November 28, 2010 from: http://aggie-horticulture.tamu.edu/archives/ parsons/publications/hydroponics.html
Pennsylvania State University. Vegetable disease identification. September 18, 2009. Retrieved September 7, 2010 from: http://vegdis.cas.psu.edu/VegDiseases/ Identification_files/lettuce.html
Reddy, Anireddy. Plant growth in microgravity. Retrieved October 15, 2010 from: http://spacegrant.engr.colostate.edu/ projects/archive/pre2003/past/plantgrowth/index.html
Resh, Howard. Hydroponic culture of lettuce. April 2006. Retrieved on August 30, 2010 from: http://betterbuyhydroponics.com/ index.php?pr=Hydroponic_Lettuce_1
Resh, Howard. Hydroponic culture of lettuce II. July 2006. Retrieved on August 30, 2010 from: http://betterbuyhydroponics.com/ index.php?pr=Hydroponic_Lettuce_2
Resh, Howard. Hydroponic culture of lettuce III. September 2006. Retrieved on August 30, 2010 from: http://betterbuyhydroponics.com/ index.php?pr=Hydroponic_Lettuce_3
Roberts, William J. Horticultural engineering. Vol. 13. No. 4. July 1998. Retrieved from: http://aesop.rutgers.edu/ ~horteng/newsletter/1998/vol13-4.pdf
Sack, Fred, Volker Kern, Guy Etheridge, and Dave Reed. Development of gravity sensitive plant cells in microgravity. Retrieved on January 24, 2011 from: http://weboflife.nasa.gov/currentResearch/currentResearchFlight/sts107SeekingTheLight.htm
Santos, B.M., W.M. Stall, R.N. Raid, and S.E. Webb. Lettuce, endive, and escarole production in Florida. Retrieved November 28, 2010 from: http://edis.ifas.ufl.edu/pdffiles/CV/CV12600.pdf
Society for Science & the Public. Intel ISEF Rules and Regulations 2010. Retrieved August 9, 2010 from: http://www.societyforscience.org/
Shrestha, Arjina and Bruce Dunn. Hydroponics. Retrieved on October 17, 2010 from: http://pods.dasnr.okstate.edu/ docushare/dsweb/Get/Document-6839/HLA-6442web.pdf
Steinburg, Susan L., Doug W. Ming, and Don Henninger. Plant production systems for microgravity: critical issues in water, air, and solute transport through unsaturated porous media. February 2002. Retrieved on October 15, 2010 from: http://ston.jsc.nasa.gov/collections/TRS/ _techrep/TM-2002-210774.pdf
Christman, Steve. Lactuca sativa. March 20, 2004. Retrieved October 19, 2010 from: http://www.floridata.com/ref/l/lact_sat.cfm
Texas A&M University Partnership for Environmental Education and Rural Health. Plant tropisms. Retrieved November 21, 2010 from: http://peer.tamu.edu/NSF_files/plant%20tropisms.ppt
Tredway, Lane P. Pythium root rot. March 23, 2009. Retrieved on August 20, 2010 from: http://www.turffiles.ncsu.edu/ diseases/Pythium_root_rot.aspx
UniProt Consortium. Lactuca sativa. Retrieved on August 27, 2010 from: http://www.uniprot.org/taxonomy/4236
University of Arizona Cooperative Extension. Lettuce. Retrieved October 29, 2010 from: http://ag.arizona.edu/pubs/garden/mg/vegetable/lettuce.html
University of Michigan Health System. Athlete’s foot. September 1, 2007. Retrieved October 6, 2010 from: http://health.med.umich.edu/healthcontent.cfm?xyzpdqabc=0&id=6&action=detail&AEProductID=hw_cam&AEArticleID=hn-1014004
University of Michigan Health System. Tea tree. September 1, 2007. Retrieved October 11, 2010 from: http://health.med.umich.edu/healthcontent.cfm?xyzpdqabc=0&id=6&action=detail&AEProductID=hw_cam&AEArticleID=hn-2173003
Vega, Everardo. Attachment and survival of viruses on lettuce (Lactuca sativa L. var. capitata L.): Role of physicochemical and biotic factors. August 2006. Retrieved on October 22, 2010 from: http://repository.tamu.edu/bitstream/handle/1969.1/4158/etd-tamu-20068-fstc-vega.pdf?sequence=1
Whiting, David, Michael Roll, and Larry Vickerman. Plant structures: roots. August 2010. Retrieved November 18, 2010 from: http://www.cmg.colostate.edu/gardennotes/132.pdf
