by Kacey, Grade 8, Florida - 2015 YNA Winner

Introduction and Background Research

Living in Florida, it is not uncommon for me to walk out of my house and grab an orange straight from the tree. My family had planted a young orange tree in our yard, and we expected it to grow big and strong and create wonderful memories, but it would not. Weeks after we planted it, we noticed that something was terribly wrong. Its leaves were wrinkled, splotchy, and yellow. The fruit was split and asymmetrical. The shoots were bright green. Within the year, it had completely died. Obviously there was some disease or infection that caused this certain death, and I began to research it.

Healthy orange tree leaves (left) and infected leaves (right)

A tree that has died from HLB disease

I came across HLB disease, or citrus greening disease. It mimicked every symptom my tree suffered from, and it is fatal to all citrus trees that are infected. An invasive insect called the Asian citrus psyllid (Diaphorina citri) transmits the disease by consuming citrus leaves. The Asian citrus psyllid carries a bacterium called Candidatus Liberibacter, which is directed through the phloem system of the tree, or the system that transports sugars. Florida, other states, and other countries will lose billions of dollars every year because the Asian citrus psyllid will spread HLB disease. Citrus companies will shut down, people will lose their jobs, and more fruit will have to be imported.

Currently, HLB is untreatable and there has been very limited success in eliminating the Asian citrus psyllid. Current research on this subject has nearly stalled because of the extremely difficult task of reproducing HLB in a lab. My interest turned to discovering an organic pesticide that could be used to repel the Asian citrus psyllid from the leaves, therefore, stopping the transmission of HLB.

Poison ivy came to my mind because I have never seen an insect eating it or even resting on it, for that matter. The problem is that poison ivy could never be manufactured into a commercial pesticide because it is exceptionally dangerous to some people. Urushiol oil is the compound that is responsible for the irritating properties in poison ivy, poison oak, and poison sumac. Surprisingly, urushiol has never been used as a pesticide or deterrent. With this newfound knowledge, I began looking for alternative sources of urushiol oil that was in lower concentrations.

The mango is another urushiol-producing plant. The skin of the mango has low levels of urushiol oil that are not harmful to humans or the environment, but it still may have deterrent properties for the Asian citrus psyllid and other pests. If this was successful, it would be a completely organic and safe pest deterrent, unlike so many others used today. A new industry opportunity could be created for the mango while saving the dying citrus industry.

Question

What is the effect of mango skin blended with distilled water on the feeding preferences of the Asian citrus psyllid (Diaphorina citri)?

Hypothesis

If mango skins blended with distilled water are applied to citrus foliage, it will deter the Asian citrus psyllid from consuming the leaves.

Materials

An orange tree (top) and the portable field enclosure cage (bottom)

150 Asian citrus psyllids

Healthy orange tree

8 mangoes

Bioassay arena Foam core Metric ruler X-Acto knife 8 film canisters Pencil

Fine mesh enclosure

Lumite portable field enclosure cage (BioQuip Products)

Aspirator

Latex gloves

Spoons

Glass bowl

Distilled water

High-powered blender

Peeling knife

Procedure

The mango skin solution

Mango Skin Study

First, I created my mango skin solution. I washed and peeled 6 mangoes and put the peel with 150 mL of distilled water in a high-powered blender. The blender ran for 5 minutes before reaching the desired viscosity, which was quite thick to ensure that it would stay on the leaves. The solution was put into a glass container and refrigerated to prevent contamination. Any contact with the solution was handled with gloves.

Next, I created my bioassay arena. I cut a 50-cm-diameter circle out of foam core using an X-Acto knife. Eight 3.5-cm circles on the outer edges held the film canisters, all of them exactly 23 cm from the center. Eight citrus shoots with two to three leaves were cut and put into a slit in the top of the film canisters. Water was added in the film canisters to ensure that they remained healthy.

The bioassay arena (left) Applying the mango skin solution (right)

Mango skin solution was applied to the treatment group using a curved spoon to get the entire leaf covered. The foliage that was not treated with the solution acted as the control. The shoots were arranged so that every other shoot is a treatment, and every other shoot is a control.

Leaves treated with the mango skin solution (left) and the untreated control leaves (right)

Kacey using the aspirator to release psyllids into the study area

The bioassay arena creates a completely unbiased chance that any insect will go to any plant, so the results are based on the psyllids’ feeding preference. The bioassay arena is put in a fine mesh enclosure within the Lumite portable field cage to completely remove the risk of the psyllids getting released into the environment.

A researcher at the University of Florida Citrus Research and Education Center sent me the Asian citrus psyllids. The 20 psyllids for the trial were removed from their enclosure using an aspirator, a contraption that gets insects into a sealed vial without the risk of accidental release. The vial containing psyllids was put into the center of the bioassay arena and opened to release the psyllids into the experiment.

After increments of 3, 6, and 24 hours, the number of psyllids on each shoot was counted and recorded. At the end of every trial, the remaining psyllids were removed from the experimentation area using the aspirator. They were returned to their initial enclosure before the next trial. Every trial used different psyllids.

Mango Flesh Study

Two mangoes are washed and peeled, but instead of blending the skin, the mango flesh (the common edible portion) was blended with 150 mL of distilled water. The same experimental procedures used before were followed using a mango flesh solution. Mango flesh contains no urushiol oil, so this study narrows down possible explanations for the results without using a mass spectrometer.

Results and Discussion

Mango Skin Study

# of psyllids after 3 hrs # of psyllids after 6 hrs # of psyllids after 24 hrs Trail 1- Control 4 2 6 Trail 1- Treatment 2 1 0 Trail 2- Control 7 8 8 Trail 2- Treatment 1 2 1 Trail 3- Control 15 12 14 Trail 3- Treatment 0 1 1 Trail 4- Control 9 10 12 Trail 4- Treatment 0 0 0 Trail 5- Control 5 8 8 Trail 5- Treatment 0 1 1

The numbers in the columns represent the number of psyllids that were counted on each citrus shoot, and whether they were on a control or treatment leaf. The psyllids that are unaccounted for were not on a leaf at the time of the counting. In every trial, the control numbers were significantly larger. The similarity between the 3-, 6-, and 24-hour tests tells me that there is no necessary time for the mango skin solution to sit before becoming effective. Additionally, the solution will stay active for more than 24 hours, but further research will be done to see exactly how long it lasts. Fortunately, the mango skin extract had no negative effects on the leaves, as it was clear after the application, and photosynthesis could occur without obstacle.

The Time Interval Tests

The Control group (purple) had a maximum of 15 psyllids on one shoot, while the maximum number of psyllids on the Treatment group (red) was only 2. Again, the mango skin solution was effective during all three time intervals, and no negative effects on the foliage from the mango skin solution occurred.

Mango Flesh Study

# of psyllids after 3 hrs # of psyllids after 6 hrs # of psyllids after 24 hrs Trail 1- Control 8 9 7 Trail 1- Treatment 6 7 8 Trail 2- Control 5 8 6 Trail 2- Treatment 10 7 9 Trail 3- Control 4 7 6 Trail 3- Treatment 8 11 6

During the mango flesh study, the numbers of psyllids on each shoot remained consistent. The mango flesh solution study suggests that urushiol, or something unique to the skin of the mango, caused the acquired results during the mango skin study. According to the data, the mango skin solution yielded much more significant results than the mango flesh solution.

Throughout the entire study, interesting observations and questions arose. For example, Trial 3 had a much higher number of psyllids on the leaves than the other trials. Unfortunately, I could not pinpoint a logical reason as to why this happened because my procedure did not deviate. It could have been a slight difference in the concentration of the mango skin extract or possibly a difference in temperature or humidity.

While counting the psyllids, I noticed that some ingestion had taken place on the control leaves. Due to the miniscule size of the insect, the ingestion was almost unnoticeable until later in the trial. I inferred that the reason that no ingestion appeared on the treatment leaf is because of the small number of psyllids that were on the treatment leaves. Additionally, the mango skin extract coats the leaf and keeps the psyllid from consuming the actual leaf.

Conclusions

My hypothesis was entirely supported. The foliage that was treated with the mango skin solution yielded a much lower number of psyllids on the leaves than the control group in every trial. The difference between the numbers of psyllids on the control and treatment group was statistically significant, with a P-value that is less than 0.00004, which was calculated using a paired t-test. This means that there is more than a 99.996% probability that my results were because of the mango skin solution. A P-value only has to be less than 0.05 to be significant.

The mango flesh solution study did not have the same outcome as the mango skin study. The difference in the numbers of psyllids on the control and treatment group in the mango flesh study was not significant. This tells me that either the urushiol oil or another compound in the mango skin caused the significant results.

Future Research

After this experiment, many ideas have come to me about how the mango skin solution’s deterrent properties could be utilized further. Different crop industries may benefit, and harsh, nasty chemicals could be replaced with a safe solution that is as, or even more, effective. Particularly, I want to experiment with the Diaprepes root weevil, another devastating pest in the citrus industry.

Furthermore, I want to produce this mango skin solution in the form of a spray for maximum efficiency when treating citrus plants. For the purposes of this experiment, I used a more viscous solution to ensure that it would stay on the leaves, and I was unsure of a spray. Presently, I am confident that a spray would work equally as well as the thicker solution I used, but more investigation will be pursued.

Can this mango skin solution become a worldwide deterrent? Will it be able to compete with the non-organic pesticides? Could it work on pesticide-resistant insects? These are all questions that I hope to find the answer to in the future. While there is still much investigation to be done, I have tapped the surface of a growing area of research that commits to the well-being of the people and environment, instead of compromising our health for a profit.

Works Cited

Armstrong. "Poison Oak: More than Scratching the Surface." (n.d.): n. pag. Wayne's Word. 16 Jan. 2011. Web. 26 Oct. 2014. <http://waynesword.palomar.edu/ww0802.pdf>.

Bove, J. M. "Huanglongbing: A Destructive, Newly-Emerging, Century-Old Disease of Citrus." Jornal of Plant Pathology. N.p., 2006. Web. 4 Nov. 2014.

"Citrus Greening (Huanglongbing)." UF/IFAS Citrus Extension: Plant Pathology. N.p., n.d. Web. 25 Nov. 2014. <http://www.crec.ifas.ufl.edu/extension/greening/index.shtml>.

Grafton-Cadwell, Elizabeth, Kris Godfrey, Michael Rogers, Carl Childers, and Philip Stansly. N.p.: n.p., n.d. ANR University of California. Web. 3 Nov. 2014.

Halbert, Susan, and Carmelo Nunez. "Distribution of the Asian Citrus Psyllid, Daphorina Citri in the Carribean Basin." Florida Department of Agriculture. N.p., Sept. 2004. Web. 6 Dec. 2014.

Halbert, Susan, and Keremane Manjunath. "Asian Citrus Psyllids (Sternorrhyncha: Psyllidae) and Greening Disease of Citrus: A Literature Review and Assessment of Risk in Florida." BioOne. N.p., Sept. 2004. Web. 25 Feb. 2015. <http://www.bioone.org/doi/full/10.1653/0015-4040%282004%29087%5B0330%3AACPSPA%5D2.0.CO%3B2>.

Knapp, Joseph, Susan Halbert, Richard Lee, Marjorie Hoy, Richard Clark, and Michael Kesinger. "Asian Citrus Psyllid and Citrus Greening Disease." IPM Florida. UF IFAS Extension, n.d. Web. 21 Dec. 2014. <http://ipm.ifas.ufl.edu/Agricultural_IPM/asian.shtml>.

"MATERIAL SAFETY DATA SHEET." Sequioa Research Products. N.p., n.d. Web. 23 Oct. 2014. <http://www.seqchem.com/safetysheet.php?SQIndex=SRP01200u>.