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RESEARCH ARTICLE
Testing the Efficacy of Nanoparticles, Biological Formulation
with the qPCR Technique to Improve HLB Management
Shloke Patel
Hillsborough Highschool, 2218 Branch Hill Street, Tampa, Florida, 33612, USA; shlokep8@[Link]
ABSTRACT: Citrus phloem bacteria, Candidatus Liberibacter asiaticus (CLas), causes the citrus greening disease,
Huanglongbing (HLB). In 2019, this devastating disease caused a 21% decrease in the fresh citrus fruit market and a 72% decline
in the commercially grown production of oranges in Florida. The number of Florida industry jobs dropped by 59% compared to
previous years. HLB has no cure; global orange juice production is expected to fall by double digits in the coming years. Typical
symptoms include yellowing of the leaves, as well as lopsided, premature, and bitter fruits. Since, CLas hides deep within the citrus
phloem, commercial antibiotic sprays often cannot reach the bacteria. A biological solution for the prevention of the detrimental
effects of HLB was used; the formulations tested were combined with copper nanoparticles which would potentially increase the
penetration and the longevity of the formulation to mitigate the effects of the greening disease. The formulations included Firewall
and Fireline, two approved and readily available antibacterial solutions, and a bioformulation, curry leaf extract.
The experiment was conducted in a greenhouse with 42 infected plants with five leaf samples being collected from each plant.
The DNA of the leaves was extracted using the GeneJET DNA purification Kit. 6 formulations: Firewall with and without
nanoparticles, Fireline with and without nanoparticles, curry leaf extracted with and without nanoparticles were prepared and
injected into six plants. After 3 months, the bacterial DNA concentration was determined, and 4 physical parameters of the plant
were measured. The formulations were injected again, and after 70 days, qPCR assay and physical parameters were measured.
Then, both the CT values and physical parameters were collected and compared.
The bacterial count remained unchanged in the treatment and control variables, the three physical parameters, trunk diameter,
plant height, and width all improved compared to the control variable.
KEYWORDS: Biology; Agriculture; Huanglongbing; Candidatus liberibacter; Nano formulation; GeneJET.
� Introduction researchers have been experimenting with the use of minuscule
The Citrus production in Florida is limited by the impact particles called nanoparticles. These 100-nanometer particles
of various biotic and abiotic factors. Of all the citrus limiting can easily penetrate a cell wall allowing the antibiotic to seep
factors, Huanglongbing (HLB) or citrus greening is the most through phloem.³ These particles are currently used in medical
prominent and rampant in spreading throughout a grove in just and technological fields but are scarcely used in the agricultural
matter of weeks. Although Florida has favorable conditions field.
and is best suited for citrus production, it is very prone to HLB. Citrus greening disease (Huanglongbing) is caused by a
Citrus greening disease has devastated millions of acres of bacterial infection (Candidatus liberibacter), which spreads
citrus crops throughout the United States and abroad.¹ Florida through an insect vector called Asian Citrus Psyllid (ACP).
is the largest orange-producing state in the United States and Psyllid adults feed on citrus greening-infected trees and acquire
the third-largest orange producer in the world behind Brazil the bacteria through the bacterial residue on the vector’s mouth.
and China. The citrus industry makes roughly 45,000 full The psyllid then carries the bacteria from an infected plant to
time and part-time jobs and contributes 8.6 billion a year to a healthy plant. Next, the psyllid injects the bacteria onto a
Florida’s economy.¹ Since HLB was first found in 1998, orange healthy plant while feeding and transfers the greening disease
acreage and yields in Florida have decreased by 26% and 42%, into the plant's phloem, affecting the vascular system and
respectively.² Orange production dropped from 242 million limiting nutrient uptake.⁴ The disease reduces yield, fruit size,
to 104.6 million boxes in 2014 and if this negative trend and quality and increases tree mortality and production costs.
continues, Florida will not have a profitable orange industry Once the orange tree is infected with the greening disease,
soon.² In the current scenario, farmers are faced with the threat the tree eventually dies within a few years.⁵ Currently, many
of losing their orange grove to this disease. Currently, the nanoparticles are being used as fungicides due to the elements’
only short-term viable solution entails antibiotics which wash antibacterial properties.⁶
away after a month or two with devastating effects on the soil. Nanoparticles are made of many conventional materials
Additionally, many unknown side effects are caused by these that change mechanical properties when they are formed into
antibiotics. However, Florida has still allowed these chemicals nanoparticles. Nanoparticles have a greater surface area per
to be sprayed on the orange plants without any proven utility of weight than larger particles, which causes them to be more
the Firewall and Fireline solutions being demonstrated. Many reactive to large particles. These small particles usually come in
© 2021 Terra Science and Education 78 DOI: 10.36838/v4i1.17
[Link]
the form of metals for the agricultural sciences because many qPCR preparation :
phloem or xylem-limited bacteria are not easily penetrable The qPCR technique provided an essential piece of data
by regular antibiotics.⁷ Nanoparticles such as copper bind to when determining the bacterial count, the CT value. There
the bacteria, and because of copper's antibacterial properties, are 3 steps in the process of the qPCR technique which in-
they eliminate the bacteria. Research shows that engineered clude the denaturation, primer annealing, and extension. The
nanoparticles that are metalloids have demonstrated an activity qPCR solution requires 10 µL of TAQMAN qPCR buf-
that kills harmful plant bacteria. Many metalloids are known fer which is comprised of 8.6 µL of sterile deionized water,
for being bactericides and or fungicides but are not used CQUL Primer and the leaves of the plants.
conventionally. Nano-fertilizers have also been very useful in Arbo Jet Injection Preparation:
improving plant health by entering the phloem of the plant. An opening in the trunk is required to inject the 6 solutions
Some nanoparticles act as antimicrobial agents, while others into the plants. A 4 mm hole was drilled into each of the
alter the nutrition of the plant. Penetration of the vascular 42 plants using the drilling machine. Once the path into the
tissue occurs when a particle is sufficiently small so that it can plant was created, 60 mL of the desired solution was injected
seep through the cell wall. During a process called adhesion, into a one-year-old plant. The injection was slowly adminis-
the nanoparticle bends the cell wall, and eventually, the cell tered to prevent the disruption of the flow of nutrients in the
wall wraps around the nanoparticle.⁸ In large quantities, the phloem and xylem. The process took approximately 8 hours
penetration can be beneficial and can play a role in mitigating for the solution to be fully administered.
disease, which might lead to a reduction in the use of heavy Psyllid Repellency:
metals and other chemicals. The psyllid repellency was determined by the CT value,
� Methods which was an indicator for the bacterial count. The physical
Analysis and Procedure: parameters also helped in validating the CT value results.
The experiment was conducted in a greenhouse with 42 � Results and Discussion
infected plants. The psyllid repellency was determined by the The psyllid repellency was tested using 7 treatments in-
prevention of the reinfection of the plant, validated through cluding the control variable. Treatment 1 was the controlled
the CT value. Since this was a short-term experiment, im- variable, and treatments 2 and 3 were the industry standards,
mediate symptoms were tested through the measurement of Fireline and Firewall, respectively. Treatments 4 and 5 were
the physical parameters. Fireline and Firewall have extreme- the industry standards with the nano formulation, and treat-
ly toxic chemicals such as oxytetracycline hydrochloride¹⁶ in ments 6 and 7 were the bioformulation, curry leaf extract,
Fireline and Streptomycin¹³ in Firewall which are helpful in without the nano formulation and with the nano formulation,
killing the bacteria. Samples were collected from each plant to respectively.
determine the concentration of the greening bacteria through The following parameters were studied during the eval-
qPCR assay. DNA was extracted using the Gene JET DNA uations of the results. The parameters monitored include,
purification Kit. increase in the height, increase in the width of the stem,
The initial PCR measurements were used to ensure that all increase in the trunk diameter, increase in the chlorophyll
the plants had the same CT value to provide consistent data. content as a measure of the overall plant health and average
Plant Measurements: increase in the plant vigor (Table 1 through Table 7).
The trunk diameter was measured using the vernier caliper Table 1: Average Increase in Plant Traits of the 42 Citrus Plants through 2
for each of the 42 plants. Plant width was determined by the Growing Seasons.
spread of the leaves that are the farthest from each side of
the plant. The plant height was measured by the trunk of the
plant that was above soil level to the tip of the plant, vertically.
The plant width and plant height were measured using a
measuring tape.
Plant Vigor:
The plant vigor is a researcher-based observation on the
total fullness of the plant. Variables such as the color of the
leaves are taken into consideration. In this experiment a 0-10
scale was used to calibrate the plant vigor.
Chlorophyll Measurements:
The chlorophyll concentrator meter used was the Apogee
Meter which measures the content in μmol per m2. To measure Table 1 shows the average increases of all the plant traits
the chlorophyll content, a leaf was placed between the arm of taken for the 7 treatments and 6 replications, through the 2
the meter and the base of the meter, and the leaf was pressed growing seasons.
against by the arm which detected the chlorophyll. The data
then was gathered in the appendix of the system and was
uploaded to excel.
79 DOI: 10.36838/v4i1.17
[Link]
Table 2: Average Plant Vigor of the 42 Citrus Plants through 2 Growing 39.10%, which was the largest increase of all the 7 treatments.
Seasons (0-10 scale).
Figure 2: Average height increase.
As shown by the Table 3 above after a period of 5.5 months
a more drastic increase was shown compared to the interval of
the start to 3 months of the experiment.
Table 4: Average Width of the 42 Citrus Plants through 2 Growing
Seasons (ft).
Table 2 shows the average plant vigor on a 0-10 scale for the
7 treatments for the 6 replications. There was a significant
increase in treatment 3 with an increase of plant vigor of 1.33.
Table 4 shows the average width of the 42 citrus plants
Figure 1: Average increase in Plant Vigor..
through the 2 growing seasons (approximately 5.5 months).
Most of the treatments started with a plant vigor of around Over the span of 2 growing seasons treatment 3 showed the
8. As the bacteria moved towards the phloem the plant vigor largest percent increase in the width of all the treatments.
decreased and at 5.5 months the plant vigor started to increase.
Treatment 3 had the highest plant vigor at 5.5 months (Figure
1).
Table 3: Average Height of the 42 Citrus Plants through 2 Growing
Seasons(ft).
Figure 3: Average Width increase.
The average width percent increase is shown in the Figure 3
of all the treatments for the 6 replications.
Table 3 and Figure 2 shows the average height of the 42
citrus plants through the two growing seasons, approximately
5.5 months. Treatment 4 had an average percent increase of Figure 4: Average Trunk Diameter increase.
DOI: 10.36838/v4i1.17 80
[Link]
There was a positive trend between the amount of time and qPCR Assay:
the increase in the trunk diameter in the 7 treatments. The controlled environment in the greenhouse provided
Table 5: Average Trunk Diameter of the 42 Citrus Plants through 2 more controlled variables than experimenting in the field be-
Growing Seasons (inches). cause the CT value did not change before and after 3 months.
The physical parameters were measured using the qPCR tech-
nique. A scale of the acceptable CT values in relation to the
bacterial population was created. A CT value below 17 indi-
cates high levels of bacterial infection and a CT value of above
32 indicates that there is little to no bacteria present
Table 7: Average CT value of the 42 Citrus Plants through 2 Growing
Seasons.
This data was significant because there was a significant
increase of growth between 3 months and 5.5 months show-
ing that the treatments had an effect, which was allowing the
phloem and xylem to expand. Treatment 4 had the highest
percent increase of the average trunk diameter of 24.28%. The difference between the starting CT value and the CT
Table 6: Average Chlorophyll Content of the 42 Citrus Plants through 2 value after 5.5 months roughly stayed between 0 and 1 show-
Growing Seasons. ing that the bacterial count remained consistent.
The CT value might have changed over time (Table 7)
because of the bacteria's behavior concerning the temperature,
but not enough time was given to access this variable. To
see more accurate results of the bacteria usually 6 months
is needed.² During the 6 months either the plant will show
significant symptoms or display no signs of symptoms. The
hypothesis was accurately tested due to the data given by
the 4 physical parameters. The physical parameters provided
an accurate method to measure the bacterial content by
examining characteristics including whether the plant was
displaying or not displaying early symptoms such as blotchy
mottle, yellowing of the leaves and veins.
The standard deviation was between 1.32 and 0.23 for the
CT values, which shows that the data varies between 1.32
Due to the large standard deviations, the abnormal produc- and 0.23 units from the mean CT value on average. In the
tion of chlorophyll and chlorophyll deficiency are seen in later experiment, the CT value of each plant was recorded before
stages of HLB so fewer conclusions can be made from this experimenting. 42 out of the 49 plants that had similar CT
data. values were selected for this experiment. The change in the CT
values should be comparable if all the plants have identical CT
values.
Many researchers have tested the effectiveness of curry leaf
extract on different vectors and the results have been positive
due to azacytidine, an active antibacterial compound found in
curry leaf.¹⁷ The expected results of this experiment were that
since copper nanoparticles are also anti-bacterial, they would
improve the efficacy. Currently, the efficiency of the addition
of nano formulation in the plant vascular system is unknown.
Figure 5: Average Chlorophyll content. The CT value in the table shows the number of cycles it
The plant is producing the required nutrients at the higher takes for the PCR to detect the bacteria. Treatment 7 had the
rate as it has more chlorophyll which is displayed by data in the highest increase of the CT value which means the bacterial
table (Figure 5 and Table 6). content decreased the most over the period of 5.5 months.
81 DOI: 10.36838/v4i1.17
[Link]
� Conclusion [Link], W., Ma, C. and White, J., 2018. Nanoparticles for plant
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CT value. According to the data collected in this experiment,
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Verma, S. K., Kumar, A., and Kharwar, R. N. (2016, February 4)
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� Acknowledgements Library of Science.
My sincere gratitude to my mentor Dr. Balaji Aglave for � Author
guiding me through this project and offering his insight and Shloke Patel is a junior at Hillsborough High School. Growing up
expertise on this topic. I could not have completed this ex- around farms, Shloke developed a deep understanding of the benefi-
periment without the help of Florida Agricultural Research. I cial effect of farm fresh food on health. From a young age Shloke took
sincerely thank them for permitting me to conduct my exper- inspiration in gardening by creating a backyard garden and enjoying
fresh vegetables and fruits. He hopes to make a positive impact in the
iment on their premises. I also thank my science teacher Ms.
field of agricultural research.
Mishell Thomas-King for taking her time out of her day to
help me with my project by giving me tips on my research plan.
I thank my mom and dad for taking me to the research center.
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DOI: 10.36838/v4i1.17 82