Methanol and Aqueous Extract of Terminalia bellirica.: A
Quorum Sensing Inhibitor and the Novel Application of
Terminalia bellirica as an Environmental and Cost-Effective
Measure to Inhibit Plant Pathogens in Crops.
A Plant Science and Environmental Paper
Presented to
Junior Science, Engineering, and Humanities Symposium
Seattle Pacific University
by
Indira Rayala
Junior
Henry M. Jackson High School
P.O. Box 1409
Mill Creek, WA 98012
September 5, 2013- January 18, 2014
Mr. Andrew Sevald
Science Research Instructor
Mill Creek, Washington 98012
2
NAME:
HOME ADDRESS:
Indira Rayala
3215 179th ST SE
Mill Creek, WA 98012
SCHOOL:
Henry M. High School
SPONSOR/TEACHER:
Mr. Andrew Sevald
TITLE
Methanol Extract of Terminalia bellirica: A Novel
Quorum Sensing Inhibitor and the Application of Terminalia bellirica as an
Environmental and Cost-Effective to Plant Pathogens in Crops.
Abstract:
3
Contents:
Page
Purpose
4
Hypothesis
5
Independent and Dependent Variables
6
Introduction
7-8
Method
9-13
Discussion of Results
Tables and Figures
Attachments
14-15
16
17-20
Statistical Analysis
21
Conclusion
22
Future Studies
23
Acknowledgements
24
Bibliography
25
4
Purpose
The purpose of this study was to determine:
1. If the methanol extract of Teminalia bellirica will show Anti-Quorum Sensing
qualities
2. If the methanol extract of Terminalia chebula Retz will inhibit Quorum Sensing in
Ralstonia Solanacearum and Xanthomonas Oryza pv. oryzae
3. If the methanol extract of Terminalia Bellirica will prevent Ralstonia Solancearum
from infecting a tomato plant.
In order to:
i.
Develop an environmental and cheap antibiotic for plant pathogens in
crops.
ii.
Develop an antibiotic that plant pathogenic bacteria will not become
resistant to.
5
Hypotheses
1. The methanol/aqueous extract from Terminalia bellirica will inhibit the bio-film
production in Ralstonia solanacearum and Xanthomonas oryzae pv. oryzae
2. The methanol/aqueous extract from Terminalia bellirica will show Anti-Quorum sensing
ability in the disc diffusion assay.
3. The methanol/aqueous extract from Terminalia bellirica will change the integrity of
AHLs molecules
Variables
1. The independent variables are the methanol and aqueous extracts of Terminalia bellirica.
2. The dependent variables in this experiment are the amount of biofilm formed and the
presence of blue colonies in the disc diffusion assay.
3. The controlled variable is the environment and time.
6
Introduction
Annually, 500 billion dollars are lost due to bacterial diseases in crops (Oerke et al., 1994). In the United
States alone, crop losses due to plant pathogens amount to $9.1 billion dollars, while worldwide, plant
diseases reduce crop productivity by 12% (Food and Agriculture Organization, 1993). Current methods to
treat plant disease are treating plants with chemicals antibiotics and heat. This is done to eradicate the
disease, but this method is not always successful. This treatment is also hazardous to the surrounding
environment, which has cause great alarm to environmentalists. A possible treatment for stopping plant
disease is shutting down the pathogen’s quorum sensing. Once thought of as simple organisms,
research now shows that bacteria exhibit complex methods of communications, thus making it
easier to spread disease. Quorum sensing is when bacteria release signaling chemical molecules
into the environment, which are called autoinducers. Quorum sensing cells produce, detect and
respond to these molecules (Taga and Bassler, 2003). This is incredibly important in pathogenic
bacteria since quorum sensing plays a role in launching virulence. Quorum sensing allows
bacteria to chemical determine when species density becomes high enough to overcome the host
cells ability to produce an effective immune response. So if quorum sensing was to be inhibited
then bacteria would be “confused”. They would not be able to launch virulence and the host’s
immune system will be able to kill of the pathogens (Kievit and Iglewski, 2000). While this has
been heavily research in human pathogens there has been little research with plant pathogens, in
comparison. One of the main autoinducers, involved in launching virulence, in gram-negative
bacteria, which envelope all of the significant plant pathogens, is N-Acyl homoserine lactones
(AHLS). If this autoinducer was inhibited then the pathogen would be able to launch virulence in
the plant thus causing the plant no harm. This allows the plant to resist the disease and produce
anti-microbial compounds to finish the pathogen off. Although, many chemical inhibitors have
7
been discovered to inhibit AHL, they raise questions on environmental safety. To avoid this issue
and to discover an environmentally friendly method to prevent plant disease through quorum
sensing, methanol and aqueous extracts will be derived from the ayurvedic plant Terminalia
bellirica. This plant has traditionally been used to treat heart problems, but the seeds of this plant
have recently been proven to be abundant with hydrolysable tannins (Sarabhai et al.).
Terminalia chebula has shown anti-quorum sensing abilities
8
Method
Plant Extract Preperation
Dr. Dee Denver, a specialist in mutation and genome evolution, suggested that the nematodes,
Caenorhabditis elegans (C.elegans) be used as a model species for producing my Model for
Sustainable Fisheries. C. elegans are eukaryotes, meaning they share cellular structures,
molecular structures, and control pathways with higher organisms. C. elegans are also
multicellular organisms, containing 959 cells. C. elegans go through a complex developmental
process that includes embryogenesis, morphogenesis, and growth to an adult (Eisenmann 2005).
Hence, C. elegans can be directly related to more complex organisms, such as fish species.
Another advantage to using C. elegans in producing my model is the speed of the C. elegan life
cycle. C. elegans develop from egg to adult in three days and produce a few hundred offspring
three days after becoming an adult. Whereas if a fish species such as Atlantic Silversides were
used, it could take years to produce enough generations so as to develop a model.
I obtained my stock of C.elegans by mail from Dr. Denver, assistant professor of zoology at
Oregon State University. Also In preparation for my study, I purchased forty NGM agar plates
seeded with the C. elegans food source, e. coli, and forty NGM agar plates not seeded with
E.coli. I also purchased a Swift* M10 Series Compound Digital Microscope with a 3-in flip up
LCD screen with USB output and camera that provides 5-megapixel still images. In addition to
9
the microscope, SwiftCam Imaging II software was purchased to measure and annotate images
taken by the microscope’s camera.
Obtaining Synchronous Populations of C. elegans
Five groups with five replicates in each were used in my study. The first two groups, A: 1-5 and
B: 1-5, were used in the verification phase of my study and the last three groups, C: 1-5, D: 1-5,
and E: 1-5, were used in the model phase of my study. Each of the 25 populations in my entire
study had to be synchronized. That is, the C. elegans in each population had to all start at the
same phase in their life cycle. In doing this, a process from www.wormbook.org; Maintenance of
C. elegans, was revised and used to acquire just the eggs from a stock population of C.elegans.
First, I pipetted distilled water across the top of the agar of a stock dish of C. elegans. This
loosened the worms and eggs stuck in the bacteria and agar. Then, I collected 3.5-mL of the
distilled water from the dish into a test tube. A solution of .5mL 5 N NaOH and 1-mL household
bleach (5% solution of sodium hypochlorite) was mixed and then added to the test tube with the
3.5-mL mixture of distilled water, worms and eggs. The mixture of bleach and 5 N NaOH is used
to kill all everything except the eggs in the mixture. The test tube was then capped and sealed in
preparation for the vortex and centrifuge. The test tube was vortexed for three seconds, every
two minutes, for ten minutes. After vortexing, the test tube was placed in the centrifuge where it
was centrifuged at 1144×g for 30 seconds. The test tube was then aspirated to 0.1-mL. This
collection of 0.1mL was then put into another clean test tube and had distilled water added to it
up to 5-mL. This second test tube was then centrifuged at the same speed for the same amount of
time and also aspirated to 0.1mL. The 0.1mL of axenized eggs released after being centrifuged
10
was pipetted across an empty, unseeded with E. coli, NGM agar dish. The eggs were kept on the
unseeded agar dish until the worms reached the L1 stage, which only took about 9 hrs. When C.
elegans are starved of their food source, E. coli, they can not grow past the L1 stage. The
unseeded agar dish of L1 C. elegans was then gridded and sliced into 25 equal 1cm x 1cm
chunks of agar. One agar chunk was then transferred to each of the 25 agar dishes seeded with E.
coli. The worms then dispersed from the unseeded agar chunks to the E. coli food on the seeded
dishes, where they could then continue growing in their life cycle. Before any sizing or
harvesting could be preformed, the C. elegans had to remain on the E. coli seeded dishes for 2.5
days to reach their adult sizes (Eisenmann 2005).
Sample Sizing
After the C. elegans had grown to the adult stage and before any harvesting could occur, sample
sizing was done to each group of C. elegans to determine what sizes were to be harvested from
each of the populations. The sample sizing consisted of photographing 20 random C. elegans
from each population and then using SwiftCam Imaging II to size each of the 20 C. elegans in
each population. Normal distribution graphs were made for each of the populations, these graphs
were to be used in determining what sizes of C. elegans would be harvested from each
population in the Verification and Model Phases. Also, with each development of a new
generation, a sample sizing of the population was taken for use in testing a difference in the
average size of C. elegans in from generation to generation. That is, after harvesting, did future
generations of C. elegans produce smaller adult C.elegans.
Developing an Innovative New Protocol For Harvesting C. elegans
11
The NGM agar dishes that the C. elegan populations grew in were seeded with lines of E. coli
bacteria (strain OP50), so as to make it easier to quickly scan through each line of bacteria for
the C. elegans. Before choosing to harvest a C. elegan, it quickly had a picture of it taken by the
digital microscope that was hooked up by USB input to a computer where the program,
SwiftCam Imaging II, could quickly size the C. elegan in the photo. If the size of the C. elegan
coordinated with the criteria for harvesting in that particular population, then it could quickly be
harvested from the agar dish.
Verification Phase for Producing a Model for Sustainable Fisheries
In the verification phase, two groups of synchronous C. elegans were used to verify that C.
elegans would follow the same evolutionary patterns as the Atlantic Silversides in Conover’s
study. That is, if 90 percent of the largest C. elegans in a population (group A:1-5) are harvested,
then future generations will genetically differ to be smaller . Also, a control population (group
B:1-5) had a random 90 percent of it’s population harvested from each generation for the
duration of the experiment (Conover and Munch 2002).
Model Phase for Producing a Model for Sustainable Fisheries
Three different groups(C:1-5, D:1-5, E:1-5) were used in developing my Model for Sustainable
Fisheries. From the normal distribution graphs made from the sample sizing, the size
requirements for the harvesting from each of the three groups were calculated. Group C had all
sizes of C. elegans that fell under a half standard deviation (50%-84%) on the distribution graph,
harvested from all five of its replicates. Group D had all sizes of C. elegans that fell under one
third a standard deviation (50%-72.66%) on the distribution graph, harvested from all five of its
12
replicates. Group E had all sizes of C. elegans that fell under one quarter (50%-67%) of a
standard deviation on the distribution graph, harvested from all five of its replicates.
Harvesting for Producing a Model for Sustainable Fisheries
After the first harvest from Generation 1 of each of the groups, all of the C. elegan populations
could only be harvested every six days. It takes three days for the remaining adults to reproduce
and three days for the eggs produced by the remaining adults, to become adults themselves
(Eisenmann 2005).
To allow for the growth and harvest from five full generations of C. elegans, use of the new
protocol for harvesting C. elegans and producing a model for sustainable fisheries is ongoing
with results expected by March 16, 2010.
13
Discussion of Results
Sample Sizing
I arranged the sample sizings of all of my populations together into a list of 50 different sizes of
C. elegans. The results found by my sample sizing were arranged into a standard distribution
table and standard distribution graph. The standard distribution graph and table were used for
representing all five populations of C.elegans.
Swift* M10 Series Compound Digital Microscope and SwiftCam Imaging II
The use of the Swift* M10 Series Compound Digital Microscope and SwiftCam Imaging II
software was a success in being able to photograph and size the microorganism, C. elegans, in
real time. The speed of sizing a C. elegan would easily allow for a quick determination of
whether or not to harvest.
Verification Phase
90 percent of the C. elegans from each of the Group A population replicates,1-5, are to be
harvested. All C. elegans harvested in the 90 percent must be larger than a half standard
deviation, or as large as the C. elegans in the top 34 % of the population distribution table and
14
graph. 90 percent of all C.elegans that meet the following criteria on the standard deviation
graph, are currently being harvested.
Model Phase
The following are requirements for harvesting from each of the groups in the Model Phase:
The size requirements for harvesting from group C:1-5 are, from 7553.512 um-7856.252 um
(one half a standard deviation, from 50%-84%).
The size requirements for harvesting from group D:1-5 are, from 7553.512 um-7755.339 um
(one third a standard deviation, from 50%-72.66%).
The size requirements for harvesting from group E:1-5 are, from 7553.512 um-7704.882 um (one
fourth a standard deviation, from 50%-67%)
The following results are being used in harvesting from the C. elegan populations, C:1-5, D:1-5,
and E:1-5, for use in discovering which harvested group would not lose genetic variability for
length in future generations. Harvesting based on this data collection is ongoing with results
expected by March 16, 2010, so as to allow for growth and harvest from five full generations of a
C. elegans.
15
Tables & Figures
Figure 1: A population distribution graph based on sample sizings from each population, adding
up to 50 different C. elegans.
Normal Distribution value
Population Distribution of Adult C. elegans
Size of Adult C.elegans in um
Table1: Displays the sizes that will be harvested from each of the C. elegan populations.
mean
st. dev.
7553.512245
605.4794962
Harvest from mean to bold
numbers for each group
1/2 SD
302.7397481
For Model Phase:
7856.251993 Harvesting for
16
(50%-84%)
1/3 SD
(50%-72.66%)
1/4 SD
(50%-67%)
201.8264987
151.369874
Group C: 1-5
7755.338744 Harvesting for
Group D: 1-5
7704.882119 Harvesting for
Group E: 1-5
For Verification Phase:
Group A: 1-5, will harvest 90 percent of the population of C. elegans that are larger than 7856.3
Group B: 1-5, will harvest a random 90 percent of its C. elegan population.
Attachments
Attachment 1:
17
Attachment 1 is a photograph of the Swift* M10 Compound Digital Microscope and the
SwiftCam Imaging II software.
18
Attachment 2:
Attachment 2 represents the agar dishes seeded with lines of E. coli. The lines of E.
coli made it faster and easier to quickly scan down each line of E. coli for the C.
elegans.
19
Attachment 3:
Attachment 3 displays the extraction of the C. elegans eggs, so as to synchronize all populations
of C. elegans.
20
Attachment 4:
Attachment 4 includes two screen shot images of two, sized C. elegans on SwiftCam Imaging II.
21
Statistical Analysis
Length (um)
8579.8
8565.5
8533.9
8521.1
8509.2
8437.1
8350.6
8266.4
8236.4
8227.9
8180.1
8124.2
8029.1
7935.3
7897.5
7865.8
7807.3
7710.4
7687.6
7653.9
7595.2
7546.4
7541.1
7522.9
7522.7
7485.3
7459.6
7341.9
7336.4
7334.5
7273.6
7245.8
7245.8
7201.6
7192.3
7188.6
7185.4
7181.6
7175.7
7136
7073.6
7006.7
6914.9
6722.9
6576.8
6535.8
6518.4
6516.1
6425.4
Normal Distribution
0.000156654
0.000163007
0.000177622
0.000183767
0.000189593
0.000227181
0.000277001
0.00032945
0.000348812
0.000354344
0.000385708
0.000422573
0.000483992
0.000540099
0.00056069
0.000576827
0.000603477
0.000637135
0.000642926
0.000649892
0.000657327
0.000658841
0.000658748
0.000658045
0.000658034
0.000654719
0.000651009
0.00061985
0.00061786
0.000617162
0.000592109
0.000579063
0.000579063
0.000556489
0.000551478
0.000549461
0.000547706
0.000545609
0.000542328
0.000519469
0.000481272
0.000438233
0.000377788
0.000257134
0.000179373
0.000160444
0.000152815
0.000151825
0.000116146
mean
st. dev.
7553.512245
605.4794962
1/2 SD
302.7397481
7856.251993
1/3 SD
201.8264987
7755.338744
1/4 SD
151.369874
7704.882119
Select out of populations from mean
To number indicated in bold
Lengths
Mean
Standard Error
Median
Mode
Standard
Deviation
Sample Variance
Kurtosis
Skewness
Range
Minimum
Maximum
Sum
Count
Confidence
Level(95.0%)
7553.512245
86.49707088
7522.7
7245.8
605.4794962
366605.4203
-0.788340823
0.046823524
2154.4
6425.4
8579.8
370122.1
50
173.9140141
22
Conclusion

In developing a method for sustainable fisheries, a quick and efficient protocol was
established for the sizing and harvesting of the microorganism, C. elegans. This
methodology can, and will, be employed to develop a model for sustainable fisheries.

The results collected from my study are currently being used for harvesting and for
discovering at which threshold (1/2 standard deviation, 1/3 standard deviation, 1/4
standard deviation) C. elegans can be harvested without producing long-term genetic
changes in a population’s size (specifically length), after harvesting from five
generations. Such a method could be used to provide a better understanding for how to
determine what size of fish in a population should be harvested so as to not produce long
term genetic changes in a harvested fish population (specifically for length).
23
Future Studies
In future studies, the model phase method for harvesting could be used with a fish species such
as the Atlantic Silversides, the fish species used in Conover’s study. If results from such study
are significant the same could be done for all species of harvested fish so as to determine at
exactly what sizes fish can be harvested so as to not genetically change a population of fish. Such
genetic changes that are occurring due to harvesting are extremely difficult to reverse. In fact, the
only way to reverse a genetic change in a population is to stop harvesting the population
completely for about twelve full generations (Conover et al. 2009). Due to the time a full
generation can take in a harvested fish species, it would not be at all possible to completely stop
the harvesting of a fish species due to the worldwide need of fishing industries for food and jobs.
24
Acknowledgements
Dr. Dee Denver, Assistant Professor of Zoology, Oregon State University
- Served as my Qualified Scientist, and donated C. elegans
Mr. Chris Reeves, Science Research Instructor
- Assisted with design of my study, provided help with all of my questions, allowed me
access to the biology lab, reviewed my paper
Miss Lori Schlatter
- Provided hours of help and insight in synchronizing C. elegan populations, also
provided a tremendous amount of moral support
Miss Anna Morgan
- Stayed very late after school to provide help and moral support
Mr. Jay Gulshen
- Stayed late to help and provide a minimal amount of support
Mrs. Kim Griggs, Science Research Instructor
- Brought a very good vibe to the Science Research environment
Mrs. Paula O’Connor, Parent
- For allowing me to stay at the school lab for long hours and extremely late nights. Also
for bringing me lunch when I was hungry.
25
Bibliography
Allendorf , F. W., England, P. R., Luikart, G., Ritchie, P. A. & Ryman, N. 2008 Genetic effects
of harvest on wild animal populations. Trends Ecol. Evol. 23, 327–337. (doi:10.1016/
j.tree.2008.02.008)
Conover D O, Munch S B. Sustaining Fisheries Yields Over Evolutionary Time Scales, Science
2002; 297: 94-96.
Conover D O, Munch S B, Arnott S A. Reversal of evolutionary downsizing caused by selective
harvest of large fish, Proceedings of the royal society 2009; 1-6. Available from:
http://www.oceanconservationscience.org/press/files/rs ....
Eisenmann, D. M., Wnt signaling (June 25, 2005), WormBook, ed. The C. elegans Research
Community, WormBook, doi/10.1895/wormbook.1.7.1, http://www.wormbook.org.
Ernande B, Dieckmann U, Heino M. Adaptive changes in harvested populations : plasticity and
evolution of age and size at maturation, The Royal Society 2004; 271: 415-423.
Jørgensen, C. et al. 2007 Ecology: managing evolving fish stocks. Science 318,1247–1248.
(doi:10.1126/science. 1148089)
Oliver R.All About: Global Fishing. In: CNN [discussion list on the Internet]. 2008 Mar. 29;
[cited 2009 Oct. 29]. 3 p. Available from:
http://edition.cnn.com/2008/WORLD/asiapcf/03/24/eco.aboutfishing/index.html