By: Charlie Ta
Mentor: Dr. Inga Zasada
United States Department of Agriculture:
Agricultural Research Services

Plant-parasitic nematodes cause $100 billion in crop
loss annually worldwide; $10 billion in the U.S.
(blueberries, red raspberries, and wine grape industry)


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Plants affected by X. americanum or nepoviruses
become(s) necrotic, yield is reduced, and plant
mortality can occur
Currently few methods exist to control nematodes or
remediate the diseases they transmit
Regulations by the U.S. Environmental Protection
Agency will soon limit/ban pre-plant fumigation
which has traditionally been used to eradicate virustransmitting nematodes

head
tail
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Microscopic
roundworm(s) that
parasitize plants
Migratory ectoparasite
Acquire and transmit
nepoviruses such as
Tomato Ringspot Virus
(ToRSV) and Tobacco
Ringspot Virus (TRSV)
with their odontostyle
Nematode-transmitted virus with
polyhedral particles
 Type IV virus under the Baltimore
classification system (positive sense

odontostyle
single-stranded RNA that directly translates into protein)
Acquisition of virus occurs during
feeding and binds to the surface of
the odontostyle
 Viruses are lost when nematodes molt

Reverse Transcriptase Quantitative Polymerase
Chain Reaction (RT-qPCR)


A RT-qPCR can be used for
the detection of ToRSV in
X. americanum at low
concentration levels.
Virus detection using RTqPCR allows for a detailed
study of nematode-virus
interactions.
Enzyme-Linked
Immunosorbent Assay (ELISA)
Steps:
1
2
3
4
5
http://homepage.usask.ca/~vim458/virology/studpages2007/Maura_Tim/For%20Maura%20%20Virology%20website%20assignment/elisa.jpg

The coloration occurs due
to adding p-nitrophenyl
phosphate.

Could a RT-qPCR method be developed to
enable detection of small concentrations of
ToRSV?

A RT-qPCR method will be proficient in
detecting low concentrations of viruses.
 X. americanum acquires ToRSV within a week of
feeding on a virus infected host.
 This time period allows for additional virus
particles to be acquired by the nematode
I.
II.
Develop and Probe:
optimize the Forward:
Reverse:
efficiency of a
RT-qPCR to detect
ToRSV
Quantify acquisition
and saturation level
of ToRSV in X.
americanum
Methodology
Objective I: Development

Develop an internal positive control (IPC) for RTqPCR by examining homogeneity of the internal
transcribed spacer (ITS) region 1 of X. americanum
 Design IPC to similar length as the ToRSV




primer/probe set for multiplex purposes
Analyze the two sets for cross reaction and non target
RNA with each other.
Examine the thermodynamic compatibility using
hybridization software and cross referencing sequence
data available on Genbank
Validate RT-qPCR method with known virus infected
samples.
Ensures that our samples have nematodes


IPC unsuccessful
Individual genetic diversity
in the group X. americanum
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
Chromatograph
illustrating the
heterogeneity within the
ITS1 region of rDNA for a
single individual X.
americanum
A single signal becomes
multiple signals; We
observed this with
individuals other than
Xiphinema as well
 Literature suggest
phylogenetic studies on
nematodes is a common
problem
Objective I: Efficiency

A 1 to 10 dilution
series of ToRSV
from leaves.
 10-1 to 10-10 all
amplified
10-1
10-2
10-8 10-9 10-10
10-3 -4
10 10-5 10-6 10-7
Detection of ToRSV in
roots

 11, 9, 6, and 5 weeks
Threshold values of
ToRSV from
amplification plot
Ct Mean of ToRSV in Roots
30
25
20
RNA

15
10
5
0
Inoculation Date
Dilution series of
ToRSV in Roots
 Detection of ToRSV
in roots was lower
than ToRSV in
leaves

10-1 10-2 10-3 10-4
 10-1 to 10-4 amplified
http://mexicanmortarandpestle.net/images/large%20mort
ar%20and%20pestle.jpg
http://www.tari.gov.tw/tarie/photos/introduction/introductio
n_PPD_17.jpg
http://www.medicine.virginia.edu/research/c
ores/biomolec/images/rt-pcr.jpg
http://2010.igem.org/wiki/images/thumb/c/cb/IC_Assay_3
_sept.jpg/300px-IC_Assay_3_sept.jpg
http://image.made-in-china.com/2f0j00dCLaFpKzrDos/96-Well-Cell-Culture-Plate.jpg

X. americanum
 Have low fecundity
 Delicate and sensitive to disturbances

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Inoculation and recovery of nematodes were
low
RNA extraction was poor
IPC did not work


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Develop and optimize a RT-qPCR method to
detect TRSV in X. americanum
Determine the persistency and duration of
ToRSV/TRSV within X. americanum by using
the developed primers/probes for RT-qPCR
Link the genetic variability of X. americanum
populations to virus vectoring capabilities as
a means to facilitate the development of
diagnostic tools
 Dr. Inga Zasada
 Amy Peetz
 Dr. Bob Martin
 Karen Keller
 Nola Mosier
 Ruth Price
 Dr. Kevin Ahern
 Howard Hughes Medical Institute
 Cripps Scholarship