Post on 31-Dec-2015
transcript
By: Charlie TaMentor: 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)
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 virus-transmitting nematodes
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
head
tail
Nematode-transmitted virus with polyhedral particles
Type IV virus under the Baltimore classification system (positive sense single-stranded RNA that directly translates into protein)
Acquisition of virus occurs during feeding and binds to the surface ofthe odontostyle
Viruses are lost when nematodes molt
odontostyle
A RT-qPCR can be used for the detection of ToRSV in X. americanum at low concentration levels.
Virus detection using RT- qPCR allows for a detailed study of nematode-virus interactions.
The coloration occurs due to adding p-nitrophenyl phosphate.
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Steps:Steps:
Reverse Transcriptase -
Quantitative Polymerase
Chain Reaction (RT-qPCR)
Enzyme-Linked Immunosorbent Assay
(ELISA)
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. Develop and optimize the efficiency of a RT-qPCR to detect ToRSV
II. Quantify acquisition and saturation level of ToRSV in X. americanum
Methodology
Develop an internal positive control (IPC) for RT-qPCR 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
Objective I: Development
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
10-110-2
10-510-3
10-4 10-710-610-8 10-9 10-10
A 1 to 10 dilution series of ToRSV from leaves. 10-1 to 10-10 all
amplified
Objective I: Efficiency
Dilution series of ToRSV in Roots
Detection of ToRSV in roots was lower than ToRSV in leaves 10-1 to 10-4
amplified
10-1 10-2 10-3 10-4
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X. americanum Have low fecundity Delicate and sensitive to disturbances
Inoculation and recovery of nematodes were low
RNA extraction was poor IPC did not work
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