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Cucumber Green Mottle Mosaic
Virus
• 6422 nucleotide genome
• Group IV +ssRNA virus
Top image from viralzone,expasy.org, produced by the Swiss Institute of Bioinformatics.
Bottom image from NCBI.NIH.gov accession NC_001801.1, National Center for Biotechnology Information.
Disease Cycle
Contaminated
seeds
1%-5% infected
seedlings
Infected
fruit
Secondary spread by
mechanical transmission,
infection rates up to 100%
San Joaquin County, 2014
Testing methods
• DAS-ELISA
• RT-PCR
• RT-qPCR
• Immunostrips
Seed Testing
Isolate
Dilution
Yolo
1
Yolo
1/10
Yolo
1/50
Yolo
1/100
Yolo
1/200
Yolo
1/500
Yolo
1/1000
Yolo
1/2000
Yolo
1/5000
Yolo
1/10000
Yolo healthy control
Yolo Buffer control
SJ
1
SJ
1/10
SJ
1/50
SJ
1/100
SJ
1/200
SJ
1/500
SJ
1/1000
SJ
1/2000
SJ
1/5000
SJ
1/10000
SJ healthy control
SJ
Buffer control
homogenate (v/v)
ELISA O.D.
RT-PCR
1.8995
positive
2.0995
positive
N/A
N/A
1.964
positive
1.7415
positive
1.9625
positive
1.3205
positive
1.0905
negative
0.6385
negative
N/A
N/A
0.085
negative
0.08
negative
1.496
positive
1.3635
positive
N/A
N/A
1.236
positive
1.09
positive
0.7245
positive
0.666
positive
0.5345
negative
0.4035
negative
N/A
N/A
0.084
negative
0.08
negative
Powdered seeds (w/w) Seeds (# contam./total)
ELISA O.D.
RT-PCR ELISA O.D.
RT-PCR
1.9255
positive
2.209
positive
1.987
positive
N/A
N/A
N/A
N/A
1.893
positive
1.403
positive
1.0745
positive
1.5725
positive
N/A
N/A
1.4985
positive
1.69
positive
0.5155
positive
0.7155
positive
0.6745
negative
0.289
negative
0.364
negative
0.244
negative
N/A
N/A
0.096
negative
0.082
negative
0.0845
negative
0.08
negative
0.07
negative
1.444
positive
1.893
positive
1.377
positive
N/A
N/A
N/A
N/A
1.0745
positive
0.9585
positive
1.69
positive
1.0435
positive
0.7155
positive
1.041
positive
N/A
N/A
0.652
positive
0.289
negative
0.4275
negative
0.244
negative
0.15645
negative
0.096
negative
N/A
N/A
0.0895
negative
0.082
negative
0.075
negative
0.08
negative
0.073
negative
Dilution: # contaminated seeds/# total seeds; healthy and contaminated seeds were watermelon
Bioassay
• To confirm infectious virus
• Rub inoculate both
Nicotiana benthamiana (Nb)
and Chenopodium album
ssp. amaranticolor (Caa)
• Reducing inoculation buffer
• carborundum
• Symptoms take up to 10
days to develop
• For Nb systemic infection,
test upper newest leaves
with RT-PCR or ELISA
• Caa local lesions, excise local
lesions and test with RT-PCR
or ELISA
Healthy
CGMMV
CGMMV
CGMMV
CGMMV
Nb 20 days post inoculation.
Symptom expression begins as
early as 5 dpi, if Nb are at 4th true
leaf when inoculated. Not all
isolates cause strong symptoms in
Nb despite systemic infection.
ATCC1067 ZGMMV no local lesions Fresno local lesions
SJ local lesion
10 days post inoculation: all
isolates have distinct local
lesions
Israel local lesion
Yolo
local
lesion
ATCC391 (Japan) local lesions
Kern local lesion
Isolate Collection
• California isolates: 3 unique isolates, 4 total
• American Type Culture Collection: 1 CGMMV
(Japan), 1 zucchini green mottle mosaic virus
• Middle East: 1 isolate
• Europe: 26 isolates
• SE Asia: 4 isolates
• 37 total isolates
China
Korea
Taiwan
Korea
India
Israel
Canada
Israel
Taiwan
Korea
Japan
Israel
Spain
Russia
Spain
Phylogenetic analysis
includes 46 full genome
sequences from GenBank,
and 36 sequences from
international collaborators
and California outbreaks.
The 36 additional sequences
were generated from NGS
sequencing of purified virion
RNA. Analyses are ongoing.
Full genome sequence alignment, North American isolates
1
2
3
4
5
1.
2.
3.
4.
5.
Canada 2013 isolate
Fresno 2014 isolate
Kern 2014 isolate
Yolo 2013 isolate
San Joaquin 2014 isolate
Continued funding/future work
• Finish CGMMV recovery plan, currently under
review with collaborators
• We received Farm Bill funding, grant #3.0323.00
“Enhancing the sensitivity, efficiency and accuracy
for detecting Cucumber green mottle mosaic virus
in cucurbit seeds.” Beginning in August
• Go forward with qRT-PCR work
• Complete sequence analyses
• Optimize seed RNA purification or other molecular
testing method
Watermelon
Cucumber
Questions?
Cantaloupe