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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