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First Report of Cucurbit Chlorotic Yellows Virus Infecting Melon in the New World

Wintermantel, W. M., Jenkins Hladky, L. L., Fashing, P., Ando, K., McCreight, J. D.
Plant disease 2019 v.103 no.4 pp. 778
Bemisia tabaci, Cucumis melo, Cucurbit chlorotic yellows virus, Cucurbit yellow stunting disorder virus, RNA-directed RNA polymerase, Squash vein yellowing virus, cDNA libraries, coat proteins, complementary DNA, crop production, cultivars, genes, germplasm, high-throughput nucleotide sequencing, leaf extracts, melons, mixed infection, nucleotides, plant diseases and disorders, plant viruses, reverse transcriptase polymerase chain reaction, ribosomal RNA, spring, summer, virus transmission, viruses, California, East Asia, Middle East, Northern Africa, Southwestern United States
During the summer of 2018, melon (Cucumis melo L.) plants from a germplasm diversity study in a field in Imperial Valley, CA, were determined to be infected with cucurbit chlorotic yellows virus (CCYV; genus Crinivirus, family Closteroviridae). Nearly all melon accessions in the study exhibited varying degrees of interveinal yellowing and chlorotic spot symptoms similar to those caused by cucurbit yellow stunting disorder virus (CYSDV; genus Crinivirus), which is prevalent in the region; however, nucleic acid extracts from leaves of two strongly symptomatic plants tested negative for CYSDV by reverse transcription polymerase chain reaction (RT-PCR) (Wintermantel et al. 2009). Extracts of both plants were then evaluated by RT-PCR for CCYV using CCYV-specific primers designed to direct amplification of nucleotides 5,305 to 5,677 of the coat protein region of CCYV RNA 2 (NC_018174). The expected 373-nt amplicon was bidirectionally sequenced at TACGen (Richmond, CA) and confirmed as CCYV. Additional CCYV-specific primers were designed to amplify the full-length CP gene (753 nt) and full-length RNA-dependent RNA polymerase (RdRp) region of RNA1 (1,515 nt) (NC_018173). RT-PCR amplification and sequencing of the CP gene (MH806868) and RdRp region (MH806867) demonstrated 99% identity with the respective genes of 20 CCYV isolates from Asia and Africa. Several archived and frozen total nucleic acid and RNA extracts from Imperial Valley melon plants, collected over the course of 9 years (2010 to 2018), were analyzed for CCYV. Nineteen of 23 samples collected between 2014 and 2018 were positive for CCYV, and many contained mixed infections of CCYV with CYSDV and/or the ipomovirus squash vein yellowing virus (SqVYV). All 18 archived samples collected from 2010 to 2013 tested negative for CCYV, but CYSDV, the virus originally identified in the samples, was successfully amplified from these archived extracts. To confirm CCYV infection, RNA extract from one of the original samples was sent to SeqMatic (Fremont, CA) for rRNA depletion (Epicentre RiboZero kit, Illumina, Madison, WI), cDNA library construction (SeqMatic TailorMix), and high-throughput sequencing (HTS; NextSeq High Output Run, Single End Read 1 × 75 bp). HTS data assembly of 175,826 reads (14,818 nt, four fragments) confirmed a 99% match between the HTS sequences and RNA1 and RNA2 of CCYV (NC_018173, NC_018174). Results suggest CCYV was introduced to Imperial Valley in 2014, about the same time that SqVYV was first observed in California (Batuman et al. 2015) but remained undetected owing to similarity with CYSDV in both symptoms on cucurbits and vector transmission. CCYV is prevalent in East Asia, the Middle East, and North Africa (Abrahamian et al. 2012; Hamed et al. 2011; Okuda et al. 2010) and is transmitted efficiently by the whitefly, Bemisia tabaci (Li et al. 2016). B. tabaci MEAM1 reaches high populations in Imperial Valley and the surrounding region beginning in the spring melon season, rapidly increasing in numbers such that all cucurbit plants in the region are exposed to whitefly feeding from July through November, which facilitates virus transmission and disease throughout the region. Coinfection of CCYV and CYSDV often results in reduced titers of both viruses, but especially CCYV (Abrahamian et al. 2013). Further studies will be necessary to evaluate epidemiology of CCYV in the southwestern U.S. desert crop production region and to determine the impact of this virus on efforts to develop crinivirus-resistant melon cultivars.