First report of cucurbit yellow stunting disorder virus and cucurbit chlorotic yellows virus in cucurbit crops in Alabama

Authors: MONDAL, SHAONPIUS - Oak Ridge Institute For Science And Education (ORISE); Hladky, Laura; MELANSON, REBECCA - Mississippi State University; SINGH, RAGUWINDER - Louisiana State University; SIKORA, EDWARD - Auburn University; Wintermantel, William - Bill

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/21/2021
Publication Date: 6/28/2021
Citation: Mondal, S., Jenkins Hladky, L.L., Melanson, R.A., Singh, R., Sikora, E., Wintermantel, W.M. 2021. First report of cucurbit yellow stunting disorder virus and cucurbit chlorotic yellows virus in cucurbit crops in Alabama. Plant Disease. https://doi.org/10.1094/PDIS-05-21-0922-PDN.
DOI: https://doi.org/10.1094/PDIS-05-21-0922-PDN

Interpretive Summary: During the summer of 2020, foliar yellowing symptoms, including leaf mottle and interveinal yellowing with green veins were observed on several melon, squash and cucumber plants in commercial fields in Alabama. Such symptoms were consistent with those caused by the whitefly transmitted yellowing viruses, Cucurbit chlorotic yellows virus (CCYV, Crinivirus, Closteroviridae) and Cucurbit yellow stunting disorder virus (CYSDV, Crinivirus, Closteroviridae). Therefore, 231 leaf samples showing yellowing and any other mosaic symptoms were collected from individual plants from 25 commercial fields in Alabama (70 watermelon, 52 melon, 34 pumpkin, 50 squash, and 25 cucumber) during the spring and fall melon seasons. Results of diagnostic testing using nucleic acid-based assays identified CYSDV in 20/114 cucurbit plants tested (17.5%) in June 2020, but CCYV was not identified from any plants. During the fall season 37/117 plants (32%) tested for positive the presence of one or both viruses. Of the 37 virus positive samples from the fall season, 26 were singly infected with CCYV (70%), seven were singly with CYSDV (19%), and four with both CYSDV and CCYV (11%). Sequence analysis of a portions of the viral replication gene from five CCYV infected plants (2 squash, 2 cucumber, 1 pumpkin) found 98% identity with the same sequence of CCYV isolates from Israel and California. Sequence analysis of portions of the viral replication gene from five 2 CYSDV infected melon plants found greater than 99% sequence identity with the comparable sequence from RNA1 of a CYSDV isolate from Arizona, USA. In addition, each CYSDV and CCYV infection was confirmed by another nucleic acid-based analysis against a portion of the coat protein gene of each virus, respectively, encoded on RNA2 of each viral genome. Furthermore, a recently developed quantitative DNA detection method was used to confirm of four representative CYSDV and CCYV infections each. CuLCrV was not detected in any of the plants sampled. This is the first report of CYSDV and CCYV in cucurbit crops from Alabama. Surprisingly, no watermelon plants were infected with either virus, and no melon plants were infected with CCYV. The identification of CCYV and CYSDV in Alabama, along with a recent report of both criniviruses from nearby Georgia illustrates the need for a more thorough sampling of cucurbit crops in Alabama and for further monitoring of the whitefly vector, Bemisia tabaci, as well as identification of alternate hosts of these viruses to better understand epidemiology of these viruses throughout the Gulf Coast region.

Technical Abstract: During the summer of 2020, foliar yellowing symptoms, including leaf mottle and interveinal yellowing with green veins were observed on several melon, squash and cucumber plants in commercial fields in Alabama. Such symptoms were consistent with those caused by the whitefly transmitted yellowing viruses, Cucurbit chlorotic yellows virus (CCYV, Crinivirus, Closteroviridae) and Cucurbit yellow stunting disorder virus (CYSDV, Crinivirus, Closteroviridae). Therefore, in June (spring melon season) and October (fall melon season) 2020, 231 leaf samples showing yellowing and any mosaic symptoms were collected from individual plants from 25 commercial fields in Alabama (70 watermelon, 52 melon, 34 pumpkin, 50 squash, and 25 cucumber). Total RNA was used in reverse transcription polymerase chain reactions with primer sets designed to amplify portions of the CCYV and CYSDV RNA-dependent RNA polymerase (RdRp) genes encoded on RNA1 of these viruses. Total DNA was used in PCR to test for the presence of the whitefly-transmitted begomovirus, cucurbit leaf crumple virus (CuLCrV) which was previously reported in cucurbit crops from the neighboring states of Georgia. During the spring season, CYSDV was identified from 20/114 total cucurbit plants tested (17.5%) in June 2020, but CCYV was not identified from any plants. During the fall season 37/117 plants (32%) tested for positive the presence of one or both criniviruses. Of the 37 virus positive samples from the fall season, 26 were singly infected with CCYV (70%), seven were singly with CYSDV (19%), and four with both CYSDV and CCYV (11%). The RdRp amplicon was sequenced from five CCYV infected plants (2 squash, 2 cucumber, 1 pumpkin) and 2 CYSDV infected plants (both melon); the 953 nt sequenced portion of the CCYV RdRp gene was found to share 98% identity with the same sequence of a CCYV RNA1 isolate from Israel (Accession No. MH477611.1) and California (MW680157), respectively; whereas the 492 nt CYSDV amplicon shared greater than 99% sequence identity with the comparable sequence from RNA1 of a CYSDV isolate from Arizona, USA (Accession No. EF547827.1). In addition, each CYSDV and CCYV infection was confirmed using a second set of primers that amplified 394 and 372 nt sections of the coat protein gene of each virus, respectively, encoded on RNA2 of each viral genome. Furthermore, a recently developed multiplex reverse transcription-quantitative PCR method was used to confirm of four representative CYSDV and CCYV infections each. CuLCrV was not detected in any of the plants sampled. This is the first report of CYSDV and CCYV in cucurbit crops from Alabama. Surprisingly, no watermelon plants were infected with either virus, and no melon plants were infected with CCYV. The identification of CCYV and CYSDV in Alabama, along with a recent report of both criniviruses from nearby Georgia illustrates the need for a more thorough sampling of cucurbit crops in Alabama and for further monitoring of the whitefly vector, Bemisia tabaci, as well as identification of alternate hosts of these viruses to better understand epidemiology of these viruses throughout the Gulf Coast region.