High throughput sequencing-aided survey reveals widespread mixed infections of whitefly-transmitted viruses in cucurbits in Georgia, USA

Authors: KAVALAPPARA, SARITHA - University Of Georgia; MILNER, HAYLEY - University Of Georgia; KONAKALLA, NAGA - University Of Georgia; MORGAN, KAELYN - University Of Georgia; SPARKS, ALTON - University Of Georgia; MCGREGOR, CECILIA - University Of Georgia; CULBREATH, ALBERT - University Of Georgia; Wintermantel, William - Bill; BAG, SUDEEP - University Of Georgia

Submitted to: Viruses
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/24/2021
Publication Date: 5/26/2021
Citation: Kavalappara, S.R., Milner, H., Konakalla, N.C., Morgan, K., Sparks, A.N., McGregor, C., Culbreath, A.K., Wintermantel, W.M., Bag, S. 2021. High throughput sequencing-aided survey reveals widespread mixed infections of whitefly-transmitted viruses in cucurbits in Georgia, USA. Viruses. 13(6). Article 988. https://doi.org/10.3390/v13060988.
DOI: https://doi.org/10.3390/v13060988

Interpretive Summary: Viruses transmitted by whiteflies (Bemisia tabaci) have caused damage and impacted yield in the Southeastern USA. Surveys were conducted in the fall of 2019 and 2020 in Georgia, a major cucurbit-producing state of the USA, to identify the viruses infecting cucurbits and their distribution. Samples with virus-like symptoms were collected, and extracts from three cantaloupe plants, four cucumber plants, two squash plants were analyzed by high throughput sequencing. Analysis of the sequences revealed the presence of cucurbit chlorotic yellows virus (CCYV), cucurbit leaf crumple virus (CuLCrV), and cucurbit yellow stunting disorder virus (CYSDV). Using additional molecular diagnostic testing (RT-PCR and PCR), 820 total cucurbit samples were tested for presence or absence of these viruses. CuLCrV was detected in 76%, CCYV in 60%, and CYSDV in 43% of the plants that were tested. The level of mixed infections was high in all the cucurbits, with most plants tested found to be infected with at least two of these viruses. Near-complete genome sequences of two criniviruses, CCYV and CYSDV were assembled from the small RNA sequences and comparison of these showed isolates were nearly identical among the different host plants. In phylogenetic analysis, the CCYV isolates from Georgia clustered with Asian isolates, while CYSDV isolates clustered with European and US isolates.

Technical Abstract: Viruses transmitted by whiteflies (Bemisia tabaci) have been detrimental to the sustainable production of cucurbits in the Southeastern USA. Surveys were conducted in the fall of 2019 and 2020 in Georgia, a major cucurbit-producing state of the USA, to identify the viruses infecting cucurbits and their distribution. Symptomatic samples were collected, and small RNA libraries were prepared and sequenced from cantaloupe (n=3), cucumber (n=4), and squash (n=2). Analysis of the sequences revealed the presence of cucurbit chlorotic yellows virus (CCYV), cucurbit leaf crumple virus (CuLCrV), and cucurbit yellow stunting disorder virus (CYSDV). PCR and RT-PCR testing of 820 cucurbit samples found that CuLCrV was detected in 76%, CCYV in 60%, and CYSDV in 43% of the total samples tested. The level of mixed infections was high in all the cucurbits, with most plants tested being infected with at least two of these viruses. Near-complete genome sequences of two criniviruses, CCYV and CYSDV were assembled from the small RNA sequences. Analysis of the coding regions showed low genetic variability among isolates from different hosts. In phylogenetic analysis, the CCYV isolates from Georgia clustered with Asian isolates, while CYSDV isolates clustered with European and US isolates.