High Throughput Sequencing-Aided Survey Reveals Widespread Mixed Infections of Whitefly-Transmitted Viruses in Cucurbits in Georgia, USA

Abstract

Viruses transmitted by the sweet potato whitefly (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 three cantaloupes, four cucumbers, and two yellow squash samples. An analysis of the sequences revealed the presence of the criniviruses cucurbit chlorotic yellows virus (CCYV), cucurbit yellow stunting disorder virus (CYSDV), and the begomovirus cucurbit leaf crumple virus (CuLCrV). CuLCrV was detected in 76%, CCYV in 60%, and CYSDV in 43% of the total samples (n = 820) 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. An 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 USA isolates. This work enhances our understanding of the distribution of viruses on cucurbits in South Georgia and will be useful to develop strategies for managing the complex of whitefly-transmitted viruses in the region.

Keywords: cucurbit chlorotic yellows virus; cucurbit leaf crumple virus; cucurbit yellow stunting disorder virus; cucurbits; high throughput sequencing; survey; whitefly transmitted viruses.

Figure 1

The different types of symptoms observed on squash plants included (A) chlorosis and crinkling, (B) vein yellowing, (C) interveinal leaf chlorosis, (D) yellow spots, and (E) severe stunting or bunching of leaves at the top of the plant. (F) Fruits, on squash plants, displayed severe bunching, were distorted, and were streaked with green patches. The main symptom observed on cantaloupe and cucumber was (G) interveinal leaf chlorosis, more prominent at the (G–I) crown region and milder on the (J) younger leaves. Foliage symptoms on zucchini including (K) crinkled leaves and a (K–L) yellow mosaic pattern.

Figure 2

(A) Viruses detected on cantaloupe (CaF1-2019, CaF1-2020, CaF2-2019), cucumber (CF1-2019, CF2-2019, CF1-2020), and squash (SF1-2019, SF1-2020, SF2-2020) in Georgia by high throughput sequencing (HTS) of small RNAs (A) and RCA-HTS (B). Viruses detected and their genomic components are presented along the Y-axis. The number of contigs of each genome detected are presented along the X-axis. Each grid in the graph represents one contig and each color represents a different sample. Abbreviations used for viruses: Cucurbit chlorotic yellows virus (CCYV), cucurbit yellow stunting virus (CYSDV), cucurbit leaf crumple virus (CuLCrV), euphorbia yellow mosaic virus (EYMV), squash mild leaf curl virus (SqMLCV), sida golden mottle virus (SiGMoV), tomato mild yellow leaf curl virus (TmYLCV), and tobacco yellow crinkle virus (TbYCV).

Figure 2

(A) Viruses detected on cantaloupe (CaF1-2019, CaF1-2020, CaF2-2019), cucumber (CF1-2019, CF2-2019, CF1-2020), and squash (SF1-2019, SF1-2020, SF2-2020) in Georgia by high throughput sequencing (HTS) of small RNAs (A) and RCA-HTS (B). Viruses detected and their genomic components are presented along the Y-axis. The number of contigs of each genome detected are presented along the X-axis. Each grid in the graph represents one contig and each color represents a different sample. Abbreviations used for viruses: Cucurbit chlorotic yellows virus (CCYV), cucurbit yellow stunting virus (CYSDV), cucurbit leaf crumple virus (CuLCrV), euphorbia yellow mosaic virus (EYMV), squash mild leaf curl virus (SqMLCV), sida golden mottle virus (SiGMoV), tomato mild yellow leaf curl virus (TmYLCV), and tobacco yellow crinkle virus (TbYCV).

Figure 3

Read coverage maps of the viruses detected by high throughput sequencing of small RNAs of symptomatic cucurbits from Georgia. Scaled genome positions of the virus are shown above the histograms and the Y-axis represents the coverage in number of reads. Within the specified aggregation bucket, from top to bottom, the colors mean: The maximum coverage value (read count), the average coverage value, and the minimum coverage value. Abbreviations used for the viruses: Cucurbit chlorotic yellows virus (CCYV), cucurbit yellow stunting virus (CYSDV), cucurbit leaf crumple virus (CuLCrV), euphorbia yellow mosaic virus (EYMV), squash mild leaf curl virus (SqMLCV), sida golden mottle virus (SiGMoV), and tomato mild yellow leaf curl virus (TmYLCV).

Figure 3

Read coverage maps of the viruses detected by high throughput sequencing of small RNAs of symptomatic cucurbits from Georgia. Scaled genome positions of the virus are shown above the histograms and the Y-axis represents the coverage in number of reads. Within the specified aggregation bucket, from top to bottom, the colors mean: The maximum coverage value (read count), the average coverage value, and the minimum coverage value. Abbreviations used for the viruses: Cucurbit chlorotic yellows virus (CCYV), cucurbit yellow stunting virus (CYSDV), cucurbit leaf crumple virus (CuLCrV), euphorbia yellow mosaic virus (EYMV), squash mild leaf curl virus (SqMLCV), sida golden mottle virus (SiGMoV), and tomato mild yellow leaf curl virus (TmYLCV).

Figure 4

Phylogenetic relationship of coding regions of (A) RNA 1 and (B) RNA 2 of cucurbit chlorotic yellows virus (CCYV) and cucurbit yellow stunting disorder virus (CYSDV) isolates from Georgia with the corresponding sequences of CCYV and CYSDV isolates available in the GenBank. The country of origin of the sequences used are shown in parentheses after the accession numbers of viruses. CCYV sequences are shaded in blue and CYSDV sequences are in green. The Beet yellows virus (BYV) was set as an outgroup. Construction of the phylogenetic trees were done by the neighbor-joining method. The number on each node shows the percentage of bootstrap values (2000 replicates) in which a given node was recovered. Nodes with lower than 50 bootstrap values were collapsed since they were insignificant.