High Prevalence of Three Potyviruses Infecting Cucurbits in Oklahoma and Phylogenetic Analysis of Cucurbit Aphid-Borne Yellows Virus Isolated from Pumpkins

Abstract

Field information about viruses infecting crops is fundamental for understanding the severity of the effects they cause in plants. To determine the status of cucurbit viruses, surveys were conducted for three consecutive years (2016-2018) in different agricultural districts of Oklahoma. A total of 1331 leaf samples from >90 fields were randomly collected from both symptomatic and asymptomatic cucurbit plants across 11 counties. All samples were tested with the dot-immunobinding assay (DIBA) against the antisera of 10 known viruses. Samples infected with papaya ringspot virus (PRSV-W), watermelon mosaic virus (WMV), zucchini yellow mosaic virus (ZYMV), and cucurbit aphid-borne-yellows virus (CABYV) were also tested by RT-PCR. Of the 10 viruses, PRSV-W was the most widespread, with an overall prevalence of 59.1%, present in all 11 counties, followed by ZYMV (27.6%), in 10 counties, and WMV (20.7%), in seven counties, while the remaining viruses were present sporadically with low incidence. Approximately 42% of the infected samples were positive, with more than one virus indicating a high proportion of mixed infections. CABYV was detected for the first time in Oklahoma, and the phylogenetic analysis of the first complete genome sequence of a CABYV isolate (BL-4) from the US showed a close relationship with Asian isolates.

Keywords: CABYV; cucurbits; phylogeny; potyviruses; survey.

Figure 1

Geographical locations of different counties of Oklahoma. The solid blue lines denote borders of different agricultural districts, and the red dots represent the counties from where samples were collected.

Figure 2

Symptoms observed in infected plants’ leaves and fruits. (A) The most common symptoms observed in the leaves of infected cucurbit plants. (a) Leaf distortion caused by mixed infection of watermelon mosaic virus (WMV) and papaya ringspot virus W strain (PRSV-W) on squash leaves. (b) Mosaic pattern on pumpkin leaves caused by WMV. (c) Yellowing in pumpkin caused by ZYMV. (d) Mottling and leaf deformation, caused by PRSV-W, on pumpkin leaves. (B) Ring spots on pumpkin fruit caused by PRSV-W and yellowing on the leaves by mixed infection of PRSV-W and ZYMV. (C) Chlorosis, mosaics, and vein thickening caused by CABYV BL-4 isolate on pumpkin leaves. The pumpkin plant was also infected by PRSV-W.

Figure 2

Symptoms observed in infected plants’ leaves and fruits. (A) The most common symptoms observed in the leaves of infected cucurbit plants. (a) Leaf distortion caused by mixed infection of watermelon mosaic virus (WMV) and papaya ringspot virus W strain (PRSV-W) on squash leaves. (b) Mosaic pattern on pumpkin leaves caused by WMV. (c) Yellowing in pumpkin caused by ZYMV. (d) Mottling and leaf deformation, caused by PRSV-W, on pumpkin leaves. (B) Ring spots on pumpkin fruit caused by PRSV-W and yellowing on the leaves by mixed infection of PRSV-W and ZYMV. (C) Chlorosis, mosaics, and vein thickening caused by CABYV BL-4 isolate on pumpkin leaves. The pumpkin plant was also infected by PRSV-W.

Figure 3

Analysis of reverse transcription polymerase chain reaction (RT-PCR) products of three potyviruses by 1% agarose gel electrophoresis. (A) Amplification from PRSVCPF and PRSVCPR primers showing DNA band of 963 bp. Lane 1: 10 kb DNA ladder; Lane 2: positive control; Lane 3: negative control; Lane 4–8: PRSV-W dot-immunobinding assay (DIBA)-positive samples; Lane 9–10: PRSV-W DIBA-negative samples. (B) Amplification from WMVCPF and WMVCPR primers showing DNA band of 979 bp. Lane 1: 10 kb DNA ladder; Lane 2: positive control; Lane 3: negative control; Lane 4–8: WMV DIBA-positive samples; Lane 9–10: WMV DIBA-negative samples. (C) Amplification from ZYMVCPF and ZYMVCPR primers showing DNA band of 902 bp. Lane 1: 10 kb DNA ladder; Lane 2: positive control; Lane 3: negative control; Lane 4–8: ZYMV DIBA-positive samples; Lane 9–10: ZYMV DIBA-negative samples.

Figure 4

Schematic representation of the genome organization of CABYV BL-4 isolate. The arrows point to the approximate start and end positions of the nucleotides in the particular protein. P1 protein also contains part of the P0 protein region, and P5 protein contains all the P3 protein. The diagram is not drawn to scale.

Figure 5

Maximum likelihood (ML) phylogenetic tree deduced in Mega7 using general time reversible (GTR) model. The ML tree is based on 45 complete genome sequences of CABYV isolates available in the NCBI database. GenBank accession number, isolate name, and country of origin are shown on each node. The tree was visualized in Figtree version 1.4.3. The bootstrap values >50 are shown at the respective nodes. The collapsed 22 South Korean isolates are denoted by a dark triangle. The phylogenetic grouping is shown on their respective branches. The US isolate is shown in red color. Isolates from phylogroup G1a are shown in blue, G1b in Torquoise, G2a (except BL-4) in black, G2b in green, and G2c in pink color.

Figure 6

Schematic representation of Recombination Detection Program (RDP) pairwise plot and possible recombination breakpoints in isolate HD118, where the BL-4 isolate from the US serves as a major parent, and C-HS1 from S. Korea serves as a minor parent. The pink area indicates the recombination region.