Prevalence of Aphid-Transmitted Potyviruses in Pumpkin and Winter Squash in Georgia, USA

Abstract

Viruses are a major pathogen challenging the sustainable production of cucurbits worldwide. Pumpkin and winter squash showed severe virus-like symptoms during the fall of 2022 and 2023 in Georgia, USA. Symptomatic leaves were collected from the field and processed for small RNA sequencing for virus identification using high-throughput sequencing (HTS). HTS analysis revealed the presence of two aphid-transmitted viruses (ATVs), zucchini yellow mosaic virus (ZYMV) and papaya ringspot virus (PRSV), along with three whitefly-transmitted viruses, cucurbit chlorotic yellows virus, cucurbit yellow stunting disorder virus, and cucurbit leaf crumple virus. The results of our study suggest a significant shift in ATV's abundance in these two crops between 2022 and 2023. According to the qPCR data in the fall of 2022, pumpkins experience an incidence of 56.25% and 31.25% of PRSV and ZYMV, respectively. Similarly, winter squash shows an incidence of 50% and 32.14% of PRSV and ZYMV, respectively. Mixed infection of both viruses was also observed in these two crops. In 2023, we observed a predominance of ZYMV in pumpkin and winter squash (61.25% and 42.50%, respectively). However, PRSV was not detected in pumpkins, and it was detected at a negligible level (0.62%) in winter squash using qPCR. Phylogenetic analysis of ZYMV-encoded coat protein (CP) and helper component-protease (HC-Pro) from Georgia suggests a close relationship with the European isolates. Conversely, PRSV-encoded CP and NIa-VPg show a more diverse evolutionary history. Overall, this research will provide valuable insights into the dynamics of ZYMV and PRSV in pumpkin and winter squash crops within the southeastern United States.

Keywords: aphids; cucurbit; high-throughput sequencing; papaya ringspot virus; prevalence; pumpkin; winter squash; zucchini yellow mosaic virus.

Figure 1

Genome organization of potyviruses; inverted triangular arrows indicate polyprotein cleavage sites. The P1 is a serine protease, an accessory factor for viral amplification. The helper-component proteinase (HC-Pro) is a cysteine protease that has a role in viral transmission, RNA silencing suppression, and systemic movement. The P3 and 6K2 are involved in viral replication and multiplication. The CI has ATPase and helicase activity; it has a role in particle disassembly and virus movement. The 6K2 anchors the replication apparatus to ER-like membranes and induces the formation of viral replication vesicles. The NIa-Vpg has a role in viral replication, RNA translation, and movement. The NIa-pro is a trypsin-like serine protease and has a role in the proteolytic cleavage of the potyviral polyprotein. It also functions in host specificity, DNase activity, RNA binding, replication, and multiplication. The NIb is RNA-dependent RNA polymerase and has a role in viral replication and multiplication. The coat protein (CP) encapsulates the virus’s RNA genome. In association with HC-Pro, CP has a role in seed transmission, cell–cell and systemic movement, virus assembly, and host adaptation. Pretty Interesting Potyviridae ORF (PIPO) has a primary role in viral movement. These virus-encoded proteins have a role in the movement, pathogenicity, symptom development, and resistance breakdown [16].

Figure 2

Different symptoms observed on pumpkin and winter squash plants included pumpkin leaf (A) marginal yellowing (PK1); (B) cupping and shoestring (PK2); (C) mosaic and vein clearing (PK3); and (D) thickening, curling, crumpling, and vein clearing (PK4). The two symptoms observed in winter squash leaves were (E) light and dark green mosaic and blistering (WS1) and (F) yellowing, thickening, and inward rolling (WS2).

Figure 3

Read coverage maps of the genome of the zucchini yellow mosaic virus (ZYMV) and papaya ringspot virus (PRSV) detected by high-throughput sequencing (HTS) of small RNAs of symptomatic pumpkin and winter squash samples. The data were analyzed using CLC Genomics workbench v23.0.2. The colored heatmap shows the coverage with several reads. A to F represent read coverage maps of the ZYMV for samples with (A) marginal yellowing (PK1), (B) cupping and shoestring (PK2), (C) mosaic and vein clearing (PK3), (D) thickening, curling, crumpling, and vein clearing (PK4), (E) light and dark green mosaic and blistering (WS1), and (F) yellowing, thickening, and inward rolling (WS2). On the other hand, G to L shows read coverage maps of the PRSV for (G) PK1, (H) PK2, (I) PK3, (J) PK4, (K) WS1, and (L) WS2. Genome positions of the virus are presented to scale above the histograms, and the coverage in the number of reads with reference genomes (GenBank accession number NC_003224 for ZYMV and NC_001785 for PRSV) is represented on the Y-axis. The colors in the specified aggregation bucket mean the maximum, average, and minimum coverage values (read counts) from top to bottom.

Figure 4

Graphs showing the prevalence of zucchini yellow mosaic virus (ZYMV) and papaya ringspot virus (PRSV): (A) bar graphs showing the prevalence of ZYMV and PRSV between the two consecutive years, 2022–2023, among the total samples collected; (B) distribution of ZYMV and PRSV in pumpkin and winter squash in 2022–2023.

Figure 5

Sequence comparisons of nucleotide sequences of zucchini yellow mosaic virus (ZYMV)-encoded coat protein (CP) and helper component proteinase (HC-Pro) and papaya ringspot virus (PRSV)-encoded CP and NIa-Vpg from pumpkin and winter squash plants in Georgia, USA, with the sequences obtained from the NCBI GenBank. Maximum likelihood (ML) phylogenetic trees deduced in MEGAX using the 1000 bootstrap method. The phylogenetic trees are based on (A) 28 sequences of ZYMV-encoded CP, (C) 29 sequences of ZYMV-encoded HC-Pro, (E) 25 sequences of PRSV CP, and (G) 28 sequences of PRSV NIa-Vpg. GenBank accession number, country name, host plant, and year of sample collection are shown on each node. Sweet potato mild mottle virus (SPMMV) [Genus: Ipomovirus, Z73124] was used as an outgroup for the tree. The final tree was created using uniform rates among the sites, and branches were condensed to 50%. The calculated trees were displayed and analyzed using Tree Explorer implemented in the MEGA-X program. Color-coded pairwise identity matrixes generated from (B) 28 ZYMV-encoded CP, (D) 29 ZYMV-encoded HC-Pro, (F) 25 PRSV-encoded CP, and (H) 28 PRSV-encoded NIa-Vpg sequences. Each colored cell represents a percentage identity score between two sequences (one indicated horizontally to the left and the other vertically at the bottom).

Figure 5

Sequence comparisons of nucleotide sequences of zucchini yellow mosaic virus (ZYMV)-encoded coat protein (CP) and helper component proteinase (HC-Pro) and papaya ringspot virus (PRSV)-encoded CP and NIa-Vpg from pumpkin and winter squash plants in Georgia, USA, with the sequences obtained from the NCBI GenBank. Maximum likelihood (ML) phylogenetic trees deduced in MEGAX using the 1000 bootstrap method. The phylogenetic trees are based on (A) 28 sequences of ZYMV-encoded CP, (C) 29 sequences of ZYMV-encoded HC-Pro, (E) 25 sequences of PRSV CP, and (G) 28 sequences of PRSV NIa-Vpg. GenBank accession number, country name, host plant, and year of sample collection are shown on each node. Sweet potato mild mottle virus (SPMMV) [Genus: Ipomovirus, Z73124] was used as an outgroup for the tree. The final tree was created using uniform rates among the sites, and branches were condensed to 50%. The calculated trees were displayed and analyzed using Tree Explorer implemented in the MEGA-X program. Color-coded pairwise identity matrixes generated from (B) 28 ZYMV-encoded CP, (D) 29 ZYMV-encoded HC-Pro, (F) 25 PRSV-encoded CP, and (H) 28 PRSV-encoded NIa-Vpg sequences. Each colored cell represents a percentage identity score between two sequences (one indicated horizontally to the left and the other vertically at the bottom).