Evaluation of Plant Introduction Lines of Yellow Squash (Cucurbita pepo) for Resistance against Single Infection of Cucurbit Chlorotic Yellows Virus and Cucurbit Leaf Crumple Virus

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Ismaila A. Adeleke Department of Plant Pathology, University of Georgia, 115 Coastal Way, Tifton, GA 31793, USA

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Saritha R. Kavalappara Department of Plant Pathology, University of Georgia, 115 Coastal Way, Tifton, GA 31793, USA

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Clarence B. Codod Department of Plant Pathology, University of Georgia, 115 Coastal Way, Tifton, GA 31793, USA

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Prasanna Kharel Department of Horticulture, University of Georgia, 2360 Rainwater Road, Tifton, GA 31793, USA

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Alex Luckew Department of Horticulture, University of Georgia, 1111 Miller Plant Sciences, Athens, GA 30602, USA

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Cecilia McGregor Department of Horticulture, University of Georgia, 1111 Miller Plant Sciences, Athens, GA 30602, USA

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Alvin M. Simmons US Vegetable Research, US Department of Agriculture–Agricultural Research Service, 2700 Savannah Highway, Charleston, SC 29414, USA

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Rajagopalbabu Srinivasan Department of Entomology, University of Georgia, 1109 Experiment Street, Redding Building, Griffin, GA 30223, USA

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Sudeep Bag Department of Plant Pathology, University of Georgia, 115 Coastal Way, Tifton, GA 31793, USA

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Abstract

Whitefly-transmitted viruses have emerged as a major threat to cucurbit production in the United States during the past several decades. Cucurbit chlorotic yellows virus (CCYV), Cucurbit yellow stunting disorder virus (CYSDV), and Cucurbit leaf crumple virus (CuLCrV) are the main arthropod-borne plant viruses in cucurbit crops, including yellow squash (Cucurbita pepo). Symptoms of these viruses include interveinal chlorosis, chlorotic spots, yellowing, and curling of the leaves. The evaluation of specific viruses affecting a plant is challenging because of the prevalence of mixed infections in naturally infected fields. To devise an efficient breeding-based management approach, two PI lines (PI 171625 and PI 171627) were screened in a greenhouse to assess their resistance to individual infections of CCYV and CuLCrV. These lines were compared against a susceptible cultivar Gentry in two separate trials. PI 171627 displayed delayed symptoms, a reduced virus load, and a smaller area under the disease progress curve (AUDPC) compared with PI 171625 and susceptible cultivar Gentry when the plants were infected with CCYV. However, the AUDPC for CuLCrV was the same for both genotypes and the commercial line. Accession PI 171627, which displayed delayed and milder symptoms, could potentially provide a source for resistance against CCYV for breeding program. Future research is needed to comprehend the underlaying mechanism to understand this response.

Infections and damage caused by whitefly-transmitted viruses (WTVs) have emerged as a major threat to global food production during the past several decades (Navas-Castillo et al. 2011). Affected crops include species belonging to the Cucurbitaceae and Solanaceae families (Adkins et al. 2009; Guzman et al. 2000; Navas-Castillo et al. 2011; Polston and Anderson 1997; Polston et al. 1999), among others. Commercial production of cucurbits, such as squash (Cucurbita pepo L.), is centered in southern Georgia and northern Florida, USA, where a high incidence of WTVs has been reported (Adeleke et al. 2022). Squash production in Georgia, USA, accounts for about 4% of total vegetable production, with a total farm gate value of USD1.3 billion (Georgia Farm Gate Value Report 2022).

Squash production under natural conditions in the field has been threatened and affected continuously as a result of whiteflies and the complex of WTVs in the southeastern United States (Devendran et al. 2023; Kavalappara et al. 2021a; LaTora et al. 2022; Moodley et al. 2019). The severity of the effect of these viruses on squash depends on the time of infection, plant vigor, and form of infection, which could be a single or mixed infection with multiple viruses simultaneously (De Barro et al. 2011; Lapidot et al. 2014). Cucurbit chlorotic yellows virus (CCYV) (Kavalappara et al. 2021b), Cucurbit yellow stunting disorder virus (CYSDV) (Gadhave et al. 2018), and Cucurbit leaf crumple virus (CuLCrV) (Larsen 2010) are major WTVs that affect cucurbit crops in the southeastern United States. Management of whiteflies and the complex of viruses transmitted by them is challenging because of the broad host range, short generation of the whitefly life cycle, and the resistance of whiteflies to insecticides (Hidayat et al. 2018). Current management of WTVs relies heavily on using insecticides and resistant varieties when available. But, unlike in some other crops, such as melons, peppers, and tomatoes, in which some host plant resistance to WTVs has been developed, host plant resistance to WTVs is not available in commercial squash cultivars (Candian et al. 2021; Codod et al. 2022; Jennings 1994; Vidavski et al. 2008). To develop commercially viable squash with resistance to WTVs, several PIs were screened for resistance under field conditions for 2 consecutive years in Florida and Georgia, USA (Luckew et al. 2022), where mixed infection of WTVs is common (Kavalappara et al. 2021a). In our study, two Cucurbita pepo PI lines (PI 171625 and PI 171627), chosen for their superior performance against WTVs under field conditions (Luckew et al. 2022), were subjected to greenhouse evaluation to assess their resistance to single infections of crinivirus CCYV and begomovirus CuLCrV, and to validate the findings from the field. This initiative is part of a broader strategy to incorporate resistance sources into breeding programs to improve sustainability and profitability by reducing yield losses caused by WTVs.

Materials and Methods

Whitefly colony and maintenance of virus isolates.

An adult whitefly (Bemisia tabaci MEAM1) colony was established and maintained on cotton (Gossypium spp.), which is a nonhost for CCYV and CuLCrV, within an insect rearing cage with 160-μm aperture (#BugDorm 2400F; Mega View, Taiwan) in a greenhouse set at 25 ± 2 °C. To sustain the whitefly population, cotton seedlings were replaced every 4 weeks, facilitating the insects’ migration onto fresh plants for continued maintenance. The CCYV isolate used in our investigation was obtained in Fall 2020 from squash cultivated in experimental fields at the University of Georgia (UGA), located in Tifton, GA, USA. The CCYV isolates from Georgia, USA (MW629381, MW629379, MW685455, MW629380, OM489401, and MW685461), exhibit more than 99% nucleotide sequence similarity with CCYV isolates documented in both the United States and Europe (Kavalappara et al 2021a). The CuLCrV used in our study was also collected from Georgia, USA, and was maintained on snap beans in the research laboratory of R. S. The CuLCrV maintained on snapbeans (Phaseolus vulgaris) was subsequently transferred to yellow squash ‘Gold Star’ for maintenance of the inoculum. Because CCYV (Tzanetakis et al. 2013) and CuLCrV (Hagen et al. 2008) are not transmitted mechanically, the cultures of both viruses were maintained by whitefly transmissions on yellow squash ‘Gold Star’ (Seedway, Hall, NY, USA).

Plant materials.

Self-pollinated seeds from two C. pepo lines, PI 171625 and PI 171627 (hereafter referred to as UGA26 and UGA28, respectively) were obtained from C. M. The PI accession PI 171625, commonly known as 6789, originates from Amasya, whereas PI 171627, known as Erken, hails from Tokat, both located in Turkey. Gentry (Seedway, Hall, NY, USA), a squash cultivar susceptible to WTVs, was used as a control. Three seeds each of UGA26, UGA28, and ‘Gentry’ were inoculated with CCYV or CuLCrV and all three plants of one line or cultivar inoculated with one virus were maintained in the same cage (Fig. 1A). Similarly, three plants of each line or cultivar were used as a mock inoculated control (nonviruliferous whitefly) and uninfected control (no whiteflies or virus) (Fig. 1B and 1C) as well. In each trial, nine insect-proof cages with a 160-μm aperture were maintained and monitored in the greenhouse. This layout was used for both CCYV and CuLCrV trials, and each trial was performed two times.

Fig. 1.
Fig. 1.

Schematic diagram of the experimental layout. (A) Infected plants treated with viruliferous whiteflies carrying infection of Cucurbit chlorotic yellows virus or Cucurbit leaf crumple virus. (B) Mock plants treated with nonviruliferous whiteflies. (C) Uninfected plants with no whiteflies or virus treatment. UGA26 and UGA28 are PI lines. Gentry is a susceptible cultivar. Plants 1, 2, and 3 are replicates. The three plant replicates from each line were maintained within the same cage.

Citation: HortScience 59, 7; 10.21273/HORTSCI17861-24

Virus transmission and maintenance of plants.

Before inoculation, a single infection of CCYV or CuLCrV was confirmed on the source plants using the SYBR Green–based quantitative polymerase chain reaction (qPCR) detection method described by Kavalappara et al. (2022). Nonviruliferous whiteflies were released onto the virus source plant (‘Gold Star’; Seedway, Hall, NY, USA) and allowed an acquisition access period of 48 h in an insect-proof cage. Approximately 50 adult viruliferous whiteflies per clip cage were transferred onto squash seedlings at the two- to three-true leaf stage 2 weeks after planting for an inoculation access period (IAP) of 48 h. Criniviruses, such as CCYV (Célix et al. 1996), are transmitted in a semipersistent manner by whiteflies (Tzanetakis et al. 2013), whereas begomoviruses, including CuLCrV, are transmitted in a persistent manner (Fiallo-Olivé et al. 2020). Whiteflies were provided with a 48-h acquisition access period and an IAP to ensure efficient acquisition and transmission of CCYV and CuLCrV, although, in theory, both viruses, particularly CCYV, could be acquired and transmitted in a shorter duration. After an IAP, plants were treated by spraying with the neonicotinoid insecticide ASSAIL (UPL, King of Prussia, PA, USA) at a rate of 0.92 g⋅L–1, with the a.i. acetamiprid to kill the whiteflies. Plants were fertilized (Miracle-Gro, Marysville, OH, USA) with 15 g⋅L–1 every 2 weeks during the duration of the experiment. A leaf sample was collected from each plant replicate 2 and 4 weeks postinoculation (wpi) for all trials. A separate, healthy squash plant maintained in an insect-proof cage was used as a negative control for the assay.

Plant evaluation and sampling.

Plant response to virus infection was monitored weekly, and the severity of symptoms was recorded at 7, 14, 21, and 28 d after inoculation. In both the CCYV and CuLCrV experiments, an overall score was assigned to individual plants using a symptoms severity rating scale as follows: 0 = no symptoms, 1 = 1% to 20%, 2 = 21% to 40%, 3 = 41% to 60%, 4 = 61% to 80%, and 5 = 81% to 100% (Luckew et al. 2022). This scale was used for both CCYV and CuLCrV based on the typical symptoms expressed by the whole plant.

For CCYV quantification, ≈0.1 g of leaf tissue was sampled from the lower two to three leaves because crinivirus concentrations, including that of CCYV, tend to be greater in these regions (Orfanidou et al. 2021; Tamang et al. 2021; Tzanetakis et al. 2013; Wintermantel et al. 2017). Similarly, for CuLCrV quantification, leaf tissue weighing 0.1 g was collected from the upper two to three leaves of CuLCrV-infected plants. Samples for CuLCrV quantification were collected from the upper leaves because young, infected squash leaf tissue has been shown to be reliable for assessing CuLCrV concentration and making comparisons (Gadhave et al. 2020). Samples were collected at two time points (2 and 4 wpi), frozen immediately in liquid nitrogen, and kept at –80 °C pending further laboratory analysis.

Nucleic acid isolation and quantitative analysis.

Nucleic acid was isolated from ≈0.1 g of leaf tissue of CCYV- and CuLCrV-inoculated plants and controls using the Trizol RNA extraction protocol (Thermo Fisher Scientific, Waltham, MA, USA) and the DNeasy plant mini kit (Qiagen, Germantown, MD, USA), respectively, according to the manufacturers’ instructions. The concentration of extracted nucleic acid was determined using a Nanodrop Spectrophotometer (Thermo Fisher Scientific).

SYBR Green qPCR was developed and standardized to determine CCYV and CuLCrV accumulation at two different times. Complementary DNA (cDNA) was synthesized from 100 ng⋅μL–1 for each sample CCYV-infected sample according to the protocol described by Adeleke et al. (2022). To calculate the copy number of the virus in each sample, the cycle threshold (Ct) value of each sample was compared with that of a standard. A fragment size of 87 bp of the conserved region of mitochondrion cytochrome oxidase subunit I (CyOXID) was amplified with the primer set (CyOXID-F and CyOXID-R) for the housekeeping gene, according to Papayiannis et al. (2011). The number of copies was estimated based on the formula described by Rotenberg et al. (2009) and Tamang et al. (2021).

The qPCR assay was carried out with 10 μL of SSOAdvanced Universal SYBR Green Supermix (BioRad, Hercules, CA, USA), 1 μL each of forward and reverse primer (10 μM), 11 μL RNAse-free water, and 2 μL cDNA or DNA to a final volume of 25 μL. Details of all primer sets used in this analysis for the detection of virus accumulation are given in Table 1. Three replicates for each biological sample were included. A plasmid carrying the targeted gene of each virus was used as the positive control, and nuclease-free water was included as a no-template control. A separate, healthy squash plant maintained in an insect-proof cage was used for the negative control. The assay was carried out using the CFX96 Touch Deep Well Real-Time PCR System® (Bio-Rad), with the runtime profile of an initial denaturation step of 3 min at 95 °C followed by 35 cycles of denaturation for 30 s at 95 °C and a combined step of annealing and extension at 62 °C for 30 s. Melt curve analysis was performed to ensure specificity of the reactions.

Table 1.

List of primers used for the real-time quantitative polymerase chain reaction analysis of criniviruses and begomovirus.

Table 1.

Statistical analysis.

A graph showing the disease progression over time was created by plotting the means and standard errors of disease severity for each experiment using the ggplot package in R (R Core Team 2019; Wickham 2016). The area under the disease progress curve (AUDPC) was calculated per replicate using the audpc function in the epifitter package in R v. 0.3.0 (Alves and Del Ponte 2021). For each trial, a one-way analysis of variance (ANOVA) was performed to test whether the AUDPC values were significantly different in CCYV- or CuLCrV-infected UGA26, UGA28, and ‘Gentry’. Post hoc mean separation was performed using Tukey’s honestly significant difference test through the HSD.test function in the Agricola package in R v. 1.4.0 (de Mendiburu and Yaseen 2020). Virus accumulation for plant lines and two sampling points was subjected to a two-way ANOVA using R v. 4.0.3 (R Core Team 2019), followed by multiple mean comparisons using Tukey’s significant difference (P < 0.05) to compare virus severity on each line.

Results

Detection of CCYV and CuLCrV in the source plants.

Before the inoculation of the experimental plants, nucleic acid was isolated from both the CCYV and CuLCrV source plants, and was tested to ensure the presence and high concentration of CCYV or CuLCrV and the absence of other WTVs prevalent in Georgia, USA. The results show a single infection of each virus on the individual plant with no contamination of other WTVs.

Cucurbit chlorotic yellows virus.

At 2 wpi, an initial symptom of chlorotic spots was observed on the lower leaves of CCYV-infected plants of ‘Gentry’ and UGA26; UGA28 did not exhibit any of these symptoms (Fig. 2A). The symptoms progressed extensively into the upper leaves and, by 4 wpi, the entire lower leaves of both UGA26 and ‘Gentry’ turned completely yellow. Symptoms were more severe in the susceptible cultivar Gentry than in UGA26. On the other hand, mild interveinal chlorosis was observed in UGA28, and the symptom remained restricted to the lower leaves of the infected plants. The mock-inoculated plants (healthy whiteflies) and healthy controls (uninoculated) did not exhibit any of these symptoms at any time (Fig. 2A) during the course of the experiment.

Fig. 2.
Fig. 2.

Phenotypic response of inoculated plants to virus infection. (A) Cucurbit chlorotic yellows virus. (B) Cucurbit leaf crumple virus. UGA26 and UGA28 are PI test lines whereas Gentry is a susceptible cultivar.

Citation: HortScience 59, 7; 10.21273/HORTSCI17861-24

Cucurbit leaf crumple virus.

In the CuLCrV experiments, typical symptoms of CuLCrV infection were observed on both PI lines as well as the susceptible cultivar at 2 wpi. These symptoms started with chlorotic spots on the youngest leaves of the infected plant that later turned thick, crumpled, and downward curling. At 4 wpi, the crumpling of the younger leaves was more pronounced in the susceptible cultivar with yellowing compared with both the PI lines (Fig. 2B). The symptoms observed in CuLCrV-infected plants were restricted to the younger leaves only. The leaves of ‘Gentry’ were more brittle, with severe crumpling and yellowing, and interveinal chlorosis. No symptoms were observed in both the mock or uninfected controls (Fig. 2A and 2B).

Disease severity and AUDPC.

Symptoms severity in individual plants was recorded for 4 wpi to estimate the AUDPC, and was analyzed by genotype (line). In the CCYV experimental trials, UGA28 and UGA26 showed less disease severity compared with the susceptible control (‘Gentry’) across all four rating times (Fig. 3A and 3B).

Fig. 3.
Fig. 3.

Mean disease progression over time of Cucurbit chlorotic yellows virus (CCYV). (A) First trial of CCYV. (B) Second trial of CCYV. The y-axis represents the severity of infection at a specific time point. The x-axis represents the day after inoculation at which disease severity was recorded. UGA26 and UGA28 are PI lines. Gentry is a susceptible cultivar. Standard errors of the means are depicted.

Citation: HortScience 59, 7; 10.21273/HORTSCI17861-24

The AUDPC was significantly different among all the plants in the first trial (F = 211.8DFn=2,DFd=6; P < 0.05) as well as the second trial (F = 402.4DFn=2,DFd=6; P < 0.001). The effect of the trials was also compared for the AUDPC. There was no significant difference between the two trials (F = 305.2DFn=2,DFd=6; P > 0.05). However, in both trials, ‘Gentry’ exhibited more disease severity and a greater AUDPC (Fig. 4).

Fig. 4.
Fig. 4.

Cucurbit chlorotic yellows virus area under disease progress curve (AUDPC). The y-axis represents the AUDPC. The x-axis represents the lines used. UGA26 and UGA28 are PI lines. Gentry is a susceptible cultivar. Different lowercase letters above the bars indicate a significant difference in AUDPC values.

Citation: HortScience 59, 7; 10.21273/HORTSCI17861-24

In the CuLCrV experiment, UGA26 had less disease severity compared with UGA28 and ‘Gentry’ (Fig. 5). The AUDPC for UGA26 was significantly less in UGA26 compared with the other two genotypes in the first (F = 10.95DFn=2,DFd=6; P < 0.001) and second (F = 20.78DFn=2,DFd=6; P < 0.001) trials (Fig. 6). There was no difference in CuLCrV AUDPC between UGA28 and either UGA26 or ‘Gentry’ (Figs. 5 and 6). There was no significant trial effect (F = 0.379DFn=1,DFd=16; P > 0.05).

Fig. 5.
Fig. 5.

Mean severity of Cucurbit leaf crumple virus (CuLCrV) infection over time: (A) First trial of CuLCrV. (B) Second trial of CuLCrV. The y-axis represents the severity of infection at a time point. The x-axis represents the day after inoculation at which disease incidence was recorded. UGA26 and UGA28 are PI lines. Gentry is a susceptible cultivar. The error bar presented here is the standard error.

Citation: HortScience 59, 7; 10.21273/HORTSCI17861-24

Fig. 6.
Fig. 6.

Cucurbit leaf crumple virus area under disease progress curve (AUDPC). The y-axis represents the AUDPC. The x-axis represents the lines used. UGA26 and UGA28 are PI lines. Gentry is a susceptible cultivar. Different lowercase letters above the bars indicate a significant difference in AUDPC values.

Citation: HortScience 59, 7; 10.21273/HORTSCI17861-24

Accumulation of target viruses in experimental plants.

The standard curve was developed by 10-fold serial dilutions using plasmid carrying the RdRp and CP fragments of CCYV and CuLCrV, respectively. The amplification efficiency was 92.3% for CCYV (Fig. 7A) and 90.1% for CuLCrV (Fig. 7B). The linearity (R2 value) of the standard curve assay for both viruses was more than 0.99, validating the reliability of the assay for the quantification.

Fig. 7.
Fig. 7.

SYBR Green quantitative polymerase chain reaction standard curve showing the assay’s application efficiency and linearity. (A) Cucurbit chlorotic yellows virus. (B) Cucurbit leaf crumple virus. Cq = quantification cycle.

Citation: HortScience 59, 7; 10.21273/HORTSCI17861-24

In both trials, the CCYV titer in ‘Gentry’ was significantly greater than both PI lines at 2 and 4 wpi. At 2 wpi, the virus titer was less (greater Ct value) in UGA28 (no symptoms) than in UGA26, although there was no significant difference between UGA26 and UGA28 (F = 5.56DFn=6,DFd=6; P < 0.05) at that time. There was a significant difference between the two repeated trials for the CCYV experiment (F = 0.579DFn=1,DFd=16; P < 0.05). At 4 wpi, there was a significant difference in CCYV titer among all lines (F = 86.57DFn=2,DFd=6; P ≤ 0.001), with ‘Gentry’ having the greatest virus accumulation (Fig. 8A). In the second trial, there was no significant difference between the PI lines at 2 and 4 wpi. However, there was a significant difference between the PI lines and ‘Gentry’ at 4 wpi (F = 119.76DFn=2,DFd=6; P < 0.001) (Fig. 8B). In case of CuLCrV, in both trials there was no significant difference between ‘Gentry’ and the PI lines in the estimated copies of the virus, both at 2 and 4 wpi (Fig. 8C and 8D).

Fig. 8.
Fig. 8.

Quantification and the virus accumulation in the inoculated plants. (A) Cucurbit chlorotic yellows virus (CCYV) first trial. (B) CCYV second trial. (C) Cucurbit leaf crumple virus (CuLCrV) first trial. (D) CuLCrV second trial. Each bar with standard errors represents an average of virus copy numbers per nanogram of DNA. The y-axis represents a logarithmic scale from the virus accumulation titer value. Significant differences between means were separated with Tukey’s honestly significant difference test at α = 0.05. ns, *, **, *** Nonsignificant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. UGA26 and UGA28 are PI test lines whereas Gentry is a susceptible cultivar.

Citation: HortScience 59, 7; 10.21273/HORTSCI17861-24

Discussion

CuLCrV and CCYV pose significant threats to cucurbit production, particularly in the southeastern United States (Adkins et al. 2011; Kavalappara et al. 2021a). CCYV, a member of the genus Crinivirus, harbors a single-strand bipartite RNA genome and is transmitted in a semipersistent manner by whiteflies (Célix et al. 1996). CCYV has been linked to notable yield losses, causing reductions in yield and marketability (Gyoutoku et al. 2009; Okuda et al. 2010; Peng and Huang 2011). On the other hand, CuLCrV is a single-strand DNA virus with a bipartite genome and belongs to the genus Begomovirus and is transmitted in a persistent manner by whiteflies (Hagen et al. 2008). CuLCrV infections have been documented to reach up to 100% in squash during the fall season in Georgia, USA (Kavalappara et al. 2021a). The significant surge in the population of B. tabaci in Florida and Georgia, USA, during the past 2 years, coupled with the incidence of CuLCrV, has resulted in extensive yield losses (Dawson 2016; Martini et al. 2016; McAvoy 2017). Despite the significant impact of CuLCrV and CCYV on squash production, there are currently no commercial varieties with resistance to any of these viruses (Candian et al. 2021).

Two C. pepo accessions, UGA26 and UGA28, were screened previously in field conditions for resistance to WTVs and they exhibited superior performance, with reduced CuLCrV accumulation in mixed infection with CYSDV (Luckew et al. 2022). However, the reliability of resistance identified in field screening is undermined by the variability in whitefly pressure, inoculation intensity, viral inoculum levels, and interactions during mixed infections (Cohen et al. 1988; Lapidot et al. 2006). Therefore, controlled greenhouse inoculations with pure cultures are important for validating field results to ensure the reliability of identified resistance sources before their integration into breeding programs. In addition, the response of these germplasm materials to CCYV infection has been previously unreported. The objective of our study was to assess the resistance of these two accessions to pure cultures of CCYV and CuLCrV under greenhouse conditions. This involved using pure cultures of the viruses to deliver greater virus titers of each virus, thereby validating the results obtained from field evaluations.

In response to CCYV infections, both UGA28 and UGA26 showed milder symptoms and accumulated significantly lower levels of CCYV RNA compared with the susceptible cultivar Gentry. In particular, UGA28 outperformed UGA26, demonstrating delayed symptom development, lower AUDPC values, and reduced virus accumulation within the infected plant tissue. Plant resistance to a virus entails reduced infection, multiplication, and invasion, resulting in lower virus titers; tolerance is manifested by mild or absent symptoms and limited yield loss (Cooper and Jones 1983; Fraser 1990; Kang et al. 2005, Pagán and García-Arenal 2018). Thus, these two PI accessions, and UGA28 in particular, have both resistance against CCYV and are tolerant to the yellowing disease caused by it.

However, no significant difference was observed between the two PI lines and ‘Gentry’ when inoculated with CuLCrV in terms of virus accumulation. Nonetheless, regarding disease severity, UGA26 exhibited reduced severity and lower AUDPC values compared with UGA28 and ‘Gentry’ in response to CuLCrV infection. The higher CuLCrV titers in UGA26 is in contrast with a previous field study (Luckew et al. 2022) in which UGA26 performed better than CuLCrV in terms of load and symptom expression. It is worth noting that the samples in the previous study contained mixed infections with CYSDV (Luckew et al. 2022) and possibly other viruses, which could have influenced the reduced CuLCrV titers observed in UGA26.

Limited efforts have been made to identify resistance in cucurbits against CCYV. Melon accession JP 138332 exhibited relatively lower CCYV titers compared with other melon accessions tested, suggesting potential inhibition of virus multiplication (Okuda et al. 2012). A single quantitative trait locus conferring resistance to CCYV in JP 138332 was identified on chromosome 1 (Kawazu et al. 2018). In addition, recent studies have identified C. pepo accessions PI 420328 and PI 458731, among others, as resistant to CCYV, showing significantly reduced CCYV titers compared with the susceptible cultivar Gentry (Kavalappara et al. 2024).

Melon PI 236355 was found to be completely resistant in field tests and controlled inoculation in the greenhouse, whereas partial resistance to CuLCrV was reported in several melon breeding lines, including MR-1, PI 124112, PI 179901, PI 234607, PI 313970, and PI 414723 (McCreight et al. 2008). A single recessive gene, culcv, was attributed to resistance in PI 313970, a C. melo accession, and likely in the other resistant accessions in different field and greenhouse experiments (McCreight et al. 2008).

Conclusion

In conclusion, we identified and characterized two C. pepo lines, UGA26 and UGA28, that have partial resistance against CCYV. Both of these PI lines did not show resistance against infection by CuLCrV. UGA26 (PI 171625) was also identified previously to be resistant to the Cucumber mosaic virus (Lebeda and Kristkova 1996). Criniviruses such as CCYV (Orfanidou et al. 2017) demonstrate minimal genetic variability in their coding regions across various isolates (Akhter et al. 2016; Orílio and Navas-Castillo 2009; Tzanetakis et al. 2013). As a result, the resistance identified in our study is expected to provide protection against a wide range of CCYV isolates. Given the lack of resistant sources available against CCYV (McCreight et al. 2020), these lines may be useful sources to introgress resistance to high-yielding commercial cultivars. Further studies need to be conducted to understand the inheritance of resistance mechanisms against CCYV in these lines.

The use of host-resistant crops is the most efficient strategy for managing the WTVs of cucurbits. Our study further elucidated WTV resistance in yellow squash to a single infection of CCYV and CuLCrV in the greenhouse. Our results show that UGA28 is more tolerant compared with UGA26 against a single infection of CCYV.

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  • Fig. 1.

    Schematic diagram of the experimental layout. (A) Infected plants treated with viruliferous whiteflies carrying infection of Cucurbit chlorotic yellows virus or Cucurbit leaf crumple virus. (B) Mock plants treated with nonviruliferous whiteflies. (C) Uninfected plants with no whiteflies or virus treatment. UGA26 and UGA28 are PI lines. Gentry is a susceptible cultivar. Plants 1, 2, and 3 are replicates. The three plant replicates from each line were maintained within the same cage.

  • Fig. 2.

    Phenotypic response of inoculated plants to virus infection. (A) Cucurbit chlorotic yellows virus. (B) Cucurbit leaf crumple virus. UGA26 and UGA28 are PI test lines whereas Gentry is a susceptible cultivar.

  • Fig. 3.

    Mean disease progression over time of Cucurbit chlorotic yellows virus (CCYV). (A) First trial of CCYV. (B) Second trial of CCYV. The y-axis represents the severity of infection at a specific time point. The x-axis represents the day after inoculation at which disease severity was recorded. UGA26 and UGA28 are PI lines. Gentry is a susceptible cultivar. Standard errors of the means are depicted.

  • Fig. 4.

    Cucurbit chlorotic yellows virus area under disease progress curve (AUDPC). The y-axis represents the AUDPC. The x-axis represents the lines used. UGA26 and UGA28 are PI lines. Gentry is a susceptible cultivar. Different lowercase letters above the bars indicate a significant difference in AUDPC values.

  • Fig. 5.

    Mean severity of Cucurbit leaf crumple virus (CuLCrV) infection over time: (A) First trial of CuLCrV. (B) Second trial of CuLCrV. The y-axis represents the severity of infection at a time point. The x-axis represents the day after inoculation at which disease incidence was recorded. UGA26 and UGA28 are PI lines. Gentry is a susceptible cultivar. The error bar presented here is the standard error.

  • Fig. 6.

    Cucurbit leaf crumple virus area under disease progress curve (AUDPC). The y-axis represents the AUDPC. The x-axis represents the lines used. UGA26 and UGA28 are PI lines. Gentry is a susceptible cultivar. Different lowercase letters above the bars indicate a significant difference in AUDPC values.

  • Fig. 7.

    SYBR Green quantitative polymerase chain reaction standard curve showing the assay’s application efficiency and linearity. (A) Cucurbit chlorotic yellows virus. (B) Cucurbit leaf crumple virus. Cq = quantification cycle.

  • Fig. 8.

    Quantification and the virus accumulation in the inoculated plants. (A) Cucurbit chlorotic yellows virus (CCYV) first trial. (B) CCYV second trial. (C) Cucurbit leaf crumple virus (CuLCrV) first trial. (D) CuLCrV second trial. Each bar with standard errors represents an average of virus copy numbers per nanogram of DNA. The y-axis represents a logarithmic scale from the virus accumulation titer value. Significant differences between means were separated with Tukey’s honestly significant difference test at α = 0.05. ns, *, **, *** Nonsignificant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. UGA26 and UGA28 are PI test lines whereas Gentry is a susceptible cultivar.

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    • Search Google Scholar
    • Export Citation
  • Alves KS, Del Ponte EM. 2021. epifitter: Analysis and simulation of plant disease progress curves. https://CRAN.R-project.org/package=epifitter.

  • Candian JS, Coolong T, Dutta B, Srinivasan R, Sparks A, Barman A, da Silva ALBR. 2021. Yellow squash and zucchini cultivar selection for resistance to Cucurbit leaf crumple virus in the southeastern United States. HortTechnology. 31:504513. https://doi.org/10.21273/HORTTECH04877-21.

    • Search Google Scholar
    • Export Citation
  • Célix A, López-Sesé A, Alwarza N, Gomez-Guillamón ML, Rodriguez-Cerezo E. 1996. Characterization of cucurbit yellow stunting disorder virus, a Bemisia tabaci–transmitted closterovirus. Phytopathology. 86:13701376. https://doi.org/10.1094/Phyto-86-1370.

    • Search Google Scholar
    • Export Citation
  • Codod CB, Severns PM, Sparks AN, Srinivasan R, Kemerait RC, Dutta B. 2022. Characterization of the spatial distribution of the whitefly-transmitted virus complex in yellow squash fields in southern Georgia, USA. Front Agron. 4:930388. https://doi.org/10.3389/fagro.2022.930388.

    • Search Google Scholar
    • Export Citation
  • Cohen S, Kern J, Harpaz I, Ben-Joseph R. 1988. Epidemiological studies of the Tomato yellow leaf curl virus (TYLCV) in the Jordan Valley, Israel. Phytoparasitica. 16:259270. https://doi.org/10.1007/BF02979527.

    • Search Google Scholar
    • Export Citation
  • Cooper J, Jones A. 1983. Responses of plants to viruses: Proposals for the use of terms. Phytopathology. 73:127128.

  • Dawson K. 2016. Whitefly populations troubling Georgia vegetable growers. https://vegetablegrowersnews.com/news/whitefly-populations-troubling-georgia-vegetable-growers/. [accessed 15 May 2024].

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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
  • Fiallo-Olivé E, Pan L-L, Liu S-S, Navas-Castillo J. 2020. Transmission of begomoviruses and other whitefly-borne viruses: Dependence on the vector species. Phytopathology. 110(1):1017. https://doi.org/10.1094/PHYTO-07-19-0273-FI.

    • Search Google Scholar
    • Export Citation
  • Fraser R. 1990. The genetics of resistance to plant viruses. Annu Rev Phytopathol. 28:179200. https://doi.org/10.1146/annurev.phyto.43.011205.141140.

    • Search Google Scholar
    • Export Citation
  • Gadhave KR, Dutta B, Coolong T, Sparks AN, Adkins S, Srinivasan R. 2018. First report of Cucurbit yellow stunting disorder virus in cucurbits in Georgia, United States. Plant Health Prog. 19:910. https://doi.org/10.1094/PHP-03-17-0016-BR.

    • Search Google Scholar
    • Export Citation
  • Gadhave KR, Gautam S, Dutta B, Coolong T, Adkins S, Srinivasan R. 2020. Low frequency of horizontal and vertical transmission of Cucurbit leaf crumple virus in whitefly Bemisia tabaci Gennadius. Phytopathology. 110:12351241. https://doi.org/10.1094/PHYTO-09-19-0337-R.

    • Search Google Scholar
    • Export Citation
  • Georgia Farm Gate Value Report. 2022. #AR-24-01. https://caed.uga.edu/publications/farm-gate-value.html. [accessed 15 May 2024].

  • Guzman P, Sudarshana MR, Seo YS, Rojas MR, Natwick E, Turini T. 2000. A new bipartite geminivirus (begomovirus) causing leaf curl and crumpling in cucurbits in the Imperial Valley of California. Plant Dis. 84:488. https://doi.org/10.1094/PDIS.2000.84.4.488C.

    • Search Google Scholar
    • Export Citation
  • Gyoutoku Y, Okazaki S, Furuta A, Etoh T, Mizobe M, Kuno K, Hayashida S, Okuda M. 2009. Chlorotic yellows disease of melon caused by Cucurbit chlorotic yellows virus, a new crinivirus. Ann Phytopathol Soc Jpn. 75:109111. https://doi.org/10.3186/jjphytopath.75.109.

    • Search Google Scholar
    • Export Citation
  • Hagen C, Rojas MR, Sudarshana MR, Xoconostle-Cazares B, Natwick ET, Turini TA, Gilbertson RL. 2008. Biology and molecular characterization of Cucurbit leaf crumple virus, an emergent cucurbit-infecting begomovirus in the Imperial Valley of California. Plant Dis. 92(5):781793. https://doi.org/10.1094/PDIS-92-5-0781.

    • Search Google Scholar
    • Export Citation
  • Hidayat P, Bintoro D, Nurulalia L, Basri M. 2018. Species, host range, and identification key of whiteflies of Bogor and surrounding area. J Trop Plant Pests Dis. 18:127150. https://doi.org/10.23960/j.hptt.218127-150.

    • Search Google Scholar
    • Export Citation
  • Jennings DL. 1994. Breeding for resistance to African cassava mosaic virus in East Africa. Trop Sci. 34:110122.

  • Kang BC, Yeam I, Jahn MM. 2005. Genetics of plant virus resistance. Annu Rev Phytopathol. 43:581621. https://doi.org/10.1146/annurev.phyto.43.011205.141140.

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Ismaila A. Adeleke Department of Plant Pathology, University of Georgia, 115 Coastal Way, Tifton, GA 31793, USA

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Saritha R. Kavalappara Department of Plant Pathology, University of Georgia, 115 Coastal Way, Tifton, GA 31793, USA

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Clarence B. Codod Department of Plant Pathology, University of Georgia, 115 Coastal Way, Tifton, GA 31793, USA

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Prasanna Kharel Department of Horticulture, University of Georgia, 2360 Rainwater Road, Tifton, GA 31793, USA

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Alex Luckew Department of Horticulture, University of Georgia, 1111 Miller Plant Sciences, Athens, GA 30602, USA

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Cecilia McGregor Department of Horticulture, University of Georgia, 1111 Miller Plant Sciences, Athens, GA 30602, USA

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Alvin M. Simmons US Vegetable Research, US Department of Agriculture–Agricultural Research Service, 2700 Savannah Highway, Charleston, SC 29414, USA

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Rajagopalbabu Srinivasan Department of Entomology, University of Georgia, 1109 Experiment Street, Redding Building, Griffin, GA 30223, USA

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Sudeep Bag Department of Plant Pathology, University of Georgia, 115 Coastal Way, Tifton, GA 31793, USA

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Contributor Notes

This study was supported by the US Department of Agriculture (USDA) Hatch grant awarded to S.B. (award no. 1020319) and the USDA–University of Georgia (UGA) (cooperative agreement no. 58-6080-9-006). The funders had no role in the study design, data collection and analysis, decision to publish or manuscript preparation.

This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation for its use by the USDA or UGA.

S.B. is the corresponding author. E-mail: sudeepbag@uga.edu.

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  • Fig. 1.

    Schematic diagram of the experimental layout. (A) Infected plants treated with viruliferous whiteflies carrying infection of Cucurbit chlorotic yellows virus or Cucurbit leaf crumple virus. (B) Mock plants treated with nonviruliferous whiteflies. (C) Uninfected plants with no whiteflies or virus treatment. UGA26 and UGA28 are PI lines. Gentry is a susceptible cultivar. Plants 1, 2, and 3 are replicates. The three plant replicates from each line were maintained within the same cage.

  • Fig. 2.

    Phenotypic response of inoculated plants to virus infection. (A) Cucurbit chlorotic yellows virus. (B) Cucurbit leaf crumple virus. UGA26 and UGA28 are PI test lines whereas Gentry is a susceptible cultivar.

  • Fig. 3.

    Mean disease progression over time of Cucurbit chlorotic yellows virus (CCYV). (A) First trial of CCYV. (B) Second trial of CCYV. The y-axis represents the severity of infection at a specific time point. The x-axis represents the day after inoculation at which disease severity was recorded. UGA26 and UGA28 are PI lines. Gentry is a susceptible cultivar. Standard errors of the means are depicted.

  • Fig. 4.

    Cucurbit chlorotic yellows virus area under disease progress curve (AUDPC). The y-axis represents the AUDPC. The x-axis represents the lines used. UGA26 and UGA28 are PI lines. Gentry is a susceptible cultivar. Different lowercase letters above the bars indicate a significant difference in AUDPC values.

  • Fig. 5.

    Mean severity of Cucurbit leaf crumple virus (CuLCrV) infection over time: (A) First trial of CuLCrV. (B) Second trial of CuLCrV. The y-axis represents the severity of infection at a time point. The x-axis represents the day after inoculation at which disease incidence was recorded. UGA26 and UGA28 are PI lines. Gentry is a susceptible cultivar. The error bar presented here is the standard error.

  • Fig. 6.

    Cucurbit leaf crumple virus area under disease progress curve (AUDPC). The y-axis represents the AUDPC. The x-axis represents the lines used. UGA26 and UGA28 are PI lines. Gentry is a susceptible cultivar. Different lowercase letters above the bars indicate a significant difference in AUDPC values.

  • Fig. 7.

    SYBR Green quantitative polymerase chain reaction standard curve showing the assay’s application efficiency and linearity. (A) Cucurbit chlorotic yellows virus. (B) Cucurbit leaf crumple virus. Cq = quantification cycle.

  • Fig. 8.

    Quantification and the virus accumulation in the inoculated plants. (A) Cucurbit chlorotic yellows virus (CCYV) first trial. (B) CCYV second trial. (C) Cucurbit leaf crumple virus (CuLCrV) first trial. (D) CuLCrV second trial. Each bar with standard errors represents an average of virus copy numbers per nanogram of DNA. The y-axis represents a logarithmic scale from the virus accumulation titer value. Significant differences between means were separated with Tukey’s honestly significant difference test at α = 0.05. ns, *, **, *** Nonsignificant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. UGA26 and UGA28 are PI test lines whereas Gentry is a susceptible cultivar.

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