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Article

Effects of Acquisition Time and Viral Load of Source Plants on Infections of Two Tomato Begomoviruses in Bemisia tabaci

by
Ya-Yu Huang
1,†,
Wei-Hua Li
1,†,
Kyeong-Yeoll Lee
2,
Wen-Shi Tsai
3 and
Chi-Wei Tsai
1,*
1
Department of Entomology, National Taiwan University, Taipei 106, Taiwan
2
Department of Plant Medicine, Kyungpook National University, Daegu 41566, Republic of Korea
3
Department of Plant Medicine, National Chiayi University, Chiayi 600, Taiwan
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agriculture 2025, 15(11), 1195; https://doi.org/10.3390/agriculture15111195
Submission received: 7 April 2025 / Revised: 28 May 2025 / Accepted: 29 May 2025 / Published: 30 May 2025
(This article belongs to the Section Crop Protection, Diseases, Pests and Weeds)
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Abstract

:
Tomato yellow leaf curl disease poses one of the most severe threats to tomato production worldwide. This disease is associated with a group of closely related tomato yellow leaf curl viruses. These viruses can be transmitted by the sweet potato whitefly (Bemisia tabaci) in a persistent-circulative mode. Virus particles can infect the midgut and filter chamber of whiteflies feeding on infected plants, circulate in the hemolymph, and eventually infect the primary salivary gland (PSG) of whiteflies. Later, the whiteflies feed on healthy plants, and viral particles are introduced into the plants through their saliva. Virus–vector interactions play a crucial role in the efficiency and dynamics of virus transmission. In this study, we assessed the effects of the acquisition time and viral load of source plants on infections of two tomato begomoviruses, tomato yellow leaf curl Thailand virus (TYLCTHV) and tomato leaf curl Taiwan virus (ToLCTV), in B. tabaci Middle East–Asia Minor 1. We found that more viruses were acquired and accumulated in the whitefly midgut and PSG before reaching a plateau when the acquisition time increased and when the source plant had a higher viral load. The midgut and PSG acquired and accumulated more TYLCTHV than ToLCTV with the same acquisition time and regardless of the viral loads in coinfected source plants. These results not only help us to understand virus–vector interactions but also help in developing integrated disease management strategies.

1. Introduction

Crops and vegetables face severe threats due to various pathogens. Among these, viruses pose significant challenges to agricultural production. Most plant viruses rely on specific vectors, including insects, mites, nematodes, and fungi, for transmission [1,2]. Hemipteran insects (e.g., aphids, whiteflies, and leafhoppers) constitute the largest group of insect vectors [3]. Among these, the sweet potato whitefly (Bemisia tabaci) is listed among 100 of the world’s worst invasive alien species [4]. In addition to causing direct damage by piercing and sucking the plant sap, B. tabaci can transmit viruses that cause widespread diseases in various crops, such as tomatoes, melons, beans, and cassava [5,6,7].
The mode of transmission of plant viruses by insect vectors can be classified into nonpersistent, semipersistent, and persistent based on various parameters, such as the acquisition access period (AAP), the retention time, the latent period, and the ability of the virus to infect the vector’s internal organs [1,8]. In the case of persistent transmission, when a vector feeds on the phloem of an infected plant, viral particles enter the digestive tract with the plant sap. These particles infect the midgut and filter chamber. They then move into the hemocoel, where they circulate via the hemolymph to the salivary glands, eventually infecting them [9,10,11]. In this process, the virus may or may not replicate within the vector, depending on the virus species [12]. When the vector feeds on a healthy plant, the viral particles are introduced into the plant through the vector’s saliva, initiating a new infection [13,14,15]. Virus–vector interactions play a crucial role in the efficiency and dynamics of transmission [16].
Tomato yellow leaf curl disease poses one of the most severe threats to tomato production worldwide [17,18]. This disease is associated with a group of closely related tomato yellow leaf curl viruses belonging to the genus Begomovirus and family Geminiviridae. This disease causes various symptoms, such as leaf curling, interveinal and marginal chlorosis, apical clustering of shoots, and stunted growth [19]. If tomato plants become infected during the early growth stages, they typically fail to produce marketable fruits. This results in 20–100% yield losses, severely impacting the tomato industry [20,21,22]. Plant viral infections cannot be treated using chemicals. Hence, control strategies primarily focus on reducing B. tabaci populations to mitigate disease spread [23].
To better understand the interactions between tomato begomoviruses and B. tabaci and to determine their impact on virus transmission, we assessed the effects of acquisition time and viral load of source plants on infections of tomato begomoviruses in B. tabaci Middle East–Asia Minor 1 (MEAM1). This study focused on tomato yellow leaf curl Thailand virus (TYLCTHV) and tomato leaf curl Taiwan virus (ToLCTV), which are prevalent in tomato fields in Taiwan [24]. We evaluated infection levels by quantifying viral titers in the whitefly midgut and primary salivary gland (PSG) using quantitative real-time PCR (qPCR). Our findings will contribute to the development of effective control strategies and the implementation of an integrated disease management (IDM) program.

2. Materials and Methods

2.1. Insects, Viruses, and Plants

The sources and maintenance conditions of B. tabaci MEAM1, TYLCTHV, and ToLCTV have been described previously [25]. A laboratory colony of MEAM1 whiteflies was maintained on Chinese kale (Brassica oleracea cv. Alboglabra Group), a nonhost plant for both viruses. The colony was housed in whitefly-proof net cages (30 × 30 × 30 cm; MegaView, Taichung, Taiwan) within a growth chamber at 28 °C under a 16 h light: 8 h dark photoperiod.
TYLCTHV and ToLCTV were maintained in tomato plants (Solanum lycopersicum var. ANT 22) through whitefly-mediated transmission. To obtain singly infected plants, 20 TYLCTHV/ToLCTV-viruliferous whiteflies were enclosed with a leaf of a tomato seedling (at the three-leaf stage) in a fine mesh bag (6 × 15 cm; 40 mesh/cm) for 48 h to inoculate the virus. To obtain coinfected plants, 10 ToLCTV-viruliferous whiteflies and 10 TYLCTHV-viruliferous whiteflies were collectively used to inoculate each tomato seedling for 48 h to inoculate the virus. Viral infection was confirmed by performing PCR (described below) three weeks after whitefly-mediated transmission. Chinese kale and tomato plants were grown from seeds and fertilized weekly. All plants were cultured in growth chambers at 28 °C under a 16 h light: 8 h dark photoperiod.

2.2. PCR

DNA was extracted from the systemically infected leaves of inoculated plants by using a fast extraction method [25]. PCR was performed as described previously [26]. The primers THCP-F and THCP-R (Table S1) were used for detecting TYLCTHV infection [27], while the primers TWCP-F and TWCP-R (Table S1) were used for detecting ToLCTV infection [26]. PCR products were analyzed by performing gel electrophoresis.

2.3. qPCR

The relative quantification of viral titers in the whitefly and plant tissues was performed as described previously [27]. The primers THqAV2-F and THqAV2-R (Table S1) were used for amplifying the partial TYLCTHV AV2 gene [27], while the primers TWqV2-F and TWqV2-R (Table S1) were used for amplifying the partial ToLCTV V2 gene [26]. To quantify the viral load in plant tissues, the tomato 25S rRNA was selected as the reference gene and amplified using the primers 25SrRNA-F and 25SrRNA-R (Table S1) [28]. To quantify the viral titer in whitefly tissues, the whitefly heat shock protein 90 gene (HSP90) was selected as the reference gene and amplified using primers HSP90-F and HSP90-R (Table S1) [29]. The PCR efficiencies of the primer pairs for ToLCTV V2, TYLCTHV AV2, tomato 25S rRNA, and whitefly HSP90 were 100%, 100%, 99%, and 98%, respectively. The qPCR preparation and cycling conditions were as described previously [27]. Melting curves were generated at the end of the reaction to confirm amplification specificity. The amount of virus relative to that of the reference gene was calculated using the 2−ΔCt method [30]. A validation experiment demonstrated similar amplification efficiencies between the viral target and reference genes, supporting the applicability of the 2−ΔCt method (Figure S1).

2.4. Source Leaves of Viruses

Plant DNA was extracted from leaf tissue (25 mg) of each virus-infected plant by using the Plant Genomic DNA Mini Kit (Geneaid, New Taipei City, Taiwan), according to the manufacturer’s instructions. Viral loads in source leaves were determined by performing qPCR. To limit viral loads in the source leaves to a narrower range, the leaves with viral loads 10–15-fold higher than the amount of tomato 25S rRNA (used as the reference gene in qPCR) were selected as source leaves for assessing the effect of the acquisition time. To assess the effect of viral load of source plants, viral loads relative to the amount of tomato 25S rRNA were categorized into four groups: extra low (0.1–2-fold higher), low (4–6-fold higher), medium (9–11-fold higher), and high (14–16-fold higher). The leaves with viral loads outside of these categories were not chosen. To assess the effect of viral load of coinfected source plants, the leaves in which the loads of both viruses were 5–20-fold higher than the amount of tomato 25S rRNA were selected as the source leaves.

2.5. Effect of Acquisition Time on Viral Infection in B. tabaci

The effect of the acquisition time on viral infection in B. tabaci was assessed by quantifying the viral titers in the midguts and PSGs of B. tabaci after various AAPs on singly infected or coinfected leaves. Approximately 100 nonviruliferous adult whiteflies (0–5 days old) were transferred (using a mouth aspirator) from the laboratory colony into 250 mL plastic cups, each enclosing a virus-infected leaf. The leaves were detached from virus-infected tomato plants, and the petioles were placed inside glass vials filled with water to prevent them from withering. Four whiteflies (two males and two females) per sample were collected at an AAP of 3, 6, 12, 24, or 48 h. The whiteflies were then anesthetized on ice and dissected in phosphate-buffered saline (pH 7.4) under a stereomicroscope. Subsequently, the midgut and PSG of each sample were excised. Each sample contained four midguts or eight PSGs from four whiteflies. The total DNA of each sample was extracted using the gSYNC DNA Extraction Kit (Geneaid). The midguts or PSGs were directly lysed in 200 μL GST buffer (with 10% proteinase K) in a 1.5 mL microcentrifuge tube at 60 °C for 30 min. The subsequent steps were performed according to the manufacturer’s instructions. The viral titer in each sample was quantified by performing qPCR. Each AAP had six biological replicates.

2.6. Effect of Viral Load of Source Plants on Viral Infection in B. tabaci

The effect of the viral load of source plants on viral infection in B. tabaci was assessed by quantifying the viral titers in the midguts and PSGs of whiteflies that fed on singly infected or coinfected source leaves with different viral loads. Approximately 100 nonviruliferous adult whiteflies (0–5 days old) were allowed to feed on virus-infected leaves with different viral loads in 250 mL plastic cups. Four whiteflies (two males and two females) per sample were collected after a 24 h AAP. The midguts and PSGs were then excised under a stereomicroscope. Each sample contained four midguts or eight PSGs from four whiteflies. The total DNA of each sample was extracted using the gSYNC DNA Extraction Kit (Geneaid). The midguts or PSGs were directly lysed in 200 μL GST buffer (with 10% proteinase K) in a 1.5 mL microcentrifuge tube at 60 °C for 30 min. The subsequent steps were performed according to the manufacturer’s instructions. The viral titer in each sample was quantified by performing qPCR. Each viral load group had six biological replicates.

2.7. Statistical Analysis

Relative viral titers in the whitefly midguts and PSGs were determined by performing qPCR, and the obtained data were log2 transformed. The effects of acquisition time on viral infection were analyzed using a linear mixed model (LMM). The viral titer was the dependent variable, while the AAP and the virus species (TYLCTHV and ToLCTV) were set as fixed factors. As the whiteflies were sampled at each AAP, the individual identity was not included as a random factor. Estimated marginal means were calculated for each AAP and virus species combination, and pairwise comparisons between virus species at each AAP were conducted within the LMM framework. Bonferroni correction was applied to account for multiple comparisons. Data were assessed for the assumption of normal distribution using the Shapiro–Wilk test. If the assumption was met, the effect of the viral load of singly infected source plants on viral infection was analyzed by analysis of variance (ANOVA) with a post hoc Tukey’s HSD test. If not, Welch’s ANOVA was performed with the post hoc Games–Howell test. To compare TYLCTHV and ToLCTV titers in whiteflies that fed on plants within the same viral load group, normally distributed data were analyzed by Student’s t-test, while non-normally distributed data were analyzed using the Mann–Whitney U test. The effect of the viral load of coinfected source plants on viral infection was analyzed by a paired samples t-test. For non-normally distributed data, the Wilcoxon signed-rank test was employed as a nonparametric alternative. All statistical analyses were performed using SAS 9.4 (SAS Institute Inc., Cary, NC, USA).

3. Results

3.1. Effect of Acquisition Time on Viral Infection in B. tabaci

Viral titers in the midgut and PSG of whiteflies that fed on TYLCTHV/ToLCTV-infected leaves with various AAPs are illustrated in Figure 1. TYLCTHV and ToLCTV titers in the midgut increased as the AAP increased from 0 to 24 h [LMM, F (4,50) = 58.8, p < 0.01] and then reached a plateau (TYLCTHV: ~211; ToLCTV: ~27.5). TYLCTHV titers in the midgut were significantly higher than ToLCTV titers throughout the feeding period (pairwise comparisons, p < 0.01). Similarly, TYLCTHV and ToLCTV titers in the PSG increased as the AAP increased from 0 to 48 h [LMM, F (4,50) = 29.5, p < 0.01]. TYLCTHV titers in the PSG were significantly higher than ToLCTV titers throughout the feeding period (pairwise comparisons, p < 0.05 or p < 0.01). These results suggest that as the AAP increases, more viruses (TYLCTHV and ToLCTV) are acquired and accumulated in the midgut and PSG of MEAM1 whiteflies until a plateau is reached. Moreover, the midgut and PSG acquire and accumulate more TYLCTHV than ToLCTV with the same acquisition time.
Viral titers in the midgut and PSG of whiteflies that fed on source leaves coinfected with TYLCTHV and ToLCTV with various AAPs are presented in Figure 2. TYLCTHV and ToLCTV titers in the midgut increased as the AAP increased from 0 to 24 h [LMM, F (4,50) = 62.4, p < 0.01] and then reached a plateau (TYLCTHV: ~210.5; ToLCTV: ~28). TYLCTHV titers in the midgut were significantly higher than ToLCTV titers throughout the feeding period (pairwise comparisons, p < 0.01). Similarly, TYLCTHV and ToLCTV titers in the PSG increased as the AAP increased from 0 to 48 h [LMM, F (4,50) = 103.5, p < 0.01]. TYLCTHV titers in the PSG were significantly higher than ToLCTV titers throughout the feeding period (pairwise comparisons, p < 0.05 or p < 0.01). Similar to the findings in whiteflies that fed on singly infected plants, as the AAP increased, more viruses (TYLCTHV and ToLCTV) were acquired and accumulated in the midgut and PSG of MEAM1 whiteflies that fed on coinfected plants until a plateau was reached. Moreover, the midgut and PSG acquired and accumulated more TYLCTHV than ToLCTV with the same acquisition time.

3.2. Effect of Viral Load of Source Plants on Viral Infection in B. tabaci

Viral titers in the midgut of whiteflies that fed on TYLCTHV/ToLCTV-infected leaves with various viral loads are illustrated in Table 1. The TYLCTHV titer in the midgut increased as the viral load of the source leaves increased; no significant difference was noted between medium and high viral load treatments (Games–Howell test, p = 0.56). Similarly, the ToLCTV titer in the midgut increased as the viral load of the source leaves increased; no significant difference was noted between low and medium viral load treatments (Games–Howell test, p = 0.94). A comparison of TYLCTHV and ToLCTV titers in whiteflies that fed on infected leaves with the same viral load revealed that the titers were significantly different in low viral load treatment (Mann–Whitney U test, p < 0.01), in medium viral load treatment (Student’s t-test, p < 0.01), and in high viral load treatments (Student’s t-test, p < 0.01). These results suggest that as the viral load of the source plants increases, more viruses (TYLCTHV and ToLCTV) are acquired and accumulated in the midgut of MEAM1 whiteflies until a plateau is reached. Moreover, the midgut acquires and accumulates more TYLCTHV than ToLCTV when the whiteflies feed on plants with the same viral load.
Viral titers in the PSG of whiteflies that fed on TYLCTHV/ToLCTV-infected leaves with different viral loads are presented in Table 2. The TYLCTHV titer in the PSG increased as the viral load of the source leaves increased; no significant difference was noted in the titers between extra low and low viral load treatments (Tukey’s HSD test, p = 0.99) and between medium and high viral load treatments (Tukey’s HSD test, p = 0.85). Similarly, the ToLCTV titer in the PSG increased as the viral load of the source leaves increased; the titer for high viral load treatment was significantly higher than those for the other treatments (Tukey’s HSD test, p < 0.01) A comparison of TYLCTHV and ToLCTV titers in whiteflies that fed on infected leaves with the same viral load revealed that the titers were significantly different only in medium viral load treatment (Student’s t-test, p < 0.01). These results suggest that as the viral load of the source plant increases, more viruses (TYLCTHV and ToLCTV) are acquired and accumulated in the PSG of MEAM1 whiteflies until a plateau is reached.
Viral titers in the midgut and PSG of whiteflies that fed on source leaves coinfected with TYLCTHV and ToLCTV are illustrated in Figure 3 and Figure 4. Viral titers in the midgut significantly differed between the TYLCTHV and ToLCTV groups (Figure 3; Wilcoxon signed-rank test, p < 0.01). Similarly, viral titers in the PSG significantly differed between the TYLCTHV and ToLCTV groups (Figure 4; paired samples t-test, p < 0.01). These results suggest that MEAM1 whiteflies acquire and accumulate more TYLCTHV than ToLCTV in the midgut and PSG regardless of the viral load of the coinfected source plants.

4. Discussion

Many plant viruses rely on specific insect vectors for transmission; they are classified into nonpersistently, semipersistently, and persistently transmitted viruses [1,8]. Tomato begomoviruses are persistently transmitted by B. tabaci. Once these viruses are acquired by whiteflies feeding on virus-infected plants, they sequentially infect the midgut and PSG of the whiteflies [15]. In this process, virus–vector interactions play a crucial role in the efficiency and dynamics of virus transmission. In this study, we assessed the intricate interactions between tomato begomoviruses and MEAM1 whiteflies, focusing on the acquisition and infection of TYLCTHV and ToLCTV in the midgut and PSG of MEAM1 whiteflies. It is important to note that TYLCTHV and ToLCTV do not replicate in B. tabaci [11]; therefore, the acquisition time and viral load of source plants are key factors influencing virus acquisition and infection in the vector.
The acquisition and infection of TYLCTHV and ToLCTV in the midgut and PSG of MEAM1 whiteflies increased with the acquisition time on singly infected plants and then reached a plateau (Figure 1). These results are in line with their transmission mode, i.e., persistent-circulative transmission [11]. The accumulation of begomoviruses in B. tabaci has been reported to increase with the acquisition time during the early phase; however, the viral titer has only been quantified in whole whiteflies. The titers of tomato leaf curl virus (TYLCV), Sri Lankan cassava mosaic virus, tomato leaf curl New Delhi virus (ToLCNDV), and chili leaf curl virus (ChiLCV) in B. tabaci have been reported to increase with the acquisition time in infected plants [31,32,33]. Our results also showed that both TYLCTHV and ToLCTV exhibited higher relative titers in the midgut than in the PSG of MEAM1 whiteflies. This is expected, as begomoviruses do not replicate within the whitefly body [11] and must traverse multiple cellular barriers to reach and infect the PSG [15], resulting in comparatively lower viral accumulation in the PSG.
Bemisia tabaci has been reported to acquire viruses with different efficiencies. In our study, MEAM1 whiteflies acquired and accumulated more TYLCTHV than ToLCTV in their midgut and PSG throughout the feeding period (Figure 1). In another study, MED whiteflies were found to acquire more TYLCV than tomato yellow leaf curl China virus (TYLCCNV) from infected tomato plants with a 48 h AAP [34]. Asia II 1 whiteflies acquired more ChiLCV from infected chili plants than ToLCNDV from infected tomato plants throughout the feeding period [32]. Moreover, MEAM 1 whiteflies acquired more watermelon chlorotic stunt virus (WmCSV) from infected watermelon plants than TYLCV from infected tomato plants [35]. The accumulation of TYLCTHV was higher than that of ToLCTV in the midgut, possibly because TYLCTHV exhibits higher binding capacity and/or binding stability to the receptor(s) of midgut cells of whiteflies [26]. Structural analyses also suggest that variations in begomovirus coat protein sequences and structures have a significant impact on virus–receptor binding affinity [36,37]. The transmission rate has been reported to correlate with the quantity of begomoviruses in B. tabaci [34,38]. In addition, compared to ToLCTV, TYLCTHV has been reported to have higher transmission efficiency by MEAM1 whiteflies [25].
We further assessed the effect of acquisition time on the acquisition and infection of TYLCTHV and ToLCTV in MEAM1 whiteflies feeding on coinfected plants. The acquisition and infection of both viruses in the midgut and PSG of MEAM1 whiteflies increased with the acquisition time on coinfected plants and then reached a plateau (Figure 2). The accumulation of TYLCTHV in the midgut and PSG was higher than that of ToLCTV (Figure 2). MEAM1 whiteflies that fed on coinfected plants had lower ToLCTV titers in their midgut than those that fed on singly infected plants (Figure 1 and Figure 2). These results are consistent with the hypothesis that TYLCTHV has better binding capacity and/or binding stability to the receptor(s) of midgut cells of whiteflies than ToLCTV. To our knowledge, only one study on begomoviruses has focused on this issue [39]. The viral titers did not differ between MEAM1 whiteflies that fed on singly infected tomato plants and those that fed on tomato plants coinfected with TYLCV and tomato mottle virus (ToMoV) [37]. Further research is warranted on this issue.
The acquisition and infection of TYLCTHV and ToLCTV in the midgut of MEAM1 whiteflies increased with the viral load of source plants (Table 1). Studies have also suggested that TYLCV accumulation in MEAM1 whiteflies is positively correlated with the viral load of source plants [38,40]. As the viral load of source plants increases, whiteflies acquire more viruses until a plateau is reached. When whiteflies fed on source plants with a TYLCTHV titer of ≥9 or a ToLCTV titer of ≥14, the accumulation of the virus in the midgut reaches the maximum level. The TYLCTHV titer in the midgut is higher than the ToLCTV titer when the viral loads of source plants are the same. These results are also consistent with the hypothesis that TYLCTHV has better binding capacity and/or binding stability to the receptor(s) of midgut cells of whiteflies than ToLCTV.
Similar results were noted in the case of PSG; the acquisition and infection of TYLCTHV and ToLCTV in the PSG of MEAM1 whiteflies increased with the viral load of source plants (Table 2). When whiteflies fed on source plants with a TYLCTHV titer of ≥9 or a ToLCTV titer of ≥14, the accumulation of the virus in the PSG reached the maximum level. The TYLCTHV titer in the PSG was higher than the ToLCTV titer when the viral loads of source plants were the same; however, the differences were not significant. It is possible that the viral titer in the PSG was relatively low and the variation was high, resulting in a nonsignificant difference in titers between the two viruses.
Plants are commonly coinfected with multiple viruses in the field [41,42]. Virus–virus interaction may affect the acquisition and infection of viruses when whiteflies feed on coinfected plants. Compared to ToLCTV, more TYLCTHV was acquired and accumulated in the midgut of MEAM1 whiteflies regardless of the viral load of coinfected plants (Figure 3). Similar results were noted in the case of PSG; compared to ToLCTV, more TYLCTHV was acquired and accumulated in the PSG of MEAM1 whiteflies regardless of the viral load of coinfected plants (Figure 4). However, the effect of the viral load of coinfected source plants on viral infection in B. tabaci remains poorly understood, and no study has been published on this topic to date. Further research on this topic is therefore warranted.
The interactions between tomato begomoviruses and B. tabaci are complicated and are not fully understood. Our findings contribute to this understanding by elucidating how the acquisition time and viral load of source plants affect virus acquisition and infection in the midgut and PSG of MEAM1 whiteflies. This knowledge provides valuable insights for the development of IDM strategies. For instance, our results suggest that reducing the viral load in source plants—through the early removal of infected plants or the cultivation of virus-resistant varieties—can limit virus acquisition by whiteflies. Additionally, understanding the time-dependent dynamics of virus acquisition and infection supports the strategic use of insecticides or repellents aimed at minimizing whitefly feeding duration during critical acquisition periods. Finally, improving cultivation practices to enhance overall plant health and resistance may reduce susceptibility to infection and limit the spread of begomoviruses. Together, these findings offer a scientific foundation for designing targeted and effective IDM programs to manage whitefly-transmitted tomato begomoviruses.

5. Conclusions

This study revealed that more viruses (TYLCTHV and ToLCTV) are acquired and accumulated in the midgut and PSG of MEAM1 whiteflies before reaching a plateau when the acquisition time increases and when the source plant has a higher viral load. The midgut and PSG acquire and accumulate more TYLCTHV than ToLCTV with the same acquisition time. In addition, the midgut and PSG of MEAM1 whiteflies acquire and accumulate more TYLCTHV than ToLCTV regardless of the viral load of coinfected source plants. These results will not only help us to understand virus–vector interaction but will also help in the development of IDM strategies.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture15111195/s1, Figure S1: Validation of the 2−ΔCt method for quantifying the titers of (A) tomato yellow leaf curl Thailand virus (TYLCTHV) and (B) tomato leaf curl Taiwan virus (ToLCTV) in Bemisia tabaci by amplifying serial dilutions of DNA extracted from three independent biological samples; Table S1: Primers used in this study.

Author Contributions

Conceptualization, C.-W.T., K.-Y.L. and W.-H.L.; formal analysis, C.-W.T. and W.-H.L.; investigation, Y.-Y.H. and W.-H.L.; writing—original draft preparation, Y.-Y.H. and W.-H.L.; writing—review and editing, C.-W.T., K.-Y.L. and W.-S.T.; project administration, C.-W.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Animal and Plant Health Inspection Agency, Ministry of Agriculture, Taiwan, grant number 112AS-5.3.3-VP-B7.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

We thank the anonymous reviewers for valuable comments and suggestions, which helped to improve the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agriculture 15 01195 g001
Figure 1. The effect of the acquisition time on viral infection in the (A) midgut and (B) primary salivary gland (PSG) of Bemisia tabaci which fed on singly infected tomato plants. Adult whiteflies fed on tomato leaves infected with either tomato yellow leaf curl Thailand virus (TYLCTHV) or tomato leaf curl Taiwan virus (ToLCTV) for various acquisition access periods (AAPs). The viral titer relative to B. tabaci HSP90 was determined by quantitative real-time PCR (qPCR). The vertical bars represent the standard deviation. The coefficient of correlation (R2) of the regression line is labeled. Pairwise comparisons between the virus species at each AAP were conducted within the linear mixed model (LMM) framework. * p < 0.05 and ** p < 0.01. Each AAP of each virus had six biological replicates.
Figure 1. The effect of the acquisition time on viral infection in the (A) midgut and (B) primary salivary gland (PSG) of Bemisia tabaci which fed on singly infected tomato plants. Adult whiteflies fed on tomato leaves infected with either tomato yellow leaf curl Thailand virus (TYLCTHV) or tomato leaf curl Taiwan virus (ToLCTV) for various acquisition access periods (AAPs). The viral titer relative to B. tabaci HSP90 was determined by quantitative real-time PCR (qPCR). The vertical bars represent the standard deviation. The coefficient of correlation (R2) of the regression line is labeled. Pairwise comparisons between the virus species at each AAP were conducted within the linear mixed model (LMM) framework. * p < 0.05 and ** p < 0.01. Each AAP of each virus had six biological replicates.
Agriculture 15 01195 g001
Agriculture 15 01195 g002
Figure 2. The effect of the acquisition time on viral infection in the (A) midgut and (B) PSG of B. tabaci which fed on coinfected tomato plants. Adult whiteflies fed on tomato leaves coinfected with TYLCTHV and ToLCTV for various AAPs. The viral titer relative to B. tabaci HSP90 was determined by qPCR. The vertical bars represent the standard deviation. The coefficient of correlation (R2) of the regression line is labeled. Pairwise comparisons between virus species at each AAP were conducted within the LMM framework. * p < 0.05 and ** p < 0.01. Each AAP of each virus had six biological replicates.
Figure 2. The effect of the acquisition time on viral infection in the (A) midgut and (B) PSG of B. tabaci which fed on coinfected tomato plants. Adult whiteflies fed on tomato leaves coinfected with TYLCTHV and ToLCTV for various AAPs. The viral titer relative to B. tabaci HSP90 was determined by qPCR. The vertical bars represent the standard deviation. The coefficient of correlation (R2) of the regression line is labeled. Pairwise comparisons between virus species at each AAP were conducted within the LMM framework. * p < 0.05 and ** p < 0.01. Each AAP of each virus had six biological replicates.
Agriculture 15 01195 g002
Agriculture 15 01195 g003
Figure 3. The effect of the viral load of coinfected source plants on the infection of (A) TYLCTHV and (B) ToLCTV in the midgut of B. tabaci. Adult whiteflies fed on tomato leaves coinfected with TYLCTHV and ToLCTV with various viral loads (relative to tomato 25S rRNA) with a 24 h AAP. The viral titer relative to B. tabaci HSP90 in whitefly tissues was determined by qPCR. Each viral load combination had three biological replicates.
Figure 3. The effect of the viral load of coinfected source plants on the infection of (A) TYLCTHV and (B) ToLCTV in the midgut of B. tabaci. Adult whiteflies fed on tomato leaves coinfected with TYLCTHV and ToLCTV with various viral loads (relative to tomato 25S rRNA) with a 24 h AAP. The viral titer relative to B. tabaci HSP90 in whitefly tissues was determined by qPCR. Each viral load combination had three biological replicates.
Agriculture 15 01195 g003
Agriculture 15 01195 g004
Figure 4. The effect of viral load of coinfected source plants on the infection of (A) TYLCTHV and (B) ToLCTV in the PSG of B. tabaci. Adult whiteflies fed on tomato leaves coinfected with TYLCTHV and ToLCTV with various viral loads (relative to tomato 25S rRNA) with a 24 h AAP. The viral titer relative to B. tabaci HSP90 in whitefly tissues was determined by qPCR. Each viral load combination had three biological replicates.
Figure 4. The effect of viral load of coinfected source plants on the infection of (A) TYLCTHV and (B) ToLCTV in the PSG of B. tabaci. Adult whiteflies fed on tomato leaves coinfected with TYLCTHV and ToLCTV with various viral loads (relative to tomato 25S rRNA) with a 24 h AAP. The viral titer relative to B. tabaci HSP90 in whitefly tissues was determined by qPCR. Each viral load combination had three biological replicates.
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Table 1. The effect of the viral load of source plants on viral infection in the midgut of Bemisia tabaci.
Table 1. The effect of the viral load of source plants on viral infection in the midgut of Bemisia tabaci.
Source Plant Viral Load 1Relative Viral Titer 2 in the Midgut of B. tabaci (Mean ± SD)
TYLCTHV 3ToLCTV 3p-Value 4
Extra low(0.1–2 folds)3.3 ± 1.8 c2.0 ± 1.3 c0.12
Low(4–6 folds)8.2 ± 2.5 b4.3 ± 1.4 b<0.01
Medium(9–11 folds)11.0 ± 1.6 a4.6 ± 1.1 b<0.01
High(14–16 folds)11.9 ± 1.3 a6.5 ± 1.4 a<0.01
1 The viral load of source plants relative to tomato 25S rRNA. 2 The viral titer relative to B. tabaci HSP90. 3 TYLCTHV, tomato yellow leaf curl Thailand virus; ToLCTV, tomato leaf curl Taiwan virus. Different letters within the same column indicate a significant difference (Games–Howell test, p < 0.05). 4 Pairwise comparisons between TYLCTHV and ToLCTV (Mann–Whitney test or Student’s t-test).
Table 2. The effect of the viral load of source plants on viral infection in the primary salivary gland (PSG) of B. tabaci.
Table 2. The effect of the viral load of source plants on viral infection in the primary salivary gland (PSG) of B. tabaci.
Source Plant Viral Load 1Relative Viral Titer 2 in the PSG of B. tabaci (Mean ± SD)
TYLCTHV 3ToLCTV 3p-Value 4
Extra low(0.1–2 folds)1.7 ± 0.7 b1.2 ± 0.5 b0.13
Low(4–6 folds)1.8 ± 0.7 b1.4 ± 0.3 b0.28
Medium(9–11 folds)3.1 ± 0.9 a1.7 ± 0.8 b<0.01
High(14–16 folds)3.4 ± 1.1 a2.7 ± 0.9 a0.17
1 The viral load of source plants relative to tomato 25S rRNA. 2 The viral titer relative to B. tabaci HSP90. 3 Different letters within the same column indicate a significant difference (Tukey’s HSD test, p < 0.05). 4 Pairwise comparisons between TYLCTHV and ToLCTV (Mann–Whitney test or Student’s t-test).
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Huang, Y.-Y.; Li, W.-H.; Lee, K.-Y.; Tsai, W.-S.; Tsai, C.-W. Effects of Acquisition Time and Viral Load of Source Plants on Infections of Two Tomato Begomoviruses in Bemisia tabaci. Agriculture 2025, 15, 1195. https://doi.org/10.3390/agriculture15111195

AMA Style

Huang Y-Y, Li W-H, Lee K-Y, Tsai W-S, Tsai C-W. Effects of Acquisition Time and Viral Load of Source Plants on Infections of Two Tomato Begomoviruses in Bemisia tabaci. Agriculture. 2025; 15(11):1195. https://doi.org/10.3390/agriculture15111195

Chicago/Turabian Style

Huang, Ya-Yu, Wei-Hua Li, Kyeong-Yeoll Lee, Wen-Shi Tsai, and Chi-Wei Tsai. 2025. "Effects of Acquisition Time and Viral Load of Source Plants on Infections of Two Tomato Begomoviruses in Bemisia tabaci" Agriculture 15, no. 11: 1195. https://doi.org/10.3390/agriculture15111195

APA Style

Huang, Y.-Y., Li, W.-H., Lee, K.-Y., Tsai, W.-S., & Tsai, C.-W. (2025). Effects of Acquisition Time and Viral Load of Source Plants on Infections of Two Tomato Begomoviruses in Bemisia tabaci. Agriculture, 15(11), 1195. https://doi.org/10.3390/agriculture15111195

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