Comprehensive Sampling and Detection Strategies for the Field Surveillance of Tomato Brown Rugose Fruit Virus

Abstract

Tomato brown rugose fruit virus (ToBRFV) poses a significant threat to tomato production. Effective and accurate detection is critical for limiting the introduction and spread of ToBRFV. In this study, the impact of tomato planting patterns, growth stages, and cultivar variability on ToBRFV levels in tomatoes from distinct greenhouses and open fields were comprehensively analyzed. The results indicated that ToBRFV is detectable in asymptomatic tissues, regardless of artificial agroinoculation or natural infection. Additionally, higher viral levels were observed in newly emerging leaves and in fruits and sepals compared to old leaves. For tomato fruits infected with ToBRFV, the viral level in the mesocarp is higher than in the other interior parts, and no correlation was found between viral levels and the color of fruit lesions. Based on these results, it is recommended that new leaves and sepals should be given priority for testing of ToBRFV from tomato seedlings to the color turning stage, and that fruits and sepals are suggested to be collected at the full ripeness stage of tomato plants. This study underscores the importance of regular detection and optimal sampling beyond symptom observation in the surveillance of ToBRFV.

1. Introduction

Tomato brown rugose fruit virus has been identified as a new species of the genus Tobamovirus. Since its first identification in Jordan in 2015 [1], tomato brown rugose fruit virus (ToBRFV) has been reported to pose a serious threat to commercial tomato and pepper production. Similar to other tobamoviruses, ToBRFV possesses a rod-shaped particle that encapsulates a positive single-stranded RNA genome of approximately 6.4 kb. The genome of ToBRFV contains four open reading frames (ORFs). ORF1 and ORF2 are divided by a leaky stop codon that allows for readthrough, encoding non-structural proteins that make up the RNA-dependent RNA polymerase (RdRP). ORF3 and ORF4 encode a movement protein (MP) with a molecular weight of 28–31 kDa and a coat protein (CP) with a molecular weight of 17–18 kDa, respectively [2,3].

Breeding resistant varieties is one of the most effective and environmentally friendly approaches to limiting the occurrence and spread of a plant virus. Tm-2², a nucleotide-binding leucine-rich repeat class immune receptor that recognizes the MP protein of tobamoviruses [4,5], has been demonstrated to confer durable resistance to several tobamoviruses, such as tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV). However, tomato varieties that carry the Tm-2² resistance gene fail to confer resistance against ToBRFV, as the Tm-2² could not recognize the MP protein of ToBRFV [1,5,6]. Extensive efforts have been therefore conducted to screen germplasm or wild tomatoes resistant to ToBRFV [7,8]; however, a single amino acid change at position 82 of the MP of ToBRFV breaks virus-specific resistance in new resistant tomato cultivars [9]. This resistant breaking ability has rendered all commercial tomato cultivars vulnerable to ToBRFV infection, causing the virus to spread rapidly to at least 37 countries across Europe, the Americas, and Asia [10].

ToBRFV is highly stable and infectious, and the virus can survive for at least seven days on surfaces commonly found in glasshouses, and on aluminum and stainless steel surfaces for at least six months [11]. Prevalence of ToBRFV was also identified in wastewater influent samples [12,13]. ToBRFV can be transmitted in almost every step of tomato production and trade via ToBRFV-contaminated seeds and fruits, or mechanical contact through working hands, tools, and infected plants, soil, and water [14,15,16]. Infection of ToBRFV often causes mosaic, yellowing, mottling, or narrowing on tomato leaves. Affected fruits may display uneven ripening, having yellow-brown spots or blister surfaces. Asymptomatic leaves were also observed in some varieties. Prophylactic biosecurity measures, such as the testing of seeds and application of hygiene best practice measures, or eradication, are crucial to safeguard tomato and pepper crops against the entry, establishment, and spread of ToBRFV. Considering its high risk to tomato and pepper production, ToBRFV has been listed as a quarantine or regulated pest by the European and Mediterranean Plant Protection Organization and several other countries [17]. In addition to the regular inspection of ToBRFV at ports of entry for quarantine purposes, it is important to conduct surveillance for ToBRFV in greenhouses and open fields. Visual monitoring of ToBRFV requires solid knowledge of the symptoms induced by ToBRFV. The symptoms vary significantly among different tomato varieties, and similar symptoms may be caused by ToMV, tomato mottle mosaic virus, and other viruses. Accurate and timely detection of ToBRFV at a low level or during latent infection is vital, as undetected infestation can lead to the introduction and further spread of ToBRFV.

Efficient sampling of ToBRFV-infected samples in combination with a low limit detection of the collected samples is fundamental for the early and accurate detection of ToBRFV. Great efforts have been made to develop diagnostic methods for ToBRFV, including: reverse transcription PCR (RT-PCR), real-time RT-PCR, loop-mediated isothermal amplification, cluster regularly interspaced short palindromic repeats, and recombinase-aided amplification, which are designed to detect viral RNA; and enzyme-linked immunosorbent assay (ELISA), dot-ELISA, and colloidal gold immunochromatographic strip (CGICS), which are used to detect the capsid protein of ToBRFV [18,19,20,21,22,23]. These methods are useful in the detection of ToBRFV for different demands. According to International Standard for Phytosanitary Measures (ISPM) No. 6 [24], the surveillance protocol should include a description of how samples are to be taken, collected, handled, and prepared. However, there is limited information on sampling strategies for the surveillance of ToBRFV. Based on the artificial inoculation experiments in hydroponic protected cropping systems, the age of the plant at the time of infection and the different plant parts sampled affect the detection of ToBRFV [25]. Nevertheless, the relevance of different types of tomatoes and growing environments have not been evaluated.

To provide more comprehensive sampling and detection strategies for ToBRFV involving the tomato growth period, planting environment, and type, different tomato samples infected with ToBRFV were collected in this study. The viral levels in different parts of tomato plants were analyzed by qRT-PCR, dot-ELISA, and ELISA. The results indicated that the viral levels in the new emerging leaves were higher at the seedling stage of tomato plants. New leaves and sepals contain higher viral levels at the color turning stage, and fruits and sepals accumulate more abundant viral levels at the full ripeness stage of tomato plants. Additionally, the viral level in the mesocarp is higher compared to the other interior parts. This study provides a better guideline for the surveillance of ToBRFV.

2. Materials and Methods

2.1. Plant Materials and Virus Inoculation

ToBRFV-infected standard tomatoes and cherry tomatoes were collected from Shandong, Ningxia, Yunnan of China during 2023–2024. Solanum lycopersicum (cv. Moneymaker at four-leaf stage, and cv. Aishenghong at three-leaf stage) were used for virus inoculation and grown in an insect-free growth room at 25 °C under a 16 h light/8 h dark cycle. Agroinoculation of tomato plants with the infectious cDNA clone of ToBRFV (pCB301-ToBRFV) was performed as previously described [26]. Agrobacterium cells were resuspended with the infiltration buffer (10 mM MgCl₂, 10 mM MES (pH 5.8), 100 μM acetosyringone) to an OD₆₀₀ of 1.2 prior to agroinoculation. Tomato plants mock-inoculated with the agrobacterium cells harboring pCB301 served as control plants.

2.2. Collection of Tomato Samples

For tomato plants (cv. Moneymaker, and cv. Aishenghong) inoculated with the infectious clone of ToBRFV, a terminal leaflet of the newly emerging branch was collected. For tomato plants collected in the greenhouses or open fields, sampling was conducted as illustrated in Section 3. Before comprehensive sampling, tomato plants suspected of ToBRFV infection were screened onsite using ToBRFV-specific colloidal gold immunochromatographic strip (CGICS), as previously developed [27]. At least three ToBRFV-infected plants were used for each experiment. To prevent cross-contamination of viruses, scissors, blades, tweezers, and other tools used for each sample were sterilized with an autoclave in advance.

2.3. RT-PCR and Quantitative RT-PCR-Based Molecular Detection of Viruses

Total RNA was extracted from tomato plants using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Samples of 0.1 g of tomato leaves, petioles, sepals, or fruits were used for RNA extraction. First-strand complementary DNA (cDNA) was synthesized from 1 μg of total RNA using Prime-Script™ II cDNA Synthesis Kit (TaKaRa, Kusatsu, Shiga, Japan). To detect the presence of ToBRFV, specific primers ToBRFV-842-F(5′-GAAGTCCCGATGTCTGTAAGG-3′) and ToBRFV-842-R (5′-GTGCCTACGGATGTGTATGA-3′) [26], which are expected to amplify a fragment of approximately 840 bp, were used for RT-PCR. To confirm the specific infection of ToBRFV, amplicons with the expected size were recovered and subjected to DNA sequencing. To exclude the coinfection of tomato spotted wilt tospovirus (TSWV) with ToBRFV, specific primers TSWV-F (5′-ATCAGTCGAAATGGTCGGCA-3′) and TSWV-R (5′-ATACGAATCTGAATCGGCGACG-3′), which are expected to amplify fragments of approximately 280 bp, were used for RT-PCR. Tomato leaves known to be infected with ToBRFV or TSWV served as the positive controls, and healthy tomato leaves were used as the negative control.

For quantitative analysis of ToBRFV, ToBRFV-specific primers, qToBRFV-F (5′-GAAGCAGGAGCAAGTGACCA-3′) and qToBRFV-R (5′-AGGACCGAAGATTGCGTTGA-3′), and internal reference primers, qActin-F (5′-AAAGACCAGCTCATCTGTTGAGAAG-3′), and qActin-R (5′-GTGGTTTCATGAATACCAGCAGC-3′), were used, and quantitative RT-PCR was conducted with a Light Cycler 96 (Roche, Basel, Switzerland). The TB Green^® Fast qPCR Mix (TaKaRa) was used, and the program of qRT-PCR was set as follows: pre denaturation: 30 s at 94 °C, 20 °C/s; PCR reaction: 40 cycles of 5 s at 94 °C, 20 °C/s, 10 s at 60 °C, 20 °C/s; melting curve analysis: 0 s at 95 °C, 20 °C/s, 15 s at 60 °C, 20 °C/s, 0 s at 95 °C, 0.1 °C/s. The relative abundance of ToBRFV was calculated using the Rel Quant program implemented in the Roche Light Cycler 96 software. cDNA synthesized from tomato leaves known to be infected with ToBRFV served as the positive control, while those extracted from healthy tomato leaves were used as the negative control.

2.4. CGICS, Dot-ELISA, and DAS-ELISA-Based Serological Detection of ToBRFV

CGICS previously developed [27] for fast onsite detection of ToBRFV was used for the primary screen of ToBRFV infection. Approximately 0.1 g of tomato samples were collected and placed in a sample mesh bag. After adding approximately 40 drops of extraction buffer, the sample was ground by rubbing the outside of the bag with a pen. Four drops of the resulting crude extracts were added to the sample port of the CGICS, and the results was observed within 5 min.

Dot-ELISA and DAS-ELISA were conducted to detect ToBRFV as previously described [27]. In brief, 0.1 g of tomato samples were ground with 1 mL of 0.01 M phosphate-buffered saline (PBS) and centrifuged at 10,000 rpm for 3 min. For dot-ELISA, 3 μL of the supernatant were mobilized onto a nitrocellulose membrane (Merck, Rahway, NJ, USA), and the membrane was blocked with the PBST buffer containing 5% skimmed milk powder for 30 min. Following this, the membrane was incubated with the anti-ToBRFV monoclonal antibody (MAb) 17B1 [27] and subsequent alkaline phosphatase-conjugated goat anti-mouse IgG (EASYBIO, Beijing, China) for 30 min, respectively. The signal was detected using the single-component nitro-blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate solution (Lablead, Beijing, China). For DAS-ELISA, wells of the 96-well microplate (Thermo Scientific, Waltham, MA, USA) were incubated with 100 μL of the supernatant overnight. The incubation with primary and secondary antibody, rinsing, and signal detection were conducted essentially as described [27]. Tomato foliar samples known to be infected with ToBRFV and healthy tomato leaves were used as the positive and negative controls, respectively.

3. Results

3.1. ToBRFV Requires an Incubation Period for Symptom Appearance

To determine whether ToBRFV requires an incubation period before inducing symptoms, two cultivars of tomatoes, Moneymaker and Aishenghong, were agroinoculated with an infectious full-length cDNA clone of ToBRFV (pCB301-ToBRFV) and monitored for symptom development. During the initial week post inoculation, systemic leaves from the newly emerging branch of tomato plants were collected daily. Thereafter, plants were sampled every two days until symptoms appeared. Plant samples were tested for the presence of ToBRFV using RT-PCR, dot-ELISA, and CGICS developed for ToBRFV. Records of symptom expression revealed that characteristic symptoms, such as leaf narrowing and mosaic, began to develop from 12 days post inoculation (dpi) (Figure 1a). Interestingly, ToBRFV was detectable in the upper non-inoculated leaves of both cultivars as early as 4 dpi according to the RT-PCR and dot-ELISA analyses (Figure 1b,c). Moreover, ToBRFV was readily detected in the upper non-inoculated leaves at 5 dpi using CGICS (Figure 1d).

3.2. Differential Distribution of ToBRFV in Naturally Infected Tomato Plants from Greenhouses

To evaluate which parts of tomato plants are optimal for ToBRFV infection analysis, greenhouse tomato plants potentially infected with the virus were initially screened using CGICS against ToBRFV. Subsequently, different parts of ToBRFV-positive tomato plants were collected from two distinct greenhouses. The first set of samples (Set 1) were collected from a greenhouse in Shandong Province, where the tomato plants had been pruned, leaving only the growing tip. The fruits from these plants were ripe and exhibited typical symptoms of ToBRFV infection (Figure S1a). In contrast, the second and third sets of samples (Set 2 and Set 3) were harvested from two separate greenhouses in Ningxia, where the plants had not been pruned. The fruits from Set 2 were at the color turning stage and did not show the typical symptoms of ToBRFV infection (Figure S1c). Set 3 comprised asymptomatic cherry tomato plants, the fruits of which were ripe (Figure S1e). For each set, samples were collected from three individual tomato plants. To further confirm the presence of ToBRFV in the collected samples, RT-PCR was conducted, yielding the expected amplicons of approximately 840 base pairs (Figure S1b,d,f). Subsequent sequencing of these amplicons validated the infection of ToBRFV. RT-PCR detection of TSWV revealed no coinfection of TSWV with ToBRFV in these samples (Figure S1b,d,f). The levels of ToBRFV in the leaves, sepals, and fruits of the tomato plants were first estimated using DAS-ELISA (Figure 2), dot-ELISA (Figure S3a), and qRT-PCR (Figure S3b). As illustrated in Figure S2a, the mesocarp was selected as the primary sampling site for fruit analysis. The results showed that the viral levels in the fruits and sepals of Set 1 samples were significantly higher compared to the old leaves (Figure 2a,b). Furthermore, higher viral accumulation was detected in the bottom older fruits and sepals compared to the top newer fruits (Figure 2a,b). In the case of Set 2 samples, a similar trend was observed, with the viral levels in the new leaves, fruits, and sepals being higher than in the old leaves (Figure 2c,d), suggesting that the viral levels in the old leaves were consistently the lowest. Furthermore, except for Fruit 1 and Sepal 1, which showed no significant difference in viral levels, the viral levels in the remaining sepals of higher clusters of fruits were higher than in the fruits themselves (Figure 2c,d).

In the case of asymptomatic cherry tomatoes from Set 3, petioles and pedicels were also individually collected for virus detection, as shown in Figure S2b. Higher levels of ToBRFV were present in the bottom fruits and sepals compared to the top fruits and sepals (Figure 3a,b). When comparing the viral levels in different parts of the axil, a higher viral abundance was detected in the petioles and pedicels of each corresponding branch of the collected tomato samples (Figure 3a,b). These results suggested a greater accumulation of the virus in new leaves and fruits and sepals of the bottom clusters.

3.3. Differential Distribution of ToBRFV in Tomato Plants from an Open Field

Tomato samples from Set 4, suspected of being infected with ToBRFV, were collected from an open field in Yunnan. These plants had not been pruned and were at the color turning stage. Although the leaves were symptomless, mild symptoms were observed in the fruits (Figure S1g). Similarly, RT-PCR was conducted to detect ToBRFV and TSWV, and the results showed that these tomato plants were only infected with ToBRFV (Figure S1h). The relative abundance of ToBRFV in various parts of the plants, including leaves, petioles, pedicels, sepals, and fruits, was analyzed, as shown in Figure S2. Consistent with the results from greenhouse tomatoes, the viral levels in new leaves were higher than in old leaves, and the virus titer in the petioles of the bottom cluster of fruits was also higher (Figure 4a,b). In addition, the viral levels in Pedicel 2 were lower than that in Pedicel 3, which was contrary to the viral levels in the fruits (Figure 4a,b). In addition, in contrast to the greenhouse tomatoes, the viral levels in the bottom fruits (Fruit 1) of the plants were the lowest (Figure 4a,b). When comparing the plants at the color turning stage, specifically Set 2 and Set 4, the results showed that viral levels in the sepals were significantly higher than those in the fruits (Figure 2d and Figure 4b). This suggested that the viral levels in the sepals are consistently higher than those in the fruits during the color turning stage.

3.4. Viral Level in Different Parts of Symptomatic Tomato Fruits

To assess the ToBRFV level in fruits, red and green fruits infected with ToBRFV were collected from Yunnan. Each fruit was dissected to obtain three distinct parts, the mesocarp, septum, and placenta (Figure 5a,c). These samples were analyzed by both DAS-ELISA and qRT-PCR. The results indicated that the viral level in the mesocarp was higher compared to the other interior parts in both red and green fruits (Figure 5b,d and Figure S4a,b).

To evaluate whether the formation of color lesions in fruits is associated with viral levels, we examined ToBRFV-infected tomato fruits with marbling symptoms from Yunnan. The mesocarp tissue corresponding to different colored lesions, specifically brown, red, and yellow, was analyzed for ToBRFV levels (Figure 5e). Both DAS-ELISA and qRT-PCR results showed no significant differences in ToBRFV levels among the various colored lesions (Figure 5f and Figure S4c). This finding suggests that the presence of color lesions does not correlate with varying levels of viral infection.

4. Discussion

ToBRFV is recognized as a highly destructive virus for tomato crops, posing a serious threat to tomato production due to the lack of effective resistant varieties, its high stability, and ease of transmission [10]. Therefore, it is essential to monitor ToBRFV so that prompt follow-up measures can be taken to mitigate economic losses in the case of endemic. This study underscores the importance of optimized sampling strategies and accurate detection methods in the effective surveillance of ToBRFV.

Our study, through agrobacterium-mediated inoculation of tomato plants with the infectious cDNA clone of ToBRFV, demonstrated that the virus was detectable in asymptomatic leaves of upper non-inoculated tomato plants belonging to two different cultivars. This suggests that naturally infected greenhouse or field tomatoes showing no obvious symptoms may still harbor ToBRFV, highlighting the necessity for regular and systemic monitoring beyond symptom-based assessments. The detection of ToBRFV in asymptomatic samples (Set 2) from Ningxia further emphasizes that symptom phenotypes should not be the sole criterion for determining whether a plant is infected with the virus or not. These results suggest a critical need for regular monitoring and detection of tomato plants for viral infections throughout the growth cycle.

To identify optimal tissues for ToBRFV analysis, we provided a comprehensive analysis of the relative viral abundance in various tissues collected from tomato plants naturally infected with ToBRFV. Our data revealed a consistent pattern across different growing environments and plant types: the viral levels in new leaves were consistently higher than in old leaves. This aligns with a previous study by Samuel [28], which demonstrated that viruses tend to move to developing leaves, potentially leaving older leaves virus-free, especially in late-stage infections. Our findings also agree with those of Skelton et al. [25], who identified young leaves as optimal sampling sites due to higher viral titers. Additionally, we observed higher viral levels in sepals and pedicels compared to fruits during the color turning stage. This phenomenon is also similar to Samuel’s findings on the influence of developing fruit trusses on the movement of virus. Nevertheless, in tomatoes that are all at the color turning stage, the viral levels in the bottom fruits of greenhouse tomatoes were higher than in other fruits. This finding differs from the lower viral levels observed in the bottom fruits of tomatoes collected from the open fields. It is speculated that this difference might be caused by coinfection of TSWV with ToBRFV, a common phenomenon in the natural conditions. However, no TSWV was detected in any of the samples, ruling out the possibility of coinfection of TSWV with ToBRFV in the samples. It would be interesting to detect whether other viruses are prevalent in the samples, and if so, whether there is any interaction between the different viruses. At full ripeness, the viral levels in sepals of both standard tomatoes and cherry tomatoes were found to be almost equivalent to those in fruits, indicating that the virus distribution is less influenced by the growth environment, but rather is greatly affected by the development stages of plants. These results reinforce the concept that ToBRFV accumulates in the stem, supporting the idea that long-distance transport of the virus is phloem-associated, as discussed by Hipper et al. [29]. In light of these findings, we recommend using both fruits and sepals as primary sampling subjects for ToBRFV detection, a strategy that aligns with the recommendations made by Skelton et al. This approach ensures a more accurate and timely diagnosis, which is crucial for implementing effective disease management strategies and safeguarding tomato production from the devastating effects of ToBRFV.

Avni et al. [30] employed the FISH technique to trace the viral genomic RNA and understand the infection processes of ToBRFV in tomato reproductive tissues, revealing that the virus can infect nearly all reproductive organs, including petals, ovaries, stamen, style, stigma, anther, and pollen grains. Our findings on the detection of viral levels in different fruit parts showed a similar pattern, confirming the widespread presence of ToBRFV across various fruit tissues. Specifically, we observed a higher viral level in the mesocarp, which develops from the ovary wall, a tissue identified by Avni, Gelbart, Sufrin-Ringwald, Zemach, Belausov, Kamenetsky-Goldstein, and Lapidot [30] as susceptible to ToBRFV infection. This may explain the higher viral level in the mesocarp compared to other fruit parts. Furthermore, our analysis of the viral level in relation to different colored fruit lesions revealed no direct correlation between the two. The color changes of tomato fruits during ripening are influenced by a complex interplay of factors, such as hormones, flavonoids, and carotenoids [31]. These findings suggest that the interaction between ToBRFV and its host involves complicated mechanisms. In addition, the presence of ToBRFV has been confirmed in the soil of ToBRFV-infected tomato plants, the titer of ToBRFV in soil serving as a monitoring indicator for virus surveillance in the field. However, Giesbers et al. [32] could not successfully inoculate the ToBRFV-positive soil into tobacco. Whether the virus in the soil is infectious remains to be determined. There is no standard process for the detection of ToBRFV in soil; therefore, the detection of ToBRFV titer in soil is the future research direction.

5. Conclusions

Overall, our results emphasize the importance of virus detection and efficient sampling from specific plant parts for accurate assessment of ToBRFV infection. The significantly high viral levels in the newly emerging leaves, as well as in fruits and sepals, suggest that these tissues should be recommended for sampling. For tomato plants at the color turning stage, new leaves and sepals are recommended for sampling and testing. Similarly, at the full ripeness stage, fruit and sepals should be prioritized for viral detection.