Abstract
During the growing season 2018–2019, a total of 199 tomato and 21 pepper asymptomatic and symptomatic samples were collected from different major regions of Saudi Arabia. Using the general Norgen Kit and universal Pospi1-RE/Pospi1-FW primers for Pospiviroid RT-PCR detection, only seven tomato samples and three pepper samples were positive, which amplified the PCR products with the expected sizes of ~364 bp and /~350 bp respectively, confirming the presence of at least one of the nine Pospiviroid species. The PCR amplified products were sent for sequencing, and the results confirmed the presence of tomato apical stunt viroid (TASVd) and pepper chat fruit viroid (PCFVd). However, further confirmation was conducted using specific primers for TASVd and PCFVd that amplify the complete genome. One positive TASVd Saudi isolate was selected to evaluate the severity and symptom responses to mechanical inoculation of four tomato cultivars that are often grown in greenhouses. According to the data obtained, each cultivar exhibited a significant change in each metric. The studied cultivars’ plant heights did not differ significantly from one another. The greatest decrease in vegetative dry weight (39.3%) was observed in the Newton cultivar; with just a 6% decrease in comparison to the healthy controls, the Alindi cultivar was the least impacted. With a 90.2% decrease in fruit output when compared to its untreated control, the Newton cultivar was the most impacted; meanwhile, with a 49.1% decrease in fruit output, the Irit cultivar was the least impacted. The infection of viroids and their economic impact on commercial tomato cultivars have been discussed.
Keywords: solanaceous, viroids, TASVd, RT-CR, sequence, cultivar response
1. Introduction
The family Solanaceae includes several economically significant crops, like tomatoes (Solanum lycopersicum), pepper (Capsicum spp.), potatoes (S. tuberosum) and eggplant (S. melongena), as well as some ornamental plants, such as many species of petunia. It also includes some wild weeds (S. nigrum), which grow within or adjacent to cultivated crops and may represent primary reservoirs and sources of infection for viruses and viroids [1,2,3].
In Saudi Arabia, the tomato is a major vegetable crop with significant economic importance, achieving a high self-sufficiency rate of 76% in 2023. It is cultivated in various regions, using both traditional open-field methods and advanced greenhouse technologies to meet year-round demand. The total area under tomato production in Saudi Arabia in 2023 was approximately 15,208 hectares. This area is designated for both open-field and protected agriculture (greenhouse) cultivation. The total quantity of tomatoes produced was over 691,875 tons. According to data from the Saudi Ministry of Environment, Water, and Agriculture in 2023, greenhouse tomato planting accounts for almost 59% of Saudi Arabia’s total tomato fruit production, although it is far smaller than open fields. This illustrates the significance of greenhouse production in contrast to tomato cultivation in open fields. Peppers are also an important crop, but the specific 76% figure applies to tomatoes (The General Authority for Statistics (GASTAT) Publishes 2023 Agricultural Statistics Results).
Unfortunately, greenhouse-grown solanaceous crops are more highly susceptible to viroid infections than those in an open field, which may lead to an epidemic for tomato planting due to important factors like mechanical transmission and infected seeds, and these infections can lead to significant economic losses and potential epidemics [4,5]. So far, no report or study has been conducted in Saudi Arabia about viroid infection in any cultivated crop, except for one report, which described the symptoms of potato spindle tuber viroid (PSTVd) on potato based on symptoms only [6]. This highlights the significance of this study, since it is the first study to provide insight into this emerging area of study in the Kingdom of Saudi Arabia and other Arabian Peninsula countries.
The smallest infectious agent ever known, the viroid, is one of the big threats to these crops, due to its diversity, stability, and the difficulty that is usually experienced in its identification, especially in greenhouse farming [7,8]. Viroids produce a wide range of symptoms, including stunting, leaf/stem chlorosis, epinasty, necrosis and foliar/fruit deformation, dwarfing, vein discoloration, cracking, and delayed growth, depending on host plant cultivars and viroid species [9,10,11].
There are currently 40 viroid species that are members of the Pospiviroidae family, and 5 viroid species that belong to the Avsunviroidae family have been identified, as documented on the ICTV website [12]. Within the family Pospiviroidae, eight species from the genus Pospiviroid have been isolated from naturally infected tomato plants [13,14]. Two viroids have been reported from naturally infected pepper crops, pepper chat fruit viroid (PCFVd) and PSTVd [13,14,15,16,17]. In addition, the citrus exocortis viroid (CEVd) was identified in naturally infected plants of eggplant [18]. Given the lack of data on Solanaceae viroid detection and epidemiology in Saudi Arabia, the primary aim of this research was to identify the presence of the important Pospiviroid infecting tomato and pepper crops. Furthermore, investigations of cultivar response analysis were undertaken against tomato apical stunt viroid (TASVd) in Saudi Arabia.
2. Materials and Methods
2.1. Field Sample Collection
During the growing survey (2018–2019), a total of 199 tomato samples from different varieties, including Newton (Syngenta), Beisuzhok (Giverech), Alindi (Enza Zaden) and Irit (Rigk Zwan), were collected from symptomatic and asymptomatic plants growing in the five greenhouses of each of the following regions, Riyadh, Al-Qassim, Eastern province, and Tabouk. Moreover, 21 pepper samples from Habhar Shaqra and Rida growing in two greenhouses in Riyadh and Al-Qassim were collected and contained both symptomatic and asymptomatic plants. The symptomatic samples showed viroid-like symptoms, such as stunting, yellowing, leaf curling, witches’ broom and apical stunting, and fruits were smaller and ripped earlier (Figure 1 and Figure 2).
Figure 1.
Naturally infected with tomato apical stunt viroid (TASVd) on tomato leaves expiating brightening (A), leaf curling (B), necrosis (C), purple leaf symptoms (D) and tomato fruits showing a pale red color, early maturity and reduced fruit size (E).
Figure 2.
Naturally infected pepper plant in the greenhouse, infected with TASVd, and showing different symptoms, including sever stunting, leaf curling, mottling and small fruits (A). Several symptoms on pepper plant infected with PCFVd, which include stunting, leaf curling, mottling and small fruits (B).
2.2. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
The total RNA was isolated from the collected samples using an Isolate II RNA Plant Kit, BIO-52077, (Bioline Ltd., London, UK) following the manufacturer’s protocol. All the tomato and pepper plant samples were tested by an RT-PCR assay using general Pospiviroid RT-PCR Detection Kit Product #53700 (Norgen Biotek Corp., Thorold, ON, Canada) following the manufacturer’s instruction to detect all the 9 different viroids that belong to the genus Pospiviroid. The general primers Pospi1-RE/Pospi1-FW [19,20] were used for confirming the occurrence of Pospiviroid, which amplifies the complete genome (~360-bp) product.
Samples that reacted positively with the initial RT-PCR were further assayed for another run of RT-PCR with each of the following specific primers, TASVd (Viroid, Vir+/Vir-) that amplifies a full genome at 360 bp [21], and PCFVd (PCF-seq-F/PCF-seq-R) that amplifies the complete genome at ~350-bp [22,23].
For the RT-PCR test, the BioScript One-Step RT-PCR kit (Bioline Ltd., UK) was used. For each sample, 2 µL of the template RNA was placed in a purified PCR test tube and mixed with 2 µL from each pair of the primers, 2 µL of the enzyme mix, and 25 µL of the PCR buffer. The mixture was then completed to 50 µL with RNAase free distilled water. All test tubes were placed in the Advanced-Primus 96 thermal cycling machine (PEQLAB Ltd., Fareham, UK), which was pre-set at the following thermal cycle protocol: 30 min at 50 °C for the reverse transcriptase, 2 min at 95 °C for initial denaturation, 30 s at 94 °C for denaturation, 30 s at 62 °C for annealing, and 45 s at 72 °C for elongation (steps 3–5 were repeated for 35 cycles, with 10 min at 72 °C for final elongation and hold at 4 °C). Then, 5 µL of the resulting product was loaded on a micro-well of 2% agarose gel prepared in 1x Tris Borate EDTA (TBE) buffer with ethidium bromide. The gel was placed under electrophoresis and allowed to run for 45 min at 120 V and room temp. The PCR Sizer 100 bp DNA Ladder (Norgen Biotek Corp., Thorold, ON, Canada) and 1KB GeneDirex DNA Marker (GeneDireX, Inc., Taoyuan City, Taiwan) were used. The amplified results of cDNA were then visualized, and photos were taken in a gel documentation system.
2.3. Sequence and Phylogenetic Analysis
Ten samples that reacted positively with the RT-PCR described earlier using universal primers were purified using the QIAquick Gel Extraction Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions and directly sequenced in both directions by Sanger sequencing and sent to the “Advanced Genetic Technologies Center” (AGTC), College of Agriculture Sciences, University of Kentucky, USA for complete genome sequencing.
The obtained sequence results were blasted using the National Center for Biotechnology Information (NCBI) after cleaning the noisiness of the obtained data using the BIOEDIT program. Three of our TASVd and one of the PCFVd Saudi isolates were compared with 26 and 21 other worldwide isolates from GenBank for each viroid species. For a better understanding of the analysis results, a phylogenetic tree was constructed to compare the phylogenetic relationship between viroid isolates. Sequences aligned with ClustalW were used to construct a phylogenetic tree using MEGA-X with the Maximum-Likelihood approach and with 1000 bootstrap replications, as implemented by the MEGA 7.0. Sequence Demarcation Tool (SDTv1.3), and MegAlign, as implemented by Lasergene, which were used for pairwise sequence comparisons [24]. The Saudi isolates’ nucleotide identity table was created using DNAStar (19.0) software and compared with other isolates that were reported in the GenBank database.
2.4. Cultivar Responses
A greenhouse experiment was conducted to evaluate the responses of selected tomato cultivars to the mechanical inoculation with the Saudi TASVd isolate. Four cultivars were selected, namely, Newton (Syngenta), Beisuzhok (Giverech), Alindi (Enza Zaden) and Irit (Rigk Zwan). The first two were the most grown cultivars in the Alkharj area (one of the most important tomato greenhouses growing locations), and the other two were recently introduced to the Saudi market and were claimed to be Begomovirus-tolerant cultivars. Twenty plants from each cultivar were grown in a controlled greenhouse (at 25–27 °C) for one month before mechanical inoculation. Each cultivar was split into two rows, with 10 pots of each. One row was inoculated treatment, and the other row was inoculated with the buffer as a control.
For mechanical inoculation, 1 g of fresh leaf tissue infected with TASVd isolate was ground in a mortar and pestle with 4 mL of potassium phosphate buffer pH 7.0. The crude sap was rubbed against the pre-dusted leaves of the test plants with ~10 at the true leaf stage to allow the plants to grow for fruit production with Carborundum powder (400 mish), and control plants were inoculated with the potassium buffer only. All plants were kept for 4 months until the most of infected plants died.
For evaluation, all fruits (both mature and immature) were picked at one time at the end of the experiment from each plant and weighed, the plant’s height was recorded and the weight of dry matter of the whole vegetative growth was taken. For vegetative dry weight evaluation, each plant was cut from the vegetative growth above soil level, chopped into smaller pieces, placed in a paper bag and kept in a greenhouse for two weeks until complete dryness. Plants were weighed after two weeks of dryness. Data was taken from each inoculated plant as well as the controlled plants for comparison. All plants that showed symptoms after inoculation were assayed with RT-PCR against TASVd infection as described earlier, to confirm the viroid infection. All data were statistically analyzed using one-way analysis of variance, and differences between control and inoculated plants were measured for each of the indicated parameters with the Least Significant Difference test (LSD) at the 5% significance level; for each parameter, the analysis was performed after the percentages of reduction were transformed to arcsine [25]. The LSD test also was used to separate the means in percent reductions among the different cultivars for the previously mentioned parameters following their inoculation with TASVd isolate.
3. Results
3.1. Field Observation
Collected samples showed a range of symptoms that vary according to plant species, cultivar, and growth stage. Leaf symptoms of tomato plants that were positively infected with TASVd (according to RT-PCR results later) vary from mild mottling to severe necrosis as well as leaf curling, discoloration, shortened apical internodes, deformation, in some cases brightening and a leaf color turned purple (Figure 1). Leaf samples of pepper plants that were infected with TASVd (according to the RT-PCR assay later) were stunted plants, and leaves showed mottling and leaf curling. Similar symptoms (Figure 2) were associated with plants that reacted positively with PCFVd in the RT-PCR assay.
3.2. RT-PCR
Out of the tested samples collected from tomato and pepper greenhouses, which were assayed with RT-PCR using the universal Pospiviroid primers, only six tomato samples and one pepper sample showed clear PCR amplification bands at the size of ~365 bp. Meanwhile, three pepper samples showed a PCR amplification band at the size equivalent to ~350 bp, which were collected from the Riyadh region (Al-Kharj area) only (Figure 3). Since the universal primers were used in this test, that confirms the presence of at least one of the nine Pospiviroid species that can be detected and amplified with these primers. On the other hand, having one sample with a band at a smaller size (approx. 350 bp) confirms the presence of one different Pospiviroid species compared to the other samples. The same bands of ~365 bp and ~350 bp were observed for these samples after the RT-PCR assay with the species-specific primers of TASVd and PCFVd respectively (Figure 4).
Figure 3.
Agarose gel (2%) electrophoresis showing RT-PCR amplified products of four samples (lanes 1, 2, 3, 8, 9, 10, 11), amplified from tomato with the size of approx. 364 bp and (lanes 4, 5, 6) amplified from pepper plants with the size of approx. 350 bp using a pair of universal primers, Pospi1-RE and Pospi1-FW. Lane 4 negative control. Lane M represents the PCR Sizer 100 bp DNA Ladder (Norgen Biotek Corp).
Figure 4.
Agarose gel (2%) electrophoresis showing RT-PCR amplified products of four samples (lanes 2, 3, 4, 5, 6, 7, 8), amplified from tomato with the size of approx. 364 bp using specific primer for TASVd and (lanes 10, 11, 12) amplified from pepper plants with the size of approx. 350 bp using a pair of specific primers for PCFVd. Lane 1 and lane 9 as negative control. Lane M represents the GeneDirex (1KB) DNA Marker (GeneDireX, Inc.).
3.3. Sequencing and Phylogenetic Analysis
For TASVd, out of the seven RT-PCR products, that were sent for sequencing, two TASVd isolates from tomato and one from pepper had convenient sequences when these sequences were blasted in the NCBI website and showed high similarity to specific viroid species registered in GenBank. Three selected sequences isolates were then submitted to GenBank and designated as SA2 and SA4, which were identical to TASVd and were collected from tomato plants, and SA5, which was also identical to TASVd and was collected from a naturally infected pepper plant; they had the following accession numbers: MK286920, MK286921 and MK286922 respectively.
Furthermore, the obtained results showed that the nucleotide sequence of the Saudi isolate that was identical to PCFVd was collected from a pepper plant and is highly similar to all PCFVd isolates that were submitted to GenBank. This isolate was designated as SA7 and submitted to GenBank under the accession no. MK286923.
When comparing the Saudi isolates of TASVd with each other, isolate SA5 (that was collected from a pepper plant) had the highest similarity with the SA2 and SA4 isolates (that was collected from a tomato plant) grown in the same greenhouse (98.3%). The high similarity between the two isolates, SA2 and SA5, which were collected from different plant species (tomato and pepper), strengthened the probability that the initial source of infection came from tomato seeds and was mechanically transmitted to pepper plants during the growing season, as discussed earlier.
The TASVd isolates from Saudi Arabia had the highest similarity (93–95.7%) with the isolates from Ghana (MG132056 and MG132057) and Senegal (EF051631 and EF551346) and the lowest similarity (91.3–92.7%) with isolate GU911351 from the Netherlands and isolate KX579067 from Taiwan. The genetic relationship and homology tree for all Saudi isolates of TASVd are shown in Figure 5 and Figure 6 and Table 1.
Figure 5.
Homology tree based on multiple sequence alignment between three isolates of TASVd from Saudi Arabia and produced with 21 different TASVd isolates of the same species from different countries obtained from GenBank. Included is a figure showing how the colors and pairwise identities correspond.
Figure 6.
SDT matrix of pairwise identity scores produced with 21 different TASVd isolates of the same species from different countries obtained from GenBank. Included is a figure showing how the colors and pairwise identities correspond.
Table 1.
Comparison of percentages of nucleotide sequences between TASVd Saudi Arabian isolates and isolates originating in different countries.
| Accession No. | Host | Isolate | Country | MK286920 | MK286921 | MK286922 |
|---|---|---|---|---|---|---|
| MK286920 | Tomato | Al-Kharj-SA2 | Saudi Arabia | 100 | 98.3 | 98.3 |
| MK286921 | Tomato | Al-Kharj-SA4 | Saudi Arabia | 98.3 | 100 | 98.3 |
| MK286922 | Pepper | Al-Kharj-SA5 | Saudi Arabia | 98.3 | 98.3 | 100 |
| AM777161 | Solanum jasminoides | Sj1 | Germany | 93.8 | 93.3 | 94.1 |
| AY062121 | Tomato | - | Palistine | 93.5 | 93 | 93.5 |
| DQ144506 | Tomato | - | Tunisia | 93.3 | 93 | 93.8 |
| EF051631 | Tomato | - | Senegal | 94.7 | 95.2 | 95.2 |
| EF192395 | Cestrum sp | 3123751 | Netherlands | 91 | 92.1 | 92.1 |
| EF551346 | Tomato | - | Senegal | 94.1 | 94.1 | 94.7 |
| FN994891 | Tomato | Brug1 | Belgium | 93.8 | 93.3 | 94.1 |
| GU911351 | Streptosolen jamesonii | 4730772 | Netherlands | 92.4 | 92.1 | 92.7 |
| HG739071 | Solanum jasminoides | 11/0197/VI.2 | Belgium | 93.8 | 93.3 | 94.1 |
| HG739072 | Solanum jasminoides | 11/0238/VI.1 | Belgium | 93 | 92.4 | 93.3 |
| HG916812 | Tomato | - | Italy | 93.5 | 93 | 93.8 |
| HQ667140 | Solanum jasminoides | sj4 | Italy | 93.3 | 92.7 | 93.5 |
| HQ667141 | Solanum jasminoides | sj10 | Italy | 93.3 | 92.7 | 93.3 |
| JN543957 | Tomato | Sj 403-Slo | Slovenia | 93.8 | 93.3 | 94.1 |
| JN872144 | Solanum jasminoides | Sj 464-Slo | Slovenia | 93.8 | 93.3 | 94.1 |
| JQ904911 | Solanum jasminoides | Sj 499-Slo | Slovenia | 93.8 | 93.3 | 94.1 |
| KX579067 | Pepper | 5458774 | Taiwan | 91.6 | 91.3 | 91.9 |
| KY810784 | Tomato | FERA_130827 | United Kingdom | 93.8 | 93.5 | 94.1 |
| MG132056 | Tomato | Touboudan | Ghana | 94.4 | 94.7 | 94.9 |
| MG132057 | Tomato | Akumadan | Ghana | 93 | 93.3 | 93.5 |
| X06390 | Tomato | indonesian | Indonesia | 93.3 | 92.7 | 93.3 |
The Saudi isolate of PCFVd, which was collected from pepper plants, had the highest similarity (100%) with the isolate no. FJ409044 collected from sweet pepper (C. annum) in the Netherlands [16]. The Saudi isolate was also highly similar (>99%) to many other isolates from tomato plants collected from different locations in Thailand, Australia and Vietnam. The genetic relationship and homology tree for the Saudi isolate of PCFVd is shown in Figure 7 and Figure 8 and Table 2.
Figure 7.
Homology tree based on multiple sequence alignment between three isolates of PCFVd from Saudi Arabia produced with 25 different PCFVd isolates of the same species from different countries obtained from GenBank. Included is a figure showing how the colors and pairwise identities correspond.
Figure 8.
SDT matrix of pairwise identity scores produced with 25 different PCFVd isolates of the same species from different countries obtained from GenBank. Included is a figure showing how the colors and pairwise identities correspond.
Table 2.
Comparison of percentages of nucleotide sequences of PCFVd Saudi Arabian isolates with isolates originating in different countries.
| Accession No. | Host | Isolate | Country | MK286923 |
|---|---|---|---|---|
| MK286923 | Pepper | Al-Kharj-SA7 | Saudi Arabia | 100 |
| FJ409044 | Pepper | - | Netherlands | 100 |
| NC_011590 | Pepper | - | Netherlands | 100 |
| JF446885 | Tomato | CR11-15c2 | Thailand | 98.6 |
| JF446887 | Tomato | CR14-8c5 | Thailand | 99.4 |
| JF446888 | Tomato | CR14-9c2 | Thailand | 99.7 |
| JF446889 | Tomato | LPng19-3c1 | Thailand | 99.7 |
| JF446906 | Tomato | LP1-15c2 | Thailand | 98.6 |
| JF446908 | Tomato | LP2-5c2 | Thailand | 99.4 |
| JF446913 | Tomato | LP4-8c1 | Thailand | 99.1 |
| JF742637 | Tomato | Aurai 2 | Thailand | 95.1 |
| JF742638 | Tomato | Aurai 10 | Thailand | 95.1 |
| KC762952 | Tomato | EMAI12/80420 | Australia | 96.3 |
| KC762953 | Tomato | EMAI12/759 | Australia | 96.6 |
| KC762954 | Tomato | EMAI12/761 | Australia | 96.3 |
| KJ635807 | Tomato | NP6-10c2 | Thailand | 99.4 |
| MW012406 | Pepper | PCFVd_NBHP | Vietnam | 96.6 |
| PQ059146 | Tomato | PCFVd_TG | Vietnam | 97.7 |
| PQ872178 | Eggplant | SB_Eg_PC43 | Thailand | 97.7 |
| PQ872179 | Eggplant | B_Eg_PC53 | Thailand | 97.7 |
| PQ872183 | Tomato | SB_Pe_PC31 | Thailand | 99.7 |
| PQ872184 | Pepper | SB_Pe_PC1 | Thailand | 97.1 |
| PQ872185 | Pepper | SB_Pe_PC3 | Thailand | 98 |
| PQ872186 | Pepper | SB_Pe_PC5 | Thailand | 96.8 |
| PQ872191 | Pepper | SB_Pe_PC13 | Thailand | 99.7 |
| PQ872192 | Pepper | SB_Pe_PC16 | Thailand | 99.7 |
3.4. Tomato Cultivar Responses
In this experiment, the first symptoms on tomato plants of all cultivars appeared at least one month after inoculation with the local isolate of TASVd (Figure 9 and Figure 10). All inoculated cultivars responded with significant reductions in plant height when compared with the non-inoculated plants, which varied from 21.8–26.9%. Meanwhile, when comparing the percentage of reduction between the different cultivars, no significant differences were observed in the plant heights of all cultivars because of their mechanical inoculation with TASVd (Table 3).
Figure 9.
Tomato cultivar responses to mechanical inoculation with TASVd-isolate in the first greenhouse experiment.
Figure 10.
The effect of mechanical inoculation with TASVd on Newton (A), Beisuzhok (B), Irit (C) and Alindi (D) tomato cultivars (to the right) compared with the control plant (to the left).
Table 3.
The impact of inoculating four different tomato cultivars with TASVd (isolate 312). Plant height means (in centimeters) and a comparison of the percentage decrease in tomato cultivars produced in greenhouses.
| * Cultivar | ** Control | ** Inoculated | *** Reduction (%) |
|---|---|---|---|
| Newton | 120 a | 94 b | 21.8% a |
| Alindi | 130 a | 95 b | 26.9% a |
| Irit | 131 a | 100 b | 23.7% a |
| Beisuzhok | 119 a | 92 b | 22.7% a |
* For each treatment, five plants were used per cultivar, and data were recorded at 90 days post-inoculation. ** For each cultivar, means followed by the same letter in the control and inoculated columns are not significantly different (p = 0.05) according to the LSD test. *** Average percent reductions in the cultivars followed by the same letter in the percent reduction column are not significantly different (p = 0.05) according to the LSD test.
Yet it was observed that all cultivars severely affected fruit weight. When the four cultivars were compared based on the effect of TASVd on their fruit weights, clearly significant differences were observed between them, and the Newton cultivar was the most affected, with a 90.2% reduction in fruit weight, while the Irit cultivar was the least affected, with 49.1% reduction (Table 4).
Table 4.
Effect of TASVd inoculation (isolate 312) on different tomato cultivars. Means of fruit weight (g/plant) and comparison of the percent reduction in four greenhouse-grown tomato cultivars inoculated with the local isolate of tomato apical stunt viroid (TASVd).
| * Cultivar | ** Control | ** Inoculated | *** Reduction (%) |
|---|---|---|---|
| Newton | 320 a | 31 b | 90.2% a |
| Alindi | 353 a | 97 b | 72.5% b |
| Irit | 417 a | 212 b | 49.1% d |
| Beisuzhok | 343 a | 136 b | 60.3% c |
* For each treatment, five plants were used per cultivar, and data were recorded 90 days after inoculation. ** For each cultivar, means followed by the same letter in the control and inoculated columns are not significantly different (p = 0.05) according to the LSD test. *** Average percent reductions in the cultivars followed by the same letter in the percent reduction column are not significantly different (p = 0.05) according to the LSD test.
Dry weights of infected plants were significantly reduced when compared with the control plants, except for the Alindi cultivar. When comparing the reduction percentages of plant vegetative dry weight of the four cultivars, the Newton cultivar, with the highest reduction percentage in dry matter (39.3%), was the only significant one compared to the other three cultivars (Table 5).
Table 5.
Effect of TASVd inoculation (isolate 312) on different tomato cultivars. Means of fruit weight (g/plant) and comparison of the percent reduction in four greenhouse-grown tomato cultivars.
| * Cultivar | ** Control | ** Inoculated | *** Reduction (%) |
|---|---|---|---|
| Newton | 112 a | 68 b | 39.3% a |
| Alindi | 119 a | 112 a | 6.0% b |
| Irit | 132 a | 116 b | 12.6% b |
| Beisuzhok | 114 a | 93 b | 18.1% ab |
* For each treatment, five plants were used per cultivar, and data were recorded 90 days after inoculation. ** For each cultivar, means followed by the same letter in the control and inoculated columns are not significantly different (p = 0.05) according to the LSD test. *** Average percent reductions in the cultivars followed by the same letter in the percent reduction column are not significantly different (p = 0.05) according to the LSD test.
The studied cultivars’ plant heights did not differ significantly from one another. The greatest decrease in vegetative dry weight (39.3%) was observed in the Newton cultivar. With a just 6% decrease in comparison to their esteemed health controls, the Alindi cultivar was the least impacted. With a 90.2% decrease in fruit output when compared to its untreated control, the Newton cultivar was the most impacted cultivar. However, with a 49.1% decrease in fruit output, the Irit cultivar was the least impacted.
4. Discussion
Viroid diseases, especially those of fruit and vegetable crops, occur widely in Middle Eastern and African countries, where the expression of viroid disease symptoms increases because of the hot or warm climates of these regions. Natural viroid infections belong to several members of the Avsunviroidae and Pospiviroidae families. Tomato apical stunt disease was initially reported in 1980 [26] from tomatoes in the northern areas of the Ivory Coast of Africa (Cote D’Ivoire), and the causal agent, TASVd, was the first viroid to be characterized on the African continent. Since then, TASVd has been reported in asymptomatic ornamentals and tomato in different parts of Africa [21,27,28,29], Asia [30,31,32,33] and various European countries [34,35,36,37,38,39].
The symptoms recorded in this study were like those described by Matthews-Berry, in 2010 [40], in the UK on tomato. Fruits of infected plants were smaller, ripped earlier, and had a pale red discoloration and early ripening. In severe cases, mummification was observed. Similar symptoms on fruits have been described [21,26,27,35,41]. However, these symptoms were less severe in PCFVd infected plants compared to TASVd infection. Similar symptoms were described earlier in pepper plants infected with PCFVd in the Netherlands [16]. However, leaf curling, which was observed in this study, was not reported in Verhoeven’s study. On the other hand, it should be mentioned that this is the first study ever that reports a natural infection of TASVd in pepper in the world; no symptoms were described for this viroid on pepper plants beforehand. We cannot distinguish between the infection of the two species (TASVd and PCFVd) on pepper plants based on the observed symptoms. Similar symptoms produced by different species of the genus Pospoviroid on the same host were recorded in studies earlier [13,14,22,42,43]. Moreover, after first being identified in Côte d’Ivoire, TASVd was later found in Indonesia, Israel, Tunisia, Senegal, Ghana, and other European nations. Reports from Senegal and Ghana, where TASVd was found in tomato crops, and Israel, where TASVd caused severe disease in greenhouse-grown tomatoes, are especially pertinent to the current investigation. The experimental host range of the Israeli TASVd strain, which was spread through mechanical inoculation and grafting, was different from that of the original Côte d’Ivoire strain, indicating that biological characteristics can differ amongst TASVd isolates [21,30].
Molecular detection methods for TASVd, and for viroids in general [44,45,46,47], include nucleic acid hybridization [48,49], RT-PCR [50,51,52,53], real-time RT-PCR [50,51] and next-generation sequencing [54]. Hence, in the present study, RT-PCR amplification was employed for the molecular detection of TASVd and PCFVd. The obtained results showed that the same bands of ~364 bp and ~350 bp were observed for these samples after the RT-PCR assay, with the specific primers of TASVd and PCFVd respectively, like [19,20,21,22,23].
On the other hand, the distribution of infection of the two viroids in one planting area, among all other areas, suggests that the initial source of infection might be through seeds, and the spread of infection during the growing season was probably via mechanical means. This possibility can be strengthened by the fact that all positive tomato samples were collected from the Beisuzhok (Giverech) cultivar, which was widely grown in the Al-Kharj area at that time. The seed transmission of many Pospiviroid species, including TASVd, PSTVd and CEVd, was already reported in several previous studies [8,10]. Using the universal Pospi1 primer pair in the RT-PCR test, no Posiviroid species were found in the remaining examined samples. Finally, only the TASVd viroid has been confirmed to infect tomatoes, while TASVd and PCFVd have been confirmed to infect pepper plants in the tested samples.
Mechanical and graft transmission of TASVd have been shown in experimental investigations. Furthermore, an epidemiological investigation in Israeli greenhouse tomato crops showed that bumble bees could spread the viroid across tomato plants during pollination, but neither Myzus persicae nor Bemisia tabaci could spread TASVd. In that study, seed transmission was also demonstrated; in the experimental conditions, transmission rates were reported to reach 80% [33].
However, in the greenhouse experiment, one positive TASVd Saudi isolate was selected to evaluate the severity and symptom responses to mechanical inoculation of four tomato cultivars. According to the data obtained, each cultivar exhibited a significant change in each metric.
5. Conclusions
In conclusion, TASVd was found for the first time on greenhouse-growing tomato plants, whereas TASVd and PCFVd were also found for the first time on greenhouse- growing pepper plants in Saudi Arabia. This is the first report of TASVd infection of pepper plants in the world. The two viroids were isolated and identified from the Al-Kharj, area and none of the other tested samples collected from other planting areas were found to have any of the Pospiviroid species. According to the results obtained in this study, seeds might be the initial source of infection, and mechanical transmission may take place later throughout the season. All Pospiviroid-infected tomato plants found in this study were from the same cultivar, Beisuzhok (Giverech). TASVd infection on tomato plants can cause severe damage and yield reduction of up to 90% in some cultivars. Different tomato cultivars have different responses to TASVd infection, and those may significantly vary with damage and production losses due to infection. Some plant species might be symptomless carriers of TASVd. Further studies to other agricultural areas in the Kingdom of Saudi Arabia are recommended to check for the presence of any of the Pospiviroid species on greenhouse-grown solanaceous crops. Testing some of the imported tomatoes seeds is highly recommended to determine the initial source of infection. Quarantine legislations for testing of Pospiviroid species are recommended to prevent the possible entrance of any other Pospiviroid species to Saudi Arabia. Developing breeding programs to produce new tomato cultivars with resistance or high tolerance to Pospivroidae species infection, including TASVd, is recommended.
Author Contributions
M.A.A., M.A.A.-S., K.H., M.A., A.H., O.A.A. and I.M.A.-S. established the project, collected the samples, and planned the research. Data analysis and manuscript drafting, M.A.A. and M.A. All authors have read and agreed to the published version of the manuscript.
Institutional Review Board Statement
Ethical review and approval were not applicable for studies not involving humans or animals.
Informed Consent Statement
Not applicable.
Data Availability Statement
The genome sequence data of viroids presented in this study are available on the NCBI database through accession numbers MK286920, MK286921, MK286922 and MK286923.
Conflicts of Interest
The authors declare no conflicts of interest.
Funding Statement
We thank an Ongoing Research Funding Program (ORF-2026-1755-3), King Saud University, Riyadh, Saudi Arabia for financial support.
Footnotes
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References
- 1.Choi H., Jo Y., Cho W.K., Yu J., Tran P.-T., Salaipeth L., Kwak H.-R., Choi H.-S., Kim K.-H. Identification of Viruses and Viroids Infecting Tomato and Pepper Plants in Vietnam by Metatranscriptomics. Int. J. Mol. Sci. 2020;21:7565. doi: 10.3390/ijms21207565. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Hančinský R., Mihálik D., Mrkvová M., Candresse T., Glasa M. Plant Viruses Infecting Solanaceae Family Members in the Cultivated and Wild Environments: A Review. Plants. 2020;9:667. doi: 10.3390/plants9050667. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Ramallo J.C., Weht S. Solanum chacoense and Solanum nigrum carrier weeds of potato virus x and potato virus y. Rev. Agronómica Noroeste Argent. 1978;15:175–184. [Google Scholar]
- 4.Kaponi M., Kyriakopoulou P.E., Hadidi A. Viroids of the Mediterranean Basin. Viruses. 2024;16:612. doi: 10.3390/v16040612. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Öksal H.D., Aydin S., Baran M., Emir C., Karanfil A., Bozdogan O., Sipahioglu H. Persimmon (Diospyros kaki L.) and Johnsongrass [Sorghum halepense (L.) Pers.] are new natural hosts of peach latent mosaic viroid. Appl. Ecol. Environ. Res. 2021;19:4629–4639. doi: 10.15666/aeer/1906_46294639. [DOI] [Google Scholar]
- 6.Al-Shahwan I. Host Index and Status of Plant Viruses and Virus-Like Disease Agents in Saudi Arabia. The Agricultural Research Center at King Saud University; Riyadh, Saudi Arabia: 2003. pp. 5–27. [Google Scholar]
- 7.Hadidi A., Czosnek H.H., Kalantidis K., Palukaitis P. Viroids and Satellites and Their Vector Interactions. Viruses. 2024;16:1598. doi: 10.3390/v16101598. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Verhoeven J.T.J. Identification and Epidemiology of Pospiviroids. Wageningen University and Research; Wageningen, The Netherlands: 2010. [Google Scholar]
- 9.Koonin E.V., Lee B.D. Diversity and evolution of viroids and viroid-like agents with circular RNA genomes revealed by metatranscriptome mining. Nucleic Acids Res. 2025;53:gkae1278. doi: 10.1093/nar/gkae1278. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Hammond R.W., Owens R.A. Viroids: New and Continuing Risks for Horticultural and Agricultural Crops. APSnet Feature. 2006;10 doi: 10.1094/APSnetFeature-2006-1106. [DOI] [Google Scholar]
- 11.Gucek T., Trdan S., Jakse J., Javornik B., Matousek J., Radisek S. Diagnostic techniques for viroids. Plant Pathol. 2017;66:339–358. doi: 10.1111/ppa.12624. [DOI] [Google Scholar]
- 12.King A.M., Lefkowitz E., Adams M.J., Carstens E.B. Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses. Volume 9 Elsevier; Amsterdam, The Netherlands: 2011. [Google Scholar]
- 13.Verhoeven J.T.J., Roenhorst J., Owens R. Mexican papita viroid and tomato planta macho viroid belong to a single species in the genus Pospiviroid. Arch. Virol. 2011;156:1433–1437. doi: 10.1007/s00705-011-0975-2. [DOI] [PubMed] [Google Scholar]
- 14.Verhoeven J.T.J., Botermans M., Jansen C., Roenhorst J. First report of Pepper chat fruit viroid in capsicum pepper in Canada. New Dis. Rep. 2011;23:15. doi: 10.5197/j.2044-0588.2011.023.015. [DOI] [Google Scholar]
- 15.Zhang Y.-H., Zhang Z.-X. A universal probe for simultaneous detection of six pospiviroids and natural infection of potato spindle tuber viroid (PSTVd) in tomato in China. J. Integr. Agric. 2023;22:790–798. doi: 10.1016/j.jia.2022.08.119. [DOI] [Google Scholar]
- 16.Verhoeven J.T.J., Jansen C., Roenhorst J., Flores R., De la Peña M. Pepper chat fruit viroid: Biological and molecular properties of a proposed new species of the genus Pospiviroid. Virus Res. 2009;144:209–214. doi: 10.1016/j.virusres.2009.05.002. [DOI] [PubMed] [Google Scholar]
- 17.Lebas B., Clover G.G., Ochoa-Corona F., Elliott D., Tang Z., Alexander B. Distribution of Potato spindle tuber viroid in New Zealand glasshouse crops of capsicum and tomato. Australas. Plant Pathol. 2005;34:129–133. doi: 10.1071/ap05002. [DOI] [Google Scholar]
- 18.Fagoaga C., Semancik J., Duran-Vila N. A citrus exocortis viroid variant from broad bean (Vicia faba L.): Infectivity and pathogenesis. J. Gen. Virol. 1995;76:2271–2277. doi: 10.1099/0022-1317-76-9-2271. [DOI] [PubMed] [Google Scholar]
- 19.Botermans M., Roenhorst J.W., Hooftman M., Verhoeven J.T.J., Metz E., van Veen E.J., Geraats B.P., Kemper M., Beugelsdijk D.C., Koenraadt H. Development and validation of a real-time RT-PCR test for screening pepper and tomato seed lots for the presence of pospiviroids. PLoS ONE. 2020;15:e0232502. doi: 10.1371/journal.pone.0232502. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Verhoeven J.T.J., Jansen C., Willemen T., Kox L., Owens R., Roenhorst J. Natural infections of tomato by Citrus exocortis viroid, Columnea latent viroid, Potato spindle tuber viroid and Tomato chlorotic dwarf viroid. Eur. J. Plant Pathol. 2004;110:823–831. doi: 10.1007/s10658-004-2493-5. [DOI] [Google Scholar]
- 21.Candresse T., Marais A., Ollivier F., Verdin E., Blancard D. First Report of the Presence of Tomato apical stunt viroid on Tomato in Sénégal. Plant Dis. 2007;91:330. doi: 10.1094/pdis-91-3-0330c. [DOI] [PubMed] [Google Scholar]
- 22.Balsak S.C. First molecular characterization of pepper chat fruit viroid infecting pepper crops in Turkey. J. Plant Dis. Prot. 2025;132:96. doi: 10.1007/s41348-025-01091-4. [DOI] [Google Scholar]
- 23.Yanagisawa H., Matsushita Y. Host ranges and seed transmission of Tomato planta macho viroid and Pepper chat fruit viroid. Eur. J. Plant Pathol. 2017;149:211–217. doi: 10.1007/s10658-017-1160-6. [DOI] [Google Scholar]
- 24.Muhire B.M., Varsani A., Martin D.P. SDT: A Virus Classification Tool Based on Pairwise Sequence Alignment and Identity Calculation. PLoS ONE. 2014;9:e108277. doi: 10.1371/journal.pone.0108277. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Little T.M., Hills F.J. Agricultural Experimentation, Design and Analysis. John Wiley and Sons; New York, NY, USA: 1978. 350p [Google Scholar]
- 26.Walter B., Thouvenel J.-C., Fauquet C. Les viroses de la Tomate en Côte-d’Ivoire. Ann. Phytopathol. 1980;12:259. [Google Scholar]
- 27.Batuman O., Çiftçi Ö.C., Osei M.K., Miller S.A., Rojas M.R., Gilbertson R.L. Rasta Disease of Tomato in Ghana is Caused by the Pospiviroids Potato spindle tuber viroid and Tomato apical stunt viroid. Plant Dis. 2019;103:1525–1535. doi: 10.1094/pdis-10-18-1751-re. [DOI] [PubMed] [Google Scholar]
- 28.Verhoeven J.T.J., Jansen C.C.C., Roenhorst J.W. First Report of Tomato apical stunt viroid in Tomato in Tunisia. Plant Dis. 2006;90:528. doi: 10.1094/pd-90-0528a. [DOI] [PubMed] [Google Scholar]
- 29.Walter B. Tomato Apical Stunt. In: Diener T.O., editor. The Viroids. Springer; Boston, MA, USA: 1987. pp. 321–327. [Google Scholar]
- 30.Antignus Y., Lachman O., Pearlsman M., Gofman R., Bar-Joseph M. A new disease of greenhouse tomatoes in Israel caused by a distinct strain of Tomato apical stunt viroid (TASVd) Phytoparasitica. 2002;30:502–510. doi: 10.1007/bf02979755. [DOI] [Google Scholar]
- 31.Candresse T., Smith D., Diener T. Nucleotide sequence of a full-length infectious clone of the Indonesian strain of tomato apical stunt viroid (TASV) Nucleic Acids Res. 1987;15:10597. doi: 10.1093/nar/15.24.10597. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Antignus Y., Pearlsman M., Lachman O., Feigelson F. Tomato apical stunt viroid (TASVd), a pathogen of greenhouse tomatoes in Israel is seedborne and transmitted by bumble bees. Phytoparasitica. 2006;34:306–307. [Google Scholar]
- 33.Antignus Y., Lachman O., Pearlsman M. Spread of Tomato apical stunt viroid (TASVd) in greenhouse tomato crops is associated with seed transmission and bumble bee activity. Plant Dis. 2007;91:47–50. doi: 10.1094/pd-91-0047. [DOI] [PubMed] [Google Scholar]
- 34.Milanović J., Kajić V., Mihaljević S. Occurrence and molecular variability of Potato spindle tuber viroid and Tomato apical stunt viroid in ornamental plants in Croatia. Eur. J. Plant Pathol. 2014;139:785–788. doi: 10.1007/s10658-014-0432-7. [DOI] [Google Scholar]
- 35.Verhoeven J., Botermans M., Meekes E., Roenhorst J. Tomato apical stunt viroid in the Netherlands: Most prevalent pospiviroid in ornamentals and first outbreak in tomatoes. Eur. J. Plant Pathol. 2012;133:803–810. doi: 10.1007/s10658-012-0005-6. [DOI] [Google Scholar]
- 36.Mojca Virscek M., Mavric Plesko I. First report of Tomato apical stunt viroid in Solanum jasminoides in Slovenia. New Dis. Rep. 2012;26:7. doi: 10.5197/j.2044-0588.2012.026.007. [DOI] [Google Scholar]
- 37.Olivier T., Demonty E., Govers J., Belkheir K., Steyer S., Jongen C. First Report of a Brugmansia sp. Infected by Tomato apical stunt viroid in Belgium. Plant Dis. 2011;95:495. doi: 10.1094/pdis-09-10-0638. [DOI] [PubMed] [Google Scholar]
- 38.Verhoeven J.T.J., Jansen C.C.C., Roenhorst J.W. First report of pospiviroids infecting ornamentals in the Netherlands: Citrus exocortis viroid in Verbena sp., Potato spindle tuber viroid in Brugmansia suaveolens and Solanum jasminoides, and Tomato apical stunt viroid in Cestrum sp. Plant Pathol. 2008;57:399. doi: 10.1111/j.1365-3059.2007.01662.x. [DOI] [Google Scholar]
- 39.Verhoeven J.T.J., Jansen C.C.C., Roenhorst J.W., Steyer S., Schwind N., Wassenegger M. First Report of Solanum jasminoides Infected by Citrus exocortis viroid in Germany and the Netherlands and Tomato apical stunt viroid in Belgium and Germany. Plant Dis. 2008;92:973. doi: 10.1094/pdis-92-6-0973a. [DOI] [PubMed] [Google Scholar]
- 40.Matthews-Berry S. Plant Disease Factsheet. The Food and Environment Research Agency (FERA); York, UK: 2010. Emerging viroid threats to UK tomato production; p. 5. [Google Scholar]
- 41.Parrella G., Numitone G. First Report of Tomato apical stunt viroid in Tomato in Italy. Plant Dis. 2014;98:1164. doi: 10.1094/pdis-02-14-0206-pdn. [DOI] [PubMed] [Google Scholar]
- 42.Sabaratnam S. Tomato Chlorotic Dwarf Viroid on Greenhouse Tomato. Abbotsford Agriculture Centre, British Columbia Ministry of Agriculture, Food and Fisheries; Abbotsford, BC, Canada: 2012. [(accessed on 9 September 2026)]. Available online: https://www2.gov.bc.ca/assets/gov/farming-natural-resources-and-industry/agriculture-and-seafood/animal-and-crops/plant-health/phu-tomatochlorotic-dwarfviroidss.pdf. [Google Scholar]
- 43.Singh R.P., Nie X., Singh M. Tomato chlorotic dwarf viroid: An evolutionary link in the origin of pospiviroids. J. Gen. Virol. 1999;80:2823–2828. doi: 10.1099/0022-1317-80-11-2823. [DOI] [PubMed] [Google Scholar]
- 44.van Brunschot S.L., Bergervoet J.H., Pagendam D.E., de Weerdt M., Geering A.D., Drenth A., van der Vlugt R.A. Development of a multiplexed bead-based suspension array for the detection and discrimination of pospiviroid plant pathogens. PLoS ONE. 2014;9:e84743. doi: 10.1371/journal.pone.0084743. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45.Tiberini A., Barba M. Optimization and improvement of oligonucleotide microarray-based detection of tomato viruses and pospiviroids. J. Virol. Methods. 2012;185:43–51. doi: 10.1016/j.jviromet.2012.05.028. [DOI] [PubMed] [Google Scholar]
- 46.Torchetti E.M., Navarro B., Di Serio F. A single polyprobe for detecting simultaneously eight pospiviroids infecting ornamentals and vegetables. J. Virol. Methods. 2012;186:141–146. doi: 10.1016/j.jviromet.2012.08.005. [DOI] [PubMed] [Google Scholar]
- 47.Nie X., Singh R.P. Viroids and Satellites. Elsevier; Amsterdam, The Netherlands: 2017. Viroid detection and identification by bioassay; pp. 347–356. [Google Scholar]
- 48.Pallás V., Sánchez-Navarro J.A., Kinard G.R., Di Serio F. Chapter 35–Molecular Hybridization Techniques for Detecting and Studying Viroids. In: Hadidi A., Flores R., Randles J.W., Palukaitis P., editors. Viroids and Satellites. Academic Press; Boston, MA, USA: 2017. pp. 369–379. [Google Scholar]
- 49.Owens R.A., Diener T.O. Sensitive and rapid diagnosis of potato spindle tuber viroid disease by nucleic Acid hybridization. Science. 1981;213:670–672. doi: 10.1126/science.213.4508.670. [DOI] [PubMed] [Google Scholar]
- 50.Faggioli F., Duran-Vila N., Tsagris M., Pallás V. Chapter 44–Geographical Distribution of Viroids in Europe. In: Hadidi A., Flores R., Randles J.W., Palukaitis P., editors. Viroids and Satellites. Academic Press; Boston, MA, USA: 2017. pp. 473–484. [Google Scholar]
- 51.Faggioli F., Luigi M., Boubourakas I.N. Chapter 36–Viroid Amplification Methods: RT-PCR, Real-Time RT-PCR, and RT-LAMP. In: Hadidi A., Flores R., Randles J.W., Palukaitis P., editors. Viroids and Satellites. Academic Press; Boston, MA, USA: 2017. pp. 381–391. [Google Scholar]
- 52.Olivier T., Šveikauskas V., Demonty E., De Jonghe K., Gentit P., Viršček-Marn M., Grausgruber-Gröger S., Morio S., Faggioli F., Visage M., et al. Inter-laboratory comparison of four RT-PCR based methods for the generic detection of pospiviroids in tomato leaves and seeds. Eur. J. Plant Pathol. 2016;144:645–654. doi: 10.1007/s10658-015-0803-8. [DOI] [Google Scholar]
- 53.Bostan H., Nie X., Singh R.P. An RT-PCR primer pair for the detection of Pospiviroid and its application in surveying ornamental plants for viroids. J. Virol. Methods. 2004;116:189–193. doi: 10.1016/j.jviromet.2003.11.014. [DOI] [PubMed] [Google Scholar]
- 54.Barba M., Hadidi A. Viroids and Satellites. Academic Press; Cambridge, MA, USA: 2017. Application of Next-Generation Sequencing Technologies to Viroids; pp. 401–412. [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The genome sequence data of viroids presented in this study are available on the NCBI database through accession numbers MK286920, MK286921, MK286922 and MK286923.