Viroids are unencapsidated, single-stranded, circular RNA consisting of 246–401 nucleotides. Among plant pathogens, they are the smallest pathogens and belong to two families (Pospiviroidae and Avsunviroidae) (Flores et al. 2005). The genus Pospiviroid in the family Pospiviroidae consists of nine species, of which one is the potato spindle tuber viroid (PSTVd) (Full ICTV Report on the Genus Pospiviroid; https://ictv.global/report/chapter/pospiviroidae/pospiviroidae/pospiviroid).
Although PSTVd was originally reported in North America, it has spread to Africa, Asia, Europe, and South America (Smith et al. 1997). In recent years, PSTVd has been identified in several countries, and new viroids are being continuously discovered (Kinoga et al. 2021; Matsushita et al. 2021; Sial and Khan 2018).
PSTVd infects various host plants and causes stunting, leaf chlorosis, leaf epinasty, fruit distortion, and stem and leaf necrosis in tomatoes (Solanum lycopersicum) and potatoes (S. tuberosum) (Hadidi et al. 2003; Singh et al. 2003). Viroids spread mechanically through plant sap, grafting, and transmission through seeds and pollen (Matsushita et al. 2018). Among these, seed transmission is an important factor responsible for the expansion of viroids as reported for PSTVd, which is transmitted through the seeds of Solanum spp., such as tomato, pepper (Capsicum annuum), and petunia (Petunia × hybrida) (Matsushita and Tsuda 2016). Moreover, PSTVd has been detected in traded tomato and pepper seeds (Constable et al. 2019), and thus, such viroids can spread to new areas through these contaminated seeds. Therefore, several countries, including Japan, are focusing on the transmission of PSTVd through contaminated seeds. To prevent the occurrence of PSTVd, plant quarantines established in each country require seed testing for viroids. Additionally, viroids have been detected in new plants and countries using these tests. In this study, we present the first report of PSTVd detected in pepper seeds produced in Vietnam, where viroids have not yet been reported. Additionally, we assessed the viability of the viroid in the seeds and its pathogenicity, and conducted phylogenetic analysis.
Pepper seeds from Vietnam were tested to confirm the absence of pospiviroid contamination during export inspections in Japan in 2022. RNA was extracted from seed samples according to the method described by Yanagisawa et al. (2012). To detect pospiviroids in each sample, we used reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), as reported by Yanagisawa et al. (2017). Detection tests were conducted in duplicates for each sample. First, primary screening was confirmed to be positive in the assay using SYBR Green with a universal primer set (6Pospi-F/R), which enabled the detection of six pospiviroids, excluding the columnea lant viroid and pepper chat fruit viroid. Ct values were 31.0 and 30.8, and both melting temperature (Tm) values were 86.4 °C. Both Tm values of the positive controls synthesized artificially based on the PSTVd sequence (GenBank/EMBL/DDBJ accession no. EU862231, Matsushita et al. 2010) were 86.0. These results estimated that the viroid in the sample was PSTVd because the Tm values of the samples and the positive control were similar. Moreover, RT-qPCR was performed using TaqMan with specific primer/probe sets for each viroid to identify the pospiviroid species. As the result, Ct values (23.4 and 22.9) were obtained in the PSTVd-specific primer/probe set (PS-F1/SM-R1/P3R), and the primer/probe sets of other viroids did not react. These results confirmed that the viroid species was PSTVd.
To determine the complete sequence of the viroid, direct sequencing analysis of PCR products obtained from the primer sets (6Pospi-F/R and PV68/87), (Yanagisawa et al. 2019) which can amplify the almost complete genome of PSTVd, was conducted in triplicate. The results showed that the complete 359 nt sequence of PSTVd (PSTVd-VN, Acc. No. LC784333) showed the highest identity (98.6%) to a PSTVd variant (No. MT663308) from tomato.
Subsequently, to investigate the phylogenetic relationship between PSTVd-VN and other known PSTVd variants isolated from pepper and other plants, and some PSTVd variants that confirmed pathogenicity, a phylogenetic tree was constructed based on full genome sequences using the Maximum Likelihood method with 1,000 replicates of bootstrapping performed in the MEGA version VII program (Fig. 1). The results showed that PSTVd-VN was grouped into a cluster that included the PSTVd variant (No. MT663308) but not the PSTVd variants previously isolated from pepper. In addition, PSTVd variants (Nos. AY518939; severe, HE575349; severe, and U23058; lethal) with strong pathogenicities existed in the cluster that contained PSTVd-VN.
Next, to confirm whether the detected PSTVd maintained the viability of viroids in seeds, the nucleic acid extracted from seeds was used as the inocula to inoculate in tomatoes (cv. Rutgers) and two varieties of pepper (bell pepper and chili pepper). PSTVd was mechanically inoculated into the young leaves of the seedlings according to the method described by Yanagisawa and Matsushita (2017). At least six seedlings of each plant were inoculated, and the plants were then grown in a greenhouse at 23–25 °C with natural light. Simultaneously, tomato plants were inoculated with two different pathogenic PSTVd variants to compare the pathogenicity. A PSTVd variant (No. AB623143, Tsushima et al. 2011) exhibiting mild pathogenicity was used. The other PSTVd variant (No. EU862231) was used for severe pathogenicity because there was only one base difference between the two PSTVd variants (Nos. EU862231 and LC523663, Matsushita et al. 2021) that showed severe pathogenicity. One month after inoculation, viroid infections were assessed in the uppermost leaves of each inoculated plant. RT-PCRs using the primer sets (6Pospi-F/R and PV68/87) were conducted to confirm viroid infection in each inoculated plant. Direct sequencing analysis was conducted using these amplicons to confirm the viroid sequences after inoculation. The inoculated tomato plants had viroid-like symptoms, such as severe stunting, leaf chlorosis, and leaf deformation, followed by necrosis of the stems and leaf veins (Fig. 2A, E). In addition, PSTVd-VN-inoculated tomato plants showed severe pathogenicity, as the PSTVd variant (No. EU862231), and both PSTVd variants caused necrosis of the stem and leaf veins. Contrastingly, the PSTVd variant (No. AB623143) caused slight stunting. However, neither cultivar of inoculated pepper plants exhibited symptoms (Fig. 3A, B). However, as the inoculated chili pepper plants continued to grow for 3 months after inoculation, the symptoms of the fruits were confirmed. The fruits collected from PSTVd-infected pepper plants were smaller than those from healthy plants, and the yield was eventually reduced (Fig. 3C). The results of direct sequencing analysis showed that the sequence of PSTVd detected in pepper seeds and the sequence of PSTVd after inoculation completely matched.
To the best of our knowledge, this is the first report of PSTVd detection in Vietnamese plants. PSTVd-VN belonged to a cluster (Fig. 1) that includes the PSTVd variant (No. U23058) exhibiting lethal pathogenicity, and PSTVd variants (Nos. AY518939 and HE575349) showing severe pathogenicity. Tomato plants inoculated with PSTVd-VN showed severe symptoms similar to those of the PSTVd variant (No. EU862231) (Fig. 2B, F). Therefore, PSTVd-VN was confirmed to be strongly pathogenic. Additionally, PSTVd-VN-infected pepper plants did not show any symptoms; however, fruit downsizing was observed (Fig. 3B, C). Verhoeven et al. (2020) reported that PSTVd-infected C. annuum grew and obtained yields similar to those of healthy plants. Therefore, fruit symptoms may depend on the pepper cultivar, and the PSTVd strain and variant. However, in the field, identifying viroid-infected plants would be difficult because no symptoms are seen in pepper plants.
We confirmed the viability of PSTVd in the pepper seeds (Fig. 2A, E). Verhoeven et al. (2021) confirmed the viability of the viroids in the pepper seeds but did not confirm the viability of the viroids in the tomato seeds. Hence, the viability of viroids in pepper seeds may be more easily maintained than in tomato seeds. However, Matsushita and Tsuda (2016) reported transmission through pepper seeds, while Verhoeven et al. (2020, 2021) did not. Many factors are responsible for the seed transmission of viroids (host plant species and cultivars, viroid species and strain, infection stage, environmental conditions, and distribution of viroids in seed parts) (Matsushita et al. 2018). Infection of the ovule is the key to establishing viroid seed transmission. Because the Ct values of RT-qPCR were low, many viroids were suggested to exist in the tested seeds. Moreover, we confirmed the viability of viroids in seeds. These results suggested that the seeds have the potential to promote transmission of viroids. Therefore, continuous testing for viroids is necessary to determine the distribution of healthy seeds.