ZHANG Yu-hong ,Ll Zhi-xin ,DU Ya-jie ,Ll Shi-fang ,ZHANG Zhi-xiang

1 State Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193,P.R.China

2 Keshan Branch of Heilongjiang Academy of Agricultural Sciences,Qiqihar 161005,P.R.China

3 College of Agronomy and Biotechnology,Hebei Normal University of Science &Technology,Qinhuangdao 066004,P.R.China

Abstract Several viroids in the genus Pospiviroid can infect tomato (Solanum lycopersicum) and cause severe diseases,posing a serious threat to tomato production. For simultaneous detection of six tomato-infecting pospiviroids -columnea latent viroid (CLVd),pepper chat fruit viroid (PCFVd),potato spindle tuber viroid (PSTVd),tomato apical stunt viroid (TASVd),tomato chlorotic dwarf viroid (TCDVd),and tomato planta macho viroid (TPMVd),we developed a universal probe based on a highly conserved 61 nt long sequence shared among them. Compared with their specific probes,the universal probe has a similar,though slightly reduced,detection sensitivity and has the advantages of simple and cost-effective preparation and simultaneous detection of the six pospiviroids. In addition,the universal probe was used in dot-blot hybridization assays for a large-scale survey of viroid(s) in tomato plantings in China. Only PSTVd was detected in a few greenhouse-planted tomato plants. Sequence analysis revealed that these tomato PSTVd isolates may have been introduced from tomato seeds imported from abroad.

Keywords: tomato,viroid,simultaneous detection,molecular hybridization,pospiviroids

1.lntroduction

Viroid infection poses a serious threat to tomato (Solanum lycopersicum) production,among which several viroids in thePospiviroidgenus,including columnea latent viroid(CLVd),pepper chat fruit viroid (PCFVd),potato spindle tuber viroid (PSTVd),tomato apical stunt viroid (TASVd),tomato chlorotic dwarf viroid (TCDVd),and tomato planta macho viroid (TPMVd) can individually or simultaneously infect tomato and cause severe disease with symptoms including plant stunting,leaf and stem necrosis,and foliar and fruit deformation (Verhoevenet al.2004;EPPO 2021). Natural infections by pospiviroids in tomato plants have been reported in many countries (Elliottet al.2001;Hailstoneset al.2003;Mumfordet al.2004;Verhoevenet al.2004,2007;Di Serio 2007;Bostanet al.2010;Ling and Sfetcu 2010;Batumanet al.2019;Choiet al.2020;Natarajamurthyet al.2021). Thus,pospiviroids are classified as plant quarantine pathogens by many countries and organizations.

Early detection,which requires accurate and rapid detection technologies,is critical for the prevention and control of viroid diseases. Many such technologies have been developed and applied for viroid detection (Guceket al.2017),among which molecular hybridization is one of the most widely used routine detection methods due to its high reliability (Pallaset al.2017). Several viroids can be simultaneously detected using polyprobes or universal probes (Pallaset al.2018). Polyprobes are synthesized from recombinant DNA fragment constructed by cloning in tandem,partial or full-length genomic sequences of different viroids. Several polyprobes have been developed for detection of viroids infecting citrus(Cohenet al.2006),grapevine (Zhanget al.2012),pome and stone fruit trees (Linet al.2011;Peiroet al.2012),tomato (Sanchez-Navarroet al.2019),ornamental plants,and vegetable crops (Torchettiet al.2012). Universal probes are synthesized from conserved sequences shared by different viroids. Such probes have been developed for detection of coleviroids infecting coleus plants based on the conserved upper sequence (32 nt) of the central conserved region (CCR) (Jianget al.2013).Unlike polyprobes,universal probes are shorter and,thus,simpler and less expensive to produce.

Thus far,for pospiviroids,only two polyprobes have been developed for the detection of four distinct tomato infecting pospiviroids (Sanchez-Navarroet al.2019)and eight ornamental and vegetable plant infecting pospiviroids (Torchettiet al.2012). Here,we have developed a universal probe for the simultaneous detection of the six pospiviroids mentioned above based on a 61-nt conserved sequence shared between them.A dot-blot hybridization detection method using the universal probe was established and applied in a largescale survey of these six pospiviroids in tomato in China.PSTVd infection was found in greenhouse-planting tomato plants. The origin of these tomato PSTVd isolates was determined through sequence comparison analysis.

2.Materials and methods

2.1.Tomato samples

To survey infection by the six pospiviroids in tomato in China,over 300 leaf samples were collected from different provinces in the past few years. Some of them showed virus-like symptoms including yellowing,leaf mottling,stunting,and deformation.

2.2.Plasmids

Each of the six recombinant pospiviroid plasmids used for synthesizing viroid RNA and RNA probe contains the full-length cDNA sequence of each viroid. The GenBank accession numbers for PSTVd,PCFVd,TCDVd,TASVd,TPMVd,and CLVd sequences are KR611334,NC011590,NC000885,NC001553,NC001558,and NC003538,respectively. PSTVd was amplified from the infected potato plants (Qiuet al.2016) and cloned into the pGEM-T vector. TCDVd in the pGEM-T vector was a gift from Prof.Teruo Sano at Hirosaki University in Japan,and sequences of the other four viroids were manually synthesized by Sangon Biotech (Shanghai,China) and cloned into the pBluescript SK II(+) vector.

For the preparation of the universal probe,a 61-nt fragment was PCR-amplified from the PSTVd recombinant plasmid using primers Pospi6-probe-F1(5′-GACAGGAGTAATCCCMGCCG-3′) and Pospi6-probe-R1 (5′-GAGGAAGGAAACCMGAAGA-3′). The amplified fragment was cloned into the pGEM-T vector,and the resulting recombinant plasmid was termed pGEMPospi6.

2.3.RNA extraction,RT-PCR,cloning,and sequencing

Total RNA was extracted using Trizol reagent (TIANGEN,Beijing) according to the manufacturer’s instructions.RNA integrity was checked by agarose gel electrophoresis and was quality checked using a Nano300 (Hangzhou Allsheng Instruments,China).

Viroid cDNA was generated by M-MLV transcriptase(Promega,China) with 6-mer random primers followed by PCR usingTaqDNA polymerase (Sangon Biotech,Shanghai) for detection of viroids in tomato plants or usingpfuDNA polymerase (TransGen Biotech,Beijing)to amplify the PSTVd genome. The generic primer Pospi1-FW/RE (Verhoevenet al.2004) were used for viroid detection,while primers PSTVd/TCDVd-3P/4P(Matsushitaet al.2010) were used for PSTVd genome amplification. The PCR products obtained using PSTVd/TCDVd-3P/4P were verified by direct sequencing of subsequent PCR products amplified using a pair of PSTVd-specific primers,PSTVd-240F/239R (Qiuet al.2016).

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PCR products were purified using the PCR Product Purification Kit (TransGen Biotech),cloned into the pGEM-T vector (Promega,China),and then transformed intoEscherichiacoliDH5α competent cells (TransGen Biotech). Positive clones were selected and sequenced by Sanger sequencing (Sangon Biotech).

2.4.In-vitro transcription

The universal and specific probes and sense RNAs of the six pospiviroids were synthesized byin-vitrotranscription.All recombinant plasmids described above were digested by corresponding restriction endonucleases with cohesive ends at the 5′-terminal. Linearized plasmids were purified using the PCR Product Purification Kit (TransGen Biotech)beforein-vitrotranscription. Digoxigenin (DIG)-labeled cRNA probes were synthesized using the DIG RNA Labeling Kit (Roche,Basel,Switzerland) according to the instruction manual. Viroid sense RNAs were transcribed by a corresponding RNA polymerase using none-labeled NTP mix (Promega,China).

2.5.Northern hybridization

Northern-blot or dot-blot hybridization was performed as previously described (Jianget al.2013). Briefly,for Northern-blot hybridization,total RNAs were separated by agarose gel electrophoresis,then transferred onto a nylon membrane;for dot-blot hybridization,RNA solutions were directly added onto the nylon membrane. RNAs were fixed by UV crossing-link (100 μJ cm-2). Prehybridization and hybridization were performed for one hour and overnight in hybridization buffer (Roche,Basel,Switzerland). Hybridization signal was visualized by incubating the membrane with an alkaline phosphataselabelled anti-DIG antibody and the disodium CSPD chemiluminescence substrate (Roche) and was read and analyzed using a ChemiDoc system (Bio-Rad,USA).

2.6.Sequence alignment and phylogenetic analysis

Obtained sequences were subjected to BLAST analysis in GenBank. Multiple alignments of these sequences with the closest identified sequence or with known PSTVd sequences from China were performed using ClustalW implemented in BioEdit Software (version 7.2.5). All 52 known PSTVd sequences from potato in China were screened against the database of NCBI Virus (https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/).

Four variants of seven PSTVd sequences obtained were also used for phylogenetic tree construction together with the 265 PSTVd sequences isolated from naturally infectedSolanumplants,which were screened against the database of NCBI Virus,with MEGA-X (Kumaret al.2018) using the neighbor-joining,minimum-evolution,and maximum likelihood methods.

3.Results

3.1. ldentification of a conserved sequence between six pospiviroids

We aligned the reference genomes of six pospiviroids(CLVd,PCFVd,PSTVd,TASVd,TCDVd,and TPMVd)and found three conserved regions: a terminal conserved region (TCR),central conserved region (CCR),and a 61-nt sequence that was designated as the terminal right conserved region (TrCR) (Fig.1). TCR and CCR are two well-known conserved motifs used for the demarcation of genera in the familyPospiviroidae(Di Serioet al.2021).Unlike TCR (17 nt) and CCR (32 nt),the TrCR sequences were longer (61 nt). TrCR sequences among these six pospiviroids shared more than 88.5% similarity and had a 50.7-52.4% GC content. Although a viroid population is heterogenous and composed of close related variants that differ from each other with one or more mutations,the TrCV sequences among most of,if not all,variants of these six pospiviroids shared high similarity (＞80%).Given that the shorter CCR of the genusColeviroidhas been used as a universal probe for detecting coleviroids(Jianget al.2013),the longer TrCR sequence should also be able to be used as a universal probe for detecting these six pospiviroids.

Fig.1 Conserved sequence motifs of pospivrioids. Three conserved sequence motifs,the terminal conserved region (TCR),central conserved region (CCR),and terminal right conserved region (TrCR),were found in the six pospiviroids,columnea latent viroid (CLVd),pepper chat fruit viroid (PCFVd),potato spindle tuber viroid (PSTVd),tomato apical stunt viroid (TASVd),tomato chlorotic dwarf viroid (TCDVd),and tomato planta macho viroid (TPMVd). Their locations within the PSTVd secondary structure are shown in (A) and their sequences are shown in (B).

3.2.Universal probe for simultaneous detection of six pospiviroids

The DIG-labeled universal probe wasin-vitrosynthesized from the TrCR sequence of PSTVd. Its hybridization with the six pospiviroids and optimal hybridization temperature were determined,and its detection specificity and sensitivity were compared with specific probes.

Sense RNA transcripts of the six pospiviroids were synthesized from linearized recombinant plasmids containing full length genomes of each viroid. Five-fold dilution series of each transcript were made and used for detection by dot-blot hybridization using the universal probe at temperatures of 50,55,60,and 65°C. As expected,the universal probe could hybridize with RNA transcripts of all six pospiviroids (Fig.2-A). Although the universal probe could detect these viroids at all four temperatures,the sensitivity was a bit higher at 55 and 60°C (Fig.2-A;Appendix A).

Fig.2 Dot-blot hybridization assays using the universal probe,Pospi6,or specific probes of the six pospiviroids. The universal probe had the highest detection sensitivity at about 55°C (A) and had slightly lower detection sensitivity than specific probes of potato spindle tuber viroid (PSTVd),pepper chat fruit viroid (PCFVd),tomato chlorotic dwarf viroid (TCDVd),and tomato apical stunt viroid (TASVd) (B). However,the universal probe could detect more viroids than the specific probes (C). The universal probe could detect PSTVd in PSTVd-inoculated tomato plants at three weeks post inoculation (D). TPMVd,tomato planta macho viroid;CLVd,columnea latent viroid;NC,negative control. a6 is positive control,a7 is healthy control,and others are tomato samples.

Detection limits of the universal probe were different for each of the six pospiviroids (Fig.2-A),reaching 5-4for TCDVd but 5-6for TPMVd. This result could be explained,to a certain extent,by the differences in sequence similarities of the conserved TrCR regions of these viroids. Compared with PSTVd,TCDVd has the most mutations and PCFVd and TPMVd have the least mutations in the TrCR (Fig.1-B). Additionally,the input concentrations of RNA transcripts of each viroid were not completely identical.

To determine the specificity of the universal probe relative to each specific probe of the six pospiviroids,RNA transcripts at a concentration of 5-3of each viroid were simultaneously detected by dot-blot hybridization using the universal probe or each corresponding specific probe.Each specific probe could detect one or more other viroids,but only the universal probe could simultaneously detect all six viroids (Fig.2-C).

To evaluate the feasibility of using the universal probe to detect viroid RNA in plant tissues,we evaluated 19 total RNA samples from tomato plants three weeks after inoculation with PSTVd. A strong hybridization signal was observed for the PSTVd positive control (a6 in Fig.2-D)but not for the healthy plant (a7 in Fig.2-D). PSTVd was detected in 11 inoculated plants,which is in line with the RT-PCR detection results. In addition,the RNA transcripts of other five pospiviroids were individually mixed with total RNA of a healthy tomato plant to mimic the infected tomato plants by these five pospiviroids. All these mimic positive samples could be detected by dot-blot hybridization using the universal probe. Altogether,the universal probe synthesized from TrCR of PSTVd can simultaneously detect the six pospiviroids used in this study.

3.3. Natural PSTVd infection in tomato plantings in China

To survey infection by these six pospiviroids in tomato plantings in China,over 300 leaf samples collected from different provinces in 2019 and 2020 were detected by dot-blot hybridization using the universal probe and RTPCR using generic primers (Verhoevenet al.2004) for most members of the genusPospiviroid. None of these samples was positive. However,two positive samples were found in several previously stored tomato leaf samples in our laboratory. These stored samples were submitted by an agricultural company for virus detection in 2017. They were collected from severely diseased tomato plants in the greenhouse of the company,which expressed obvious symptoms including plant stunting,leaf crinkling,necrosis,and yellowing (Fig.3). Pospiviroid(s)infection in these samples was confirmed by RT-PCR using the generic primers (Fig.4-A). Cloning and sequencing of amplified products only obtained PSTVd sequences,indicating PSTVd infection in these diseased plants. PSTVd infection was also supported by RT-PCR using PSTVd-specific primers (Fig.4-B) and Northern-blot hybridization using PSTVd-specific probe (Fig.4-C).

Fig.3 Symptoms observed on the potato spindle tuber viroid (PSTVd)-infected tomato plants growing in the greenhouse. PSTVdinfected plants were obviously stunted and dwarfed (A and D) and their leaves became yellow,especially for upper young leaves(compare the upper and lower plant parts). Older leaves showed necrosis,yellowing,and crinkling (B and C).

Fig.4 Natural infection of potato spindle tuber viroid (PSTVd) in tomato plants in China. Two diseased tomato plants (lanes 1 and 2) were detected by RT-PCR using generic primers for the genus Pospiviroid (A),PSTVd-specific primers (B),and by Northern-blot hybridization using PSTVd-specific probe (C). M,DNA marker;NC,negative control;H,healthy tomato.

3.4.PSTVd in tomato plantings in China may be introduced from abroad

To trace the origin of PSTVd in tomato in China,seven genomic sequences were obtained by cloning and sequencing. They represented four different variants (V1-4 in Fig.5),all of which were the closest to a potato isolate,6033567 (KY936876),from the Netherlands (Verhoevenet al.2004). The master variant (V1) had four mutations,including one deletion and three insertions;another variant (V2) had one more substitution at position 67. To check the genetic relationships of these four variants with all known Chinese PSTVd sequences,their sequences were aligned using the sequence of potato isolate DN27 from the northern China (Qiuet al.2016) as a reference(Appendix B). Two substitutions at positions 67 and 221 were only found in the four variants of the tomato isolate,but not in all other known sequences. However,one of these two substitutions at position 221 exists in the potato isolate 6033567 from the Netherlands (Fig.5) and some other isolates from abroad.

Fig.5 Sequence alignment of the obtained seven potato spindle tuber viroid (PSTVd) sequences with their closest sequence(KY936876). These seven sequences represented four different variants (V1-4). Two distinctive mutations of the seven PSTVd sequences,not found in all known PSTVd sequences in China,are marked with rectangles.

3.5.PSTVd in tomato in China were not phylogenetically relate with that in potato

To exclude the possibility that PSTVd in tomato in China was transmitted from potato,the obtained four PSTVd variants from diseased tomato plants were used to construct phylogenetic trees together with the 265 PSTVd sequences isolated from naturally infectedSolanumplants. They did not cluster with the known PSTVd sequences from potato in China,regardless of which method was used for tree construction. This result indicates that PSTVd in tomato in China was unlikely transmitted from potato.

4.Discussion

For simultaneous detection of pospiviroids in tomato or other Solanaceae plants,we developed a new dotblot hybridization detection method using a universal pospiviroid probe. High reliability and low-cost make this a beneficial complement to regular detection toolkits for pospiviroids. Although molecular hybridization has lower detection sensitivity than PCR and derived methods,it has higher reliability in preventing false-positive results. Thus,this method can verify the detection results of pospiviroids by multiplexed RT-PCR/qPCR assays (Matsushitaet al.2010;van Brunschotet al.2014a;Olivieret al.2014;Yanagisawaet al.2017;Botermanset al.2020) or RTPCR/qPCR using universal primers (Verhoevenet al.2004;Botermanset al.2013;Luigiet al.2014;Tsenget al.2021). Compared with polyprobes of pospiviroids developed previously (Torchettiet al.2012),preparation of the universal probe requires only one cloning step,and thus,is much simpler and more cost-effective.

In addition to verifying the results of PCR and derived methods,dot-blot hybridization using the universal probe is suitable for large-scale survey of the six pospiviroids in tomato or other Solanaceae plants. Using this method,we finished the primary screening for more than 300 tomato leaf samples in a short time,and the results were consistent with those of RT-PCR. Although the universal probe had a slightly lower detection sensitivity than specific probes of the six pospiviroids,this sensitivity was deemed acceptable because the universal probe could detect low accumulation levels of PSTVd in inoculated tomato plants at three weeks post inoculation. Moreover,the detection sensitivity of the universal probe could be increased by adding one or more copies to elongate the size of the probe,as was done with the universal probe for coleviroids (Jianget al.2013).

Use of our developed universal probe for simultaneous detection of the six pospiviroids evaluated in this study confirms the practicality of this universal probe,synthesized from conserved sequences for simultaneous detection of viroids within a genus (Jianget al.2013).Future research should focus on developing similar universal probes for members in the genusApscaviroid,which contains the most members of the familyPospiviroidae(Di Serioet al.2021),despite the availability of the ployprobes to detect four apscaviroids that have been developed (Linet al.2011).

This is the first report,to our knowledge,of natural infection of PSTVd in tomato in China,despite PSTVd having been previously found in potato in many provinces(Songet al.2013;Qiuet al.2016). This raises the question of the origin of PSTVd in tomato in China.Although local origination cannot be completely excluded,it seems improbable. PSTVd has been present in China,including the province where the PSTVd-infected tomato plants were planted for many years and PSTVd-infected tomato plants were collected two years prior to the survey performed in this study. However,no additional positive samples were found,indicating that the probability of PSTVd transmission from other plants to tomato is extremely low,if it exists,in China. Moreover,the seven PSTVd sequences obtained from infected tomato plants contained two mutations not found in all known PSTVd sequences from potato in China,and these sequences did not cluster with the known PSTVd sequences from potato in China,regardless of which method was used for tree construction.

The PSTVd sequences of tomato isolates in China were the closest to that of a potato isolate from the Netherlands,suggesting it may have originated abroad.PSTVd can be transmitted by tomato seeds at markedly different rates for different cultivars (Faggioliet al.2015;Simmonset al.2015;Matsushita and Tsuda 2016).Recently,several PSTVd outbreaks and natural infections in commercial tomato were found to be related to seed transmission (van Brunschotet al.2014b;Dallet al.2019). Thus,seed transmission is considered as an important pathway for the introduction of pospiviroids.Indeed,the seeds of the PSTVd-infected tomato plants were imported from a French company. Therefore,PSTVd may have been introduced into China from abroad by imported tomato seeds. However,the role of seed transmission in the spread of pospiviroids in practice is possibly overestimated based on the finding that PSTVd was not successfully transmitted from seeds to seedlings/plants in large-scale grow-out trials of infested tomato seed lots (Verhoevenet al.2021). Altogether,the origin of tomato infecting PSTVd isolates in China is still an open question. In the future,more work should be done to monitor the incidence and spread of PSTVd in tomato in China.

5.Conclusion

In this study,we developed a universal probe based on a highly conserved 61-nt long sequence shared amongst six viroids,including CLVd,PCFVd,PSTVd,TASVd,TCDVd,and TPMVd. The universal probe had similar,although slightly reduced,detection sensitivity as the specific probes,but could simultaneous detect six different pospiviroid. Additionally,in using it to survey the incidence of tomato viroid(s) in China,we were able to detect PSTVd in a few greenhouse-grown tomato plants. According to the sequence analysis,these PSTVd isolates may have been introduced by imported tomato seed. Thus,future action should be taken to monitor viroid incidence in China.

Acknowledgements

This research was funded by the National Natural Science Foundation of China (31670149).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm