Tomato Planta Macho Viroid Research Articles

Reverse-transcription recombinase-aided amplification and CRISPR/Cas12a-based universal detection for fast screening and accurate identification of six pospiviroids infecting Solanaceae crops.

Pospiviroids, members of the genus Pospiviroid, can cause severe diseases in tomato and other Solanaceae crops, causing considerable economic losses worldwide. Six pospiviroids including potato spindle tuber viroid (PSTVd), tomato chlorotic dwarf viroid (TCDVd), tomato planta macho viroid (TPMVd), Columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), and tomato apical stunt viroid (TASVd) are regulated in many countries and organizations. Rapid, accurate detection is thus crucial for controlling the spread of these pospiviroids. For simultaneous detection of these six pospiviroids, we developed a rapid, visual method that uses a reverse transcription recombinase-aided amplification (RT-RAA) assay coupled with a clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 12a (CRISPR/Cas12a) system. In particular, this technique could achieve both universal detection and specific identification of the six target pospiviroids within 40 min. The universal detection could diagnose the six target pospiviroids in a single reaction, and the specific identification could identify each target pospiviroid without cross-reactivity of other pospiviroids. The sensitivity limits for the target pospiviroids detection with the proposed detection method were higher than those of the conventional reverse transcription-polymerase chain reaction (RT-PCR) method. We designed an RT-RAA-CRISPR/Cas12a-based universal detection method for both large-scale screening and accurate identification of the six target pospiviroids, which is appropriate for on-site detection. Our study results can aid in performing rapid, large-scale screening of multiple pests simultaneously. © 2024 Society of Chemical Industry.

A universal probe for simultaneous detection of six pospiviroids and natural infection of potato spindle tuber viroid (PSTVd) in tomato in China

Several viroid species 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 had 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.

Universal Primers for Rapid Detection of Six Pospiviroids in Solanaceae Plants Using One-Step Reverse-Transcription PCR and Reverse-Transcription Loop-Mediated Isothermal Amplification.

A number of viruses and viroids infect solanaceous plants causing severe yield losses. Several seed-borne viroids are listed as quarantine pathogens in many countries. Among them, columnea latent viroid, pepper chat fruit viroid, potato spindle tuber viroid, tomato apical stunt viroid, tomato chlorotic dwarf viroid, and tomato planta macho viroid are of major concerns. The objective of this study was to design and test universal primers that could be used to detect six viroids in solanaceous plants using one-step reverse transcription PCR (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Results revealed that a pair of degenerate primers could be used in a one-step RT-PCR to amplify six pospiviroids from Solanaceae seeds and plants. Moreover, five primers were designed and used in RT-LAMP to amplify six pospiviroids. The minimal concentration of viroid RNA required for a successful detection varied, ranging from 1 fg to 10 ng, depending on the species of viroid and detection method. In general, RT-LAMP was more sensitive than RT-PCR, but both assays were rapid and highly sensitive tools to detect six pospiviroids. Detection methods in use for these viroids require at least two different sets of primers. The assays developed in this research could facilitate the ability to screen a large number of solanaceous plants and seeds intended for import and export.

Development and validation of a real-time RT-PCR test for screening pepper and tomato seed lots for the presence of pospiviroids

Potato spindle tuber viroid and other pospiviroids can cause serious diseases in potato and tomato crops. Consequently, pospiviroids are regulated in several countries. Since seed transmission is considered as a pathway for the introduction and spread of pospiviroids, some countries demand for the testing of seed lots of solanaceous crops for the presence of pospiviroids. A real-time RT-PCR test, named PospiSense, was developed for testing pepper (Capsicum annuum) and tomato (Solanum lycopersicum) seeds for seven pospiviroid species known to occur naturally in these crops. The test consists of two multiplex reactions running in parallel, PospiSense 1 and PospiSense 2, that target Citrus exocortis viroid (CEVd), 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, including the former Mexican papita viroid). Dahlia latent viroid (DLVd) is used as an internal isolation control. Validation of the test showed that for both pepper and tomato seeds the current requirements of a routine screening test are fulfilled, i.e. the ability to detect one infested seed in a sample of c.1000 seeds for each of these seven pospiviroids. Additionally, the PospiSense test performed well in an inter-laboratory comparison, which included two routine seed-testing laboratories, and as such provides a relatively easy alternative to the currently used tests.

Vertical and Horizontal Transmission of Pospiviroids.

Viroids are highly structured, single-stranded, non-protein-coding circular RNA pathogens. Some viroids are vertically transmitted through both viroid-infected ovule and pollen. For example, potato spindle tuber viroid, a species that belongs to Pospiviroidae family, is delivered to the embryo through the ovule or pollen during the development of reproductive tissues before embryogenesis. In addition, some of Pospiviroidae are also horizontally transmitted by pollen. Tomato planta macho viroid in pollen infects to the ovary from pollen tube during pollen tube elongation and eventually causes systemic infection, resulting in the establishment of horizontal transmission. Furthermore, fertilization is not required to accomplish the horizontal transmission. In this review, we will overview the recent research progress in vertical and horizontal transmission of viroids, mainly by focusing on histopathological studies, and also discuss the impact of seed transmission on viroid dissemination and seed health.

Influence of the terminal left domain on horizontal and vertical transmissions of tomato planta macho viroid and potato spindle tuber viroid through pollen

Viroids can be transmitted vertically and/or horizontally by pollen. Tomato planta macho viroid (TPMVd) has a high rate of horizontal transmission by pollen, whereas potato spindle tuber viroid (PSTVd) does not. To specify the domain(s) involved in horizontal transmission, four viroid chimeras were created by exchanging the terminal left (TL) and/or pathogenicity (P) domains between PSTVd and TPMVd. PSTVd-based chimeras containing TPMVd-TL and P, or TPMVd-TL alone, displayed a high rate of horizontal transmission. TPMVd-based chimeras containing PSTVd-TL and P lost infectivity, and those containing PSTVd-TL alone displayed a low rate of horizontal transmission. In addition, the vertical transmission rate was also higher in the mutants containing TPMVd-TL than in the others. These findings indicate that the sequences or structures in the TL and P (although the role is limited) domains are important not only for horizontal but also for vertical transmission by pollen.

Pospiviroid Infection of Tomato Regulates the Expression of Genes Involved in Flower and Fruit Development.

Viroids are unencapsidated, single-stranded, covalently-closed circular, highly structured, noncoding RNAs of 239–401 nucleotides that cause disease in several economically important crop plants. In tomato (Solanum lycopersicum cv. Rutgers), symptoms of pospiviroid infection include stunting, reduced vigor, flower abortion, and reduced size and number of fruits, resulting in significant crop losses. Dramatic alterations in plant development triggered by viroid infection are the result of differential gene expression; in our study, we focused on the effect of tomato planta macho viroid (TPMVd) and Mexican papita viroid (MPVd) infection on gene networks associated with the regulation of flower and fruit development. The expression of several of the genes were previously reported to be affected by viroid infection, but two genes not previously studied were included. Changes in gene expression of SlBIGPETAL1 (bHLH transcription factor) and SlOVA6 (proline-like tRNA synthetase) are involved in petal morphology and fertility, respectively. Expression of SlOVA6 was down-regulated in flowers of TPMVd- and MPVd-infected plants, while expression of SlBIGPETAL1 was up-regulated in flowers. Up-regulation of SlBIGPETAL1 and down-regulation of SlOVA6 were positively correlated with symptoms such as reduced petal size and flower abortion. Expression analysis of additional tomato genes and a prediction of a global network association of genes involved in flower and fruit development and impacted by viroid infection may further elucidate the pathways underlying viroid pathogenicity.

Differences in dynamics of horizontal transmission of Tomato planta macho viroid and Potato spindle tuber viroid after pollination with viroid-infected pollen

For viroids, pollen transmission is an important transmission pathway to progeny seeds and new hosts. In the current study, we found that Tomato planta macho viroid (TPMVd)—but not Potato spindle tuber viroid (PSTVd)—was horizontally transmitted by pollen from petunia plants. Using tissue-printing hybridization to track the changes in viroid distribution after pollination, we noted that TPMVd was present in petunia stigma, styles, and eventually ovaries, whereas PSTVd was detected in the stigma and upper style but not the ovary. These findings suggest that horizontal transmission of viroids depends on the infection of the lower style and ovary during the elongation of pollen tubes after pollination. Additionally, TPMVd was transmitted horizontally, leading to systematic infection, when we used TPMVd-infected petunia pollen to pollinate the flowers of healthy tomato plants. Fertilization typically does not occur after heterologous pollination and thus likely is not required to accomplish horizontal transmission of viroids.

Distribution of Tomato planta macho viroid in germinating pollen and transmitting tract.

Vertical and horizontal pollen transmission is important for efficient infection by viroids. Vertical pollen transmission of viroids is attributed to the infection by viroid in the embryo sac through infected pollen. To identify the viroid infection in pollen and pollen tubes elongating through the transmitting tract, we used in situ hybridization to histochemically analyze the distribution of Tomato planta macho viroid (TPMVd) in pollen grains, the stigma, and style of petunia plants. TPMVd was present in the generative nucleus and vegetative nucleus of mature infected pollen grains and germinating pollen grains. During pollen tube growth, TPMVd was present in the vegetative nucleus and two sperm nuclei, which were generated by division of the generative nucleus in the style transmitting tract. These findings indicated that viroid infection in sperm nuclei is responsible for vertical pollen transmission of viroids. TPMVd infection from TPMVd-infected pollen tubes to the transmitting tract was not observed. In addition, TPMVd signals were not confirmed in the stigma and transmitting tract of TPMVd-infected petunia plants, suggesting that viroids may not replicate in these tissues at the stage of mature style. Therefore, TPMVd may leak from the pollen tube somewhere in the ovary, except in the transmitting tract, during the horizontal transmission of TPMVd.

Host ranges and seed transmission of Tomato planta macho viroid and Pepper chat fruit viroid

Host ranges of Tomato planta macho viroid (TPMVd) and Pepper chat fruit viroid (PCFVd) were investigated across 32 species representing 10 genera, including horticultural plants that are frequently imported in large numbers by Japan. Plants were infected by mechanical sap inoculation, and viroid presence was checked using reverse-transcription polymerase chain reaction and back-inoculation of tomato. Among the 32 species, 16 and 15 were susceptible to TPMVd and PCFVd, respectively; most belonged to Solanaceae, and symptoms were only observed in tomato, potato, green pepper, and Solanum muricatum. Seed transmission of TPMVd and PCFVd was carried out using seeds obtained from infected tomato, Capsicum annuum, and Petunia × hybrida plants, and from a series of crosses involving healthy or infected Petunia × hybrida parental material. The seed transmission rate of TPMVd was 0–4.4% in tomato and 17.5 or 43.3% in Petunia × hybrida, while that of PCFVd was 0–1.4% in tomato and 0 or 16.8% in Petunia × hybrida. Seed transmission via the viroid-infected pollen parent or infected seed parent was investigated in Petunia × hybrida; rates were 91.8% and 100% for TPMVd and 69.2% and 65.3% for PCFVd, respectively. These data indicated a risk of unexpected worldwide viroid spread via international trade of asymptomatic plants and viroid-infected seeds and pollen.

A single base pair in the right terminal domain of tomato planta macho viroid is a virulence determinant factor on tomato

Tomato planta macho viroid (TPMVd), including isolates previously designated as Mexican papita viroid (MPVd), causes serious disease on tomatoes in North America. Two predominant variants, sharing 93.8% sequence identity, incited distinct severe (MPVd-S) or mild (MPVd-M) symptoms on tomato. To identify virulence determinant factor, a series of chimeric infectious clones were generated using synthetic DNA approach to progressively replace each structural domain between the two variants. In bioassays on tomato ‘Rutgers’, three chimeras containing Terminal Left and Pathogenicity (MPVd-H1), Central (MPVd-H2), or Variable (MPVd-H3) of MPVd-S, incited mild to intermediate symptoms. However, a chimera containing Terminal Right (TR) of MPVd-S (MPVd-H4) incited severe symptoms. Only one base-pair mutation in the TR domain between MPVd-M (176U:A185) and MPVd-S (174G:C183) was identified. A reciprocal mutant (MPVd-H5) rendered the chimeric viroid mild on tomato. This single base-pair in the TR domain was determined as the virulence determinant factor for TPMVd.

In silico prediction and validation of potential gene targets for pospiviroid-derived small RNAs during tomato infection

Viroids are small, covalently closed, circular non-coding RNA pathogens of flowering plants. It is proposed that the symptoms of viroid pathogenesis result from a direct interaction between the viroid genomic RNA and unknown host plant factors. Using a comparative genomic approach we took advantage of the detailed annotation of the Arabidopsis thaliana (Arabidopsis) genome to identify sequence homologies between putative viroid-derived small RNAs (vd-sRNAs) and coding regions in the plant genome. A pool of sequence homologies among 29 species of the Pospiviroidae family and the Arabidopsis genome was analyzed. Using this strategy we identified putative host gene targets that may be involved in symptom expression in viroid-infected plants. In this communication, we report the in silico prediction and the experimental validation of pospiviroid-derived sRNAs conserved in the lower strand of the pathogenicity domain of seven viroid species infecting tomato; those vd-sRNAs targeted for cleavage the host mRNA encoding a conserved tomato WD40-repeat protein (SolWD40-repeat; SGN_U563134). Analysis of SolWD40-repeat expression indicated that this gene is down-regulated in tomato plants infected with tomato planta macho viroid (TPMVd). Furthermore, 5′ RLM-RACE revealed that the SolWD40-repeat mRNA is cleaved at the predicted target site showing complementarity to a corresponding TPMVd-sRNA identified in silico. Our approach proved to be useful for the identification of natural host genes containing sequence homologies with segments of the Pospiviroidae genome. Using this strategy we identified a functionally conserved gene in Arabidopsis and tomato, whose expression was modified during viroid infection in the host genome; regulation of this gene expression could be guided by vd-sRNA:mRNA complementarity, suggesting that the comparison of the Arabidopsis genome to viroid sequences could lead to the identification of unexpected interactions between viroid RNAs and their host.

Mexican papita viroid and tomato planta macho viroid belong to a single species in the genus Pospiviroid

Tomato planta macho viroid (TPMVd) and Mexican papita viroid (MPVd) are two closely related (>90% sequence identity) members of the genus Pospiviroid. Their current status as members of separate species is based upon the reported ability of TPMVd to replicate in Gomphrena globosa and the inability of this viroid to evoke flower break in N. glutinosa. Characterization of a viroid recently isolated from diseased tomato plants grown in Mexico (identical to GenBank accession GQ131573) casts doubt on this earlier report and indicates that these viroids should be classified as members of a single species. Giving priority to the older name, we propose including both of these viroids in the current species Tomato planta macho viroid.

First Report of Mexican papita viroid Infecting Greenhouse Tomato in Canada.

In the summer of 2008, tomato (Solanum lycopersicum) plants in a large greenhouse tomato facility located in Delta, British Columbia, Canada exhibited general stunting, chlorosis, and purple-leaf symptoms that were distinct from those of Pepino mosaic virus (PepMV) (1). Diseased plants were localized mainly in two rows in a section of the greenhouse and produced no fruits or only fruits with reduced size. Leaf samples were collected from four individuals among numerous diseased plants in this greenhouse. Screening samples by ELISA, PCR, or reverse transcription (RT)-PCR for PepMV, Tomato spotted wilt virus, Tomato yellow leaf curl virus, Tomato torrado virus, Tomato apex necrosis virus, and Begomovirus, Tobamovirus, and Pospiviroid species showed that all four plants had a mixed infection of both PepMV and a pospiviroid. RT-PCR with the pospiviroid-specific primers Pospil-RE and Pospil-FW (3) amplified the expected 196-bp products from these four samples. Each amplicon was cloned into the pCR4-TOPO vector (Invitrogen, Carlsbad, CA) and one individual cDNA clone from each isolate was sequenced. BLASTN analyses of nucleotide sequences of these clones showed 97 to 99% identity to Mexican papita viroid (MPVd) isolates currently in the NCBI Genbank. These four newly identified MPVd isolates were not identical; seven nucleotide substitutions or indels were identified in this region. The full viroid genome was obtained by RT-PCR in isolate VF2 with a new reverse primer MPVd-RE (5' GATCCCTGAAGCGCTCCT 3') in combination with the forward primer Pospil-FW (3). Using the same approach as stated above, this amplicon was cloned and sequenced. The nucleotide sequence of the 196-nt amplicon previously amplified and cloned from the isolate VF2 genome was identical to this region in the genomic clone. BLASTN analysis showed that the VF2 genome (GenBank Accession No. FJ824844) had >98% sequence identity to each of nine MPVd isolates (GenBank Accession Nos. L78454 and L78456-L78463), 94% identity to Tomato planta macho viroid (TPMVd) (GenBank Accession No. K00817) and ~80% identity to Tomato chlorotic dwarf viroid (GenBank Accession Nos. EF582392-EF582393). Prior to this find, MPVd had been identified only in papita (Solanum cardiophyllum) in Mexico and is considered a possible ancestor of TPMVd, Potato spindle tuber viroid (PSTVd), and possibly of other PSTVd-group viroids now infecting crop plants (2). The origin of MPVd in this greenhouse facility in Delta, British Columbia is unknown. The infected plants were destroyed by the grower. The pathogenicity of MPVd isolates characterized in this study was not evaluated on tomato because of quarantine regulations governing this viroid in the United States. The identification of MPVd infecting an important agricultural crop (tomato) outside its center of origin in Mexico indicates a potentially important major shift in the epidemiology of MPVd. To our knowledge this is the first report of MPVd from tomato in Canada.

Precisely full length, circularizable, complementary RNA: an infectious form of potato spindle tuber viroid.

The replication of many viral and subviral pathogens as well as the amplification of certain cellular genes proceeds via a rolling circle mechanism. For potato spindle tuber (PSTVd) and related viroids, the possible role of a circular (-)strand RNA as a template for synthesis of (+)strand progeny is unclear. Infected plants appear to contain only multimeric linear (-)strand RNAs, and attempts to initiate infection with multimeric (-)PSTVd RNAs generally have failed. To examine critically the infectivity of monomeric (-)strand viroid RNAs, we have developed a ribozyme-based expression system for the production of precisely full length (-)strand RNAs whose termini are capable of undergoing facile circularization in vitro. Mechanical inoculation of tomato seedlings with electrophoretically purified (-)PSTVd RNA led to a small fraction of plants becoming infected whereas parallel assays with an analogous tomato planta macho viroid (-)RNA resulted in a much larger fraction of infected plants. Ribozyme-mediated production of (-)PSTVd RNA in transgenic plants led to the appearance of monomeric circular (-)PSTVd RNA and large amounts of (+)PSTVd progeny. No monomeric circular (-)PSTVd RNA could be detected in naturally infected plants by using either ribonuclease protection or electrophoresis under partially denaturing conditions. Although not a component of the normal replicative pathway, precisely full length (-)PSTVd RNA appears to contain all of the structural and regulatory elements necessary for initiation of viroid replication.

Mexican papita viroid: putative ancestor of crop viroids.

The potato spindle tuber disease was first observed early in the 20th century in the northeastern United States and shown, in 1971, to be incited by a viroid, potato spindle tuber viroid (PSTVd). No wild-plant PSTVd reservoirs have been identified; thus, the initial source of PSTVd infecting potatoes has remained a mystery. Several variants of a novel viroid, designated Mexican papita viroid (MPVd), have now been isolated from Solanum cardiophyllum Lindl. (papita güera, cimantli) plants growing wild in the Mexican state of Aguascalientes. MPVd's nucleotide sequence is most closely related to those of the tomato planta macho viroid (TPMVd) and PSTVd. From TPMVd, MPVd may be distinguished on the basis of biological properties, such as replication and symptom formation in certain differential hosts. Phylogenetic and ecological data indicate that MPVd and certain viroids now affecting crop plants, such as TPMVd, PSTVd, and possibly others, have a common ancestor. We hypothesize that commercial potatoes grown in the United States have become viroid-infected by chance transfer of MPVd or a similar viroid from endemically infected wild solanaceous plants imported from Mexico as germplasm, conceivably from plants known to have been introduced from Mexico to the United States late in the 19th century in efforts to identify genetic resistance to the potato late blight fungus, Phytophthora infestans.

A viroid from Solanum pseudocapsicum closely related to the tomato apical stunt viroid.

A viroid was isolated from symptomless Solanum pseudocapsicum cultivar (cv) 'New Patterson' plants using the bidirectional PAGE method for analysis of small circular RNA molecules. The viroid was transmitted to tomato by mechanical inoculation. Infected tomato plants developed symptoms similar to those caused by potato spindle tuber viroid (PSTVd). Cloning and sequencing revealed that the viroid from Solanum pseudocapsicum is closely related to the tomato apical stunt viroid (TASVd). Therefore, the new viroid sequence variant has been named TASVd-S. The circular RNA of TASVd-S consists of 360 nucleotides which can potentially form a rod-like structure with a high degree of base-pairing like all the other known viroids. The nucleotide composition is 99 G, 72 A, 101 C and 88 U, corresponding to 200 G + C, 160 A + U with a GC content of 55.6%. In total 71% of the residues are base-paired and the basepaired residues consist of 73 G:C (57%), 43 A:U (34%) and 11 G:U (9%) base pairs. The most stable rod-like secondary structure of this viroid has 80 G:C, 39 A:U and 11 G:U base pairs with a minimum free energy of -147.5 kcal/mol (-616.6 kJ/mol). The sequence similarity of the left terminal (T1) domain of TASVd-S and the T1 domain of tomato planta macho viroid (TPMVd) is higher than the sequence similarity of these domains of TASVd-S and TASV-d.

Grapevine viroid 1B, a new member of the apple scar skin viroid group contains the left terminal region of tomato planta macho viroid

GV1 B is one of five viroids that have recently been purified from grapevines. GV1 B has now been sequenced and its 363 nucleotide residues can potentially form the typical rod-like structure of viroids with 67% of nucleotides basepaired. GV1B has highest sequence similarity with grapevine yellow speckle viroid (GYSV; 73%) and has a central sequence which is conserved in GYSV and apple scar skin viroid (ASSV) which have been reported to constitute the ASSV group. Therefore, we have placed GV1 B into the ASSV group. GV1 B contains a direct repeat sequence at the terminal portions of its T1 and T2 regions. GV1 B also contains a sequence of 69 nucleotides in the terminal portion of its Ti region which is almost identical to the corresponding region in tomato planta macho viroid (TPMV). This provides further evidence of the importance of RNA recombination in viroid evolution.