Abstract
Citrus hosts various phytopathogens that have impacted productivity, including viroids. Missing data on the status of viroids in citrus in Palestine were not reported. This study was aimed to detect any of Citrus exocortis viroid (CEVd), Citrus viroid-III (CVd-III), and Citrus viroid-IV (CVd-IV) in the Palestinian National Agricultural Research Center (NARC) germplasm collection Field inspections found symptoms such as leaf epinasty; vein discoloration, and bark cracking on various citrus varieties. RT-PCR revealed a significant prevalence of CVd-IV; CEVd and CVd-III (47%, 31%, and 22%; respectively). CVd-III variants with 91.3% nucleic acid sequence homology have been reported. The sequence of each viroid were deposited in GenBank as (OP925746 for CEVd, OP902248 and OP902249 for CVd-III-PS-1 and -PS-2 isolates, and OP902247 for CVd-IV). This was the first to report three of citrus viroids in Palestine, appealing to apply of phytosanitary measures to disseminate healthy propagating materials free from viroids.
Similar content being viewed by others
Introduction
Citrus is a host to several pathogens, many of which have an economic impact on the crop. Viroids are among of these induced annual worldwide losses in fruit trees1,2. They are known as causal agents of many diseases on herbaceous and woody plants3 causing symptoms on leaves (deformation, vein discoloration, epinasty, mottling, chlorotic and necrosis), stems (cankers, bark scaling and/or cracking), fruits malformation, and in severe cases plants death4.
The Potato spindle tuber viroid was the first detected on potato plants in 1971 by Danier5, before many viroids were reported infecting several plant crops worldwide, particularly in warm climate regions6.
To date, eight viroid species belonging to the Pospiviroidae family have been reported in citrus: Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd), Citrus viroid-III (CVd-III), Citrus viroid-IV (CVd-IV; syn. Citrus bark cracking viroid (CBCVd), Citrus viroid-V (CVdV), Citrus viroid-VI (CVd-VI), Citrus viroid-VII (CVd-VII), Citrus dwarfing viroid (CDVd), and Citrus bent leaf viroid (CBLVd)7,8,9,10,11,12.
The main constraints on the use of PCR in diagnosis are the cost and availability of sufficient sample materials which multiplex PCR can overcome13. Multiplex PCR enables the amplification of multiple target sequences by employing more than one pair of primers in the reaction. Multiplex PCR, with no loss of test utility, has the potential to result in significant time and labor savings in the laboratory. It has been successfully used in a variety of nucleic acid diagnostic applications, including RNA detection, quantitative analysis, mutation and polymorphism analysis, gene deletion analysis, and many others14. The technique has been demonstrated to be a valuable method for identifying viruses, bacteria, fungi, and other microbes in the field of infectious diseases. CVd-IV (syn. CBCVd) was considered as minor pathogen in the genus Citrus11, but it was associated with severe bark cracking on trifoliate orange rootstock15,16,17. CEVd was known to have a wide host range6,18,19, and symptoms in different species of citrus, such as stunting, bark sloughing and cracking, leaf epinasty and cracks in the petiole20,21,22,23,24,25. CVd-III was reported to have of particular interest due to its genomic variability beside its significant reduction of growth and yield26,27,28,29.
Since no data on the existence of citrus viroids in the Palestinian territories was available, research on their existence and prevalence was initiated as part of a collaboration with the National Agricultural Research Center for eight viroids. The purpose of this study was to determine the presence and prevalence of three viroids (CEVd; CVd-III; and CVd-IV) in Palestinian germplasm collection and to be the first report in this region.
Results and discussion
Recognition of putatively viroid’s symptoms
No data was available about the existence of citrus viroids in the country. However, many reported in the region6,30,31,32,33. Lacking specific symptoms; which are sometimes symptomless or similar to those of virus sources, it could be hard to specifically determine the viroid-infected trees34. Field inspections were carried out on citrus germplasm collection at the National Agriculture Research Center (NARC), Jenin-Palestine. Putative viroid like symptoms such as epinasty, stunting, vein clearing, discoloration, distortion of leaves, mottling, necrotic or chlorotic spots, scaling, cankers, and bark cracking were reported (Fig. 1). These observations nudge the believe the existence of viroid infections in the germplasm collection which might be virus free/tested ones.
RT-PCR detection
All these selected three viroids were ensured through molecular detection in a single RT-PCRs as well as multiplex PCR, which successfully applied to all tested viroids. multiplex PCR was performed successfully, and thus it could be recommended as quick and efficient tool for simultaneous detection of viroids at one reaction (Fig. 2).
Surprisingly, a relatively high total prevalence of infection of these three citrus viroids was reported (52.4%), of which CVd-IV was having the highest incidence (Fig. 3), even though it was found to be less frequent in mixed infections (Table 1).
Interestingly, samples from citrus rootstocks (Volkameriana and Trifoliata) were found infected with CVd-IV. This might explain high prevalence of the viroid among citrus in Palestine, as well as it has a wide host range35. CVd-IV was reported to be one of the main viroids circulating in all citrus-growing areas worldwide34,36.
CEVd, one of the most well-known viroid in citrus, was detected in 23.8% of tested samples (Fig. 3). This result was expected since it was found in many countries in Africa (South Africa, Ghana, Sudan, Morocco, Algeria, Tunisia, Libya and Egypt) and in Middle East (Turkey, Cyprus, Israel, Saudi Arabia and Oman)30,31,33,37,38,39.
These viroids were found in single and mixed infections. However, mixed infections could be the most likely cause, which magnifies the severity of the symptoms and reduce the productivity of citrus trees36.
Sequence analysis
The obtained sequences from the viroids (CVd-IV, CEVd, and CVd-III) were BLASTn searched to reveal high similarity with each corresponding viroids in the GenBank data. The sequences were deposited at GenBank under the accession numbers: OP902247 (CVd-IV), OP925746 (CEVd), OP902248 (CVd-III-PS-isolate-1) and OP902249 (CVd-III-PS-isolate-2).
Even though BLASTn searching of the CVd-IV revealed high similarity to those from Turkey (MZ995261) and Oman (KC121568), in accordance with what was proposed for their middle East origin31. A phylogenetic tree, constructed using molecular evolutionary genetics analysis across computing platforms (MEGA-11)40, revealed that CVd-IV clustered with other similar viroids available in GenBank records (Fig. 4a).
Nucleotide sequences obtained from CVd-III infected samples, two isolates were revealed to have 91.3 percent similarity by Pairwise Sequence Alignment-EMBOSS. Water provided by the European Molecular Biology Laboratory (EMBL); suggesting they were putative locally mutated. Thus, these two isolate considered varied within the viroid genetic variation in the country and designated as CVd-III-PS-isolate-1 and CVd-III-PS-isolate-2. They were clustered together in a phylogenetic tree with other GenBank deposited isolates from the Middle East region, as well as others isolated worldwide (Fig. 4b). The same can be noticed for the CEVd as it aligns with those isolate accessions retrieved from the GenBank (Fig. 4b,c).
Although, this study was the first to report citrus viroid in Palestine, their existence in relatively high prevalence is alarming, especially once rootstocks were detected infected. Knowing their main route of dissemination throughout propagating materials21,41; phytosanitary actions are highly recommended, which is the only way to prevent further spread of viroids42,43. Certification program and sanitary actions to exclude these viroids side by side with viruses are highly appealing. National germplasm collections must be subjected to strictly measurement to ensure distributing propagating plant materials free of viruses as well as virus like pathogens.
Methods
Field inspections
Several field visits to the germplasm collection of the National Agricultural Research Center were achieved since it was established in 2014 and intensified in the last 3 years (2020–2022). Putative virus—like disorders were inspected in 11 out of 91 samples, including leaf deformation and/or discoloration; as well as growth abnormalities and fruit deformation. Leaves from different forty-two citrus plants (Clementine, Grapefruit, Lemon, Orange, Kumquats, Pomelo, and two rootstocks, Volkameriana and Trifoliata) were collected and conserved for molecular assays.
Molecular assays
Two-step RT-PCR was applied to detect any of citrus viroids: CEVd, CVd-IV, and CVd-III, using specific sets of primers as shown in Table 2.
Total nucleic acid (TNA) was extracted from 100 mg of citrus tissues according to Foissac et al.48 procedures. Briefly, leaf tissues were homogenized in grinding buffer (pH 5.6–5.8) [4M Guanidine thiosianate, 0.2 M sodium acetate, pH 5.2, 25 mM EDTA, 1.0 M potassium acetate, 2.5% PVP-40 and 2% Sodium bisulphate] with addition of 0.5% of sodium metabisulfite and 150 µl of 10% Sodium lauroyl sarcosinate (Sarkosyl) before incubating at 70°C for 10 min. On ice, 500 µl of extraction were mixed with binding solution (250 μl ethanol absolute, 500 μl NaI 6M and 35μl of re-suspended silica) and gently agitated for 10 min at room temperature. Pellets were then collected and subjected to two washing with [10.0 mM Tris–HCl, pH7.5; 0.5 mM EDTA, 50.0 mM NaCl, 50% Ethanol]. 150 μl TNA were liberated by distilled water, and NanoDrop 2000/2000c UV–Vis spectrophotometers (JENWAY, Genova Nano, Fisher Scientific UK company, England.) was used to measure their quantity and quality.
A reverse transcription reaction was carried out from 500 ng TNA and 200 units of SuperScript™ III RT (Life Technologies Corporation) in a final volume of 20 µl. Firstly, TNA were mixed with 0.5µl of random hexamers primer (50ng/µl), 0.5 µl oligodT (50 µM), and 1 µL dNTPs (10 μM) in a final volume of 10 µl. The mixture was incubated at 65 °C for 5 min. On ice, 2 µl of 10 × RT buffer, 2 µl of 0.1M DTT, 4µl of 25mM MgCl2, 1 µL RNaseOUT(40U/µL), and 1µL SuperScriptIII RT(200U/µL) were added to the mixture, before incubation at 25 °C/10 min followed by 50 °C/50 min. The reaction was terminated at 85 °C for 5 min and cooled at 4 °C for next use in PCR reaction.
A single PCR was performed for each viroid with its pair of primers separately (Table2). 2 μl cDNA were added to a final volume of 25 μl PCR mix composed of: 2.5μl of 10 × Taq polymerase buffer; 2 μl of 50 μM MgCl2; 0.5 μl of 10 μM dNTPs and 0.25 μl of Taq polymerase (5 unit/μl) (Promega Corporation, USA)), with primer pairs final concentrations of 0.2 μM for CVd-III and CVd-IV, and 0.5 μM for CEVd, meanwhile 18s primers were reached up to 0.14 μM. After a 5-min denaturation stage at 94 °C, 45 cycles of cDNA amplification were performed. Each cycle consisted of a 50-s denaturation stage at 94 °C followed by a 55 °C annealing step for 50 s, and 2 min extension at 72 °C. The elongation stage was at 72 °C for10 min. PCR products were visualized under UV light detector in 1.2% agarose gel stained with GelRed (Biotium) according the manufacturer’s recommended protocol.
For quick detection of several viroids with one reaction with high sensitivity, specificity and cost-effective, multiplex PCR was tested according to Wang et al.49. The mix of all viroid primer pairs at a final concentration of 0.2 μM except for CEVd (0.5 μM), and 18s (0.14 μM). Briefly, 2 μl cDNA mixture were used in PCR amplification with 2.5μl of 10 × Taq polymerase buffer (Promega Corporation, USA), 2 μl of 50 μM MgCl2, 0.5 μl of 10 μM dNTPs, and 0.25 μl of Taq polymerase (5 unit/μl) in 25 μl final volume. The cDNA amplification was done with 45 cycles after initial denaturation at 94 °C for 5 min. Each cycle consisted of: denaturation at 94 °C for 50 s, annealing at 58 °C for 50 s and extension at 72 °C for 2 min. The final extension was done at 72 °C for 10 min.
Sequencing and data analysis
Selected isolates of the viroids (CVd-IV, CEVd, and CVd-III) (as part of a collaboration work with the National Agricultural Research Center for detecting eight of citrus viroids) were sequenced using the sequencing facilities at An-Najah National Hospital-Nablus. The sequences were searched with databases using BLASTn on the National Center for Biotechnology Information–NCBI web server. The obtained sequences were deposited at GenBank (NCBI) and their accession numbers were obtained for each. Nucleotide sequence similarity was achieved using by Pairwise Sequence Alignment-EMBOSS Water provided by European Molecular Biology Laboratory (EMBL).
Ethics approval and consent to participate
Authors confirm that the use of plants in the present study complies with international, national and/or institutional guidelines.
Conclusion
Three of citrus viroids (CEVd, CVd-III, and CVd-IV) had been detected in Palestinian germplasm collection, reporting their existence for the first time in the country. For quick detection of several viroids with one reaction with high sensitivity, specificity and cost-effective. Multiplex RT-PCR was recommended. Applying phytosanitary measurements including viroids was highly demanded.
Data availability
Adequate and clear descriptions of the applied materials and tools are provided in the materials and method section of manuscript. In addition, the obtained data is clearly justified by mentioning the figures and tables in the manuscript. The datasets generated and/or analyzed during the current study are available in the [Citrus Viroids] repository, in the link:https://drive.google.com/drive/folders/1Bv0wpKKxkRCW1C_0NXHMbiX8zZfKtBf8?usp=drive_link. The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. All data generated or analyzed during this study are included in this published article [and its Supplementary Information files].
References
Nemeth M, Virus, mycoplasma and Rickettsia Diseases of fruit trees. Akademiai kiado. Budapest, Hungary, 840 pp. (1986)
Roistacher, C. N. Graft Transmissible Disease of Citrus Handbook for Detection and Diagnosis 286 (FAO Publication Division, 1992).
Flores, R., Hernández, C., Martínez de Alba, A. E., Daròs, J. A. & Serio, F. D. Viroids and viroid-host interactions. Annu. Rev. Phytopathol. 43, 117–139 (2005).
Kovalskaya, N. & Hammond, R. W. Molecular biology of viroid–host interactions and disease control strategies. Plant Sci. 228, 48–60 (2014).
Owens, R. A., Sano, T. & Duran-Vila, N. Plant viroids: Isolation, characterization/detection, and analysis. Methods Mol. Biol. 894, 253–271. https://doi.org/10.1007/978-1-61779-882-5_17 (2012).
El-Dougdoug, K. A. et al. Geographical distribution of viroids in Africa and the Middle East. In Viroids and Satellites (eds Hadidi, A. et al.) 485–496 (Academic Press, 2017).
Serra, P., Barbosa, C. J., Daròs, J. A., Flores, R. & Duranvila, N. Citrus viroid V: Molecular characterization and synergistic interactions with other members of the genus Apscaviroid. Virology 370(1), 102–112 (2008).
Ito, T., Ieki, H., Ozaki, K. & Ito, T. Characterization of a new citrus viroid species tentatively termed Citrus viroid OS. Arch. Virol. 146(5), 975–982 (2001).
Di Serio F, Li Sh, Pallás V, Randles J, Viroid Taxonomy. https://doi.org/10.1016/B978-0-12-801498-1.00013-9 (2017).
Seigner, L., Liebrecht, M., Keckel, L., Einberger, K. & Absmeier, C. Real-time RT-PCR detection of Citrus bark cracking viroid (CBCVd) in hops including an mRNA-based internal positive control. J. Plant Dis. Protect. 127, 763–767 (2020).
Duran-Vila, N., Roistacher, C. N., Riverabustamante, R. & Semancik, J. S. A definition of citrus viroid groups and their relationship to the exocortis disease. J. Gen. Virol. 69(12), 3069–3080 (1988).
Chambers, G. A., Donovan, N. J., Bodaghi, S., Jelinek, S. M. & Vidalakis, G. A novel citrus viroid found in Australia, tentatively named Citrus viroid VII. Arch. Virol. 163(1), 215–218 (2018).
Elnifro, E. M., Ashshi, A. M., Cooper, R. J. & Klapper, P. E. Multiplex PCR: Optimization and application in diagnostic virology. Clin. Microbiol. Rev. 13(4), 559–570. https://doi.org/10.1128/CMR.13.4.559 (2000).
Walker, B., Powers-Fletcher, M. V., Schmidt, R. L. & Hanson, K. E. Cost-effectiveness analysis of multiplex PCR with magnetic resonance detection versus empiric or blood culture-directed therapy for management of suspected Candidemia. J. Clin. Microbiol. 54, 718–726 (2016).
Vernière, C. et al. Citrus viroids: Symptom expression and effect on vegetative growth and yield of clementine trees grafted on trifoliate orange. Plant. Dis 88, 1189–1197 (2004).
Ito, T. et al. Multiple citrus viroids in citrus from Japan and their ability to produce exocortis-like symptoms in citron. Phytopathology 92(5), 542–547 (2002).
Vernière, C. et al. Interactions between citrus viroids affect symptom expression and field performance of clementine trees grafted on trifoliate orange. Phytopathology 96, 356–368 (2006).
Duran-Vila, N., and J. S. Semancik. "Citrus viroids." Viroids (2003): 178–194.
Duran-Vila, N. Citrus exocortis viroid. In Viroids and satellites (eds Hadidi, A. et al.) 169–179 (Academic Press, 2017).
Bernard, L., Duran, V. N. & Elena, S. F. Effect of citrus hosts on the generation, maintenance and evolutionary fate of genetic variability of citrus exocortis viroid. J. Gen. Virol. 90, 2040–2049 (2009).
Lin, C. Y., Wu, M. L., Shen, T. L., Yen, H. H. & Hung, T. H. Multiplex detection, distribution, and genetic diversity of Hop stunt viroid and Citrus exocortis viroid infecting citrus in Taiwan. Virol. J. 12, 11. https://doi.org/10.1186/s12985-015-0247-y (2015).
Škorić, D., Conerly, M., Szychowski, J. A. & Semancik, J. S. CEVd induced symptom modification as a response to a host specific temperature sensitive reaction. Virology 280, 115–123 (2001).
Singh, R. P., Dilworth, A. D., Ao, X., Sing, M. & Baranwal, V. K. Citrus exocortis viroid transmission through commercially-distributed seeds of Impatiens and Verbena plants. Eur. J. Plant Pathol. 124, 691–694 (2009).
Palukaitis, P. What has been happening with viroids?. Virus Gen. 49, 175–184 (2014).
Van Brunschot, S. L., Persley, D. M., Roberts, A. & Thomas, J. E. First report of pospiviroids infecting ornamental plants in Australia: Potato spindle tuber viroid in Solanum laxum (synonym S. jasminoides) and Citrus exocortis viroid in Petunia spp.. New Dis. Rep. 29, 3 (2014).
Vernière, C. et al. Citrus viroids: Symptom expression and effect on vegetative growth and yield of clementine trees grafted on trifoliate orange. Plant Dis. 88(11), 1189–1197. https://doi.org/10.1094/PDIS.2004.88.11.1189 (2004).
Tessitori M, La Rosa R, Di Serio F, Albanese G, Catara A. Molecular characterization of a Citrus viroid III (CVd-III) associated with citrus dwarfing in Italy. In Proc Conf Int Org Citrus Virol 15th IOCV, pp. 387–389. Edited by N. Duran-Vila, R. G. Milne & J. V. da Grac¸a. Riverside, CA: IOCV.(2002).
Semancik, J. S., Rakowski, A. G., Bash, J. A. & Gumpf, D. J. Application of selected viroids for dwarfing and enhancement of production of Valencia orange. J. Hort. Sci. 72, 563–570 (1997).
Owens, R. A. et al. Both point mutation and RNA recombination contribute to the sequence diversity of citrus viroid III. Virus Genes 20, 243–252 (2000).
Abou Kubaa, R., Saponari, M., El-Khateeb, A. & Djelouah, K. First identification of Citrus exocortis viroid (CEVd) and Citrus dwarf viroid (CVd-III) in citrus orchards in Syria. J. Plant Pathol. 98(1), 171. https://doi.org/10.4454/JPP.V98I1.045 (2016).
Al-Harthi, S. A., Al-Sadi, A. M. & Al-Saady, A. A. Potential of citrus budlings originating in the Middle East as sources of citrus viroids. Crop Protect. 48, 13–15 (2013).
Ashulin, L., Lachman, O., Hadas, O. & Bar-Joseph, M. Nucleotide sequence of a new viroid species, Citrus bent leaf viroid (CBLVd) isolated from grapefruit in Israel. Nucleic Acids Res. 19, 4767. https://doi.org/10.1093/nar/19.17.4767 (1991).
Hadidi A, Mazyad HH, Madkour MA, Bar-Joseph M, (2003) Viroids in the Middle East. In: Hadidi, A., Flores, R., Randles, J.W., Semancik, J.S (eds) Viroids. (CSIRO Publishing, UK) 275–278.
Aviña-Padilla, K. et al. Mexico: A landscape of viroid origin and epidemiological relevance of endemic species. Cells 11, 3487 (2022).
Semancik, J. S. & Vidalakis, G. The question of Citrus viroid IV as a Cocadviroid. Arch. Virol. 150(6), 1059–1067 (2005).
Belabess, Z., Radouane, N., Sagouti, T., Tahiri, A. & Lahlali, R. A current overview of two viroids prevailing in citrus orchards: Citrus exocortis viroid and hop stunt viroid. In Citrus—Research, Development and Biotechnology (eds Khan, M. S. & Khan, I. A.) (IntechOpen, 2021). https://doi.org/10.5772/intechopen.95914.
Da Graca, J. V. & van Vuuren, S. P. Viroids in Africa. In Viroids (eds Hadidi, A. et al.) 290–292 (CSIRO Publishing, 2003).
Opoku, A. A. Incidence of exocortis virus disease of citrus in Ghana. Ghana J. Agric. Sci. 5, 65–71 (1972).
Najar, A., Hamdi, I., Varsani, A. & Durán-Vila, N. Citrus viroids in Tunisia: Prevalence and molecular characterization. J. Plant Pathol. 99, 787–792 (2017).
Tamura, K., Stecher, G. & Kumar, S. MEGA11: Molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 38(7), 3022–3027. https://doi.org/10.1093/molbev/msab120 (2021).
Alkowni, R. Phytoviruses in Palestine: Status and Future Perspectives. Najah Univ. J. Res. (Nat. Sci.) 31(1), 11–34 (2017).
Papayiannis, L. C. Diagnostic real time RT-PCR for the simultaneous detection of Citrus exocortis viroid and Hop stunt viroid. J. Virol. Methods 196, 93–99 (2014).
Gergerich, R. C. et al. Safeguarding fruit crops in the age of agricultural globalization. Plant Dis. 99, 176–187 (2015).
Gross, H. J. et al. Nucleotide sequence and secondary structure of citrus exocortis and Chrysanthemum stunt viroid. Eur. J. Biochem. 121, 249–257 (1982).
Sieburth, P. J. et al. The Use of RT-PCR in the Florida citrus viroid indexing program. Proc. Fifteenth Conf. Int. Organ. Citrus Virol. Riverside California 15, 230–238 (2002).
Puchta, H. et al. Primary and secondary structure of Citrus viroid IV (CVd IV), a new chimeric viroid presents in dwarfed grapefruit in Israel. Nucleic Acids Res. 19, 6640. https://doi.org/10.1093/nar/19.23.6640 (1991).
Gambino, G. & Gribaudo, I. Simultaneous detection of nine grapevine viruses by multiplex reverse transcription-polymerase chain reaction with coamplification of a plant RNA as internal control. Phytopathology 96(11), 1223–1229 (2006).
Foissac, X. et al. Polyvalent degenerate oligonucleotides reverse transcription-polymerase chain reaction: A polyvalent detection and characterization tool for Trichoviruses, Capilloviruses, and Foveaviruses. Phythopathology 95, 617–625 (2005).
Wang, X., Zhou, C., Tang, K., Yan, Z. & Zhongan, L. A rapid one-step multiplex RT-PCR assay for the simultaneous detection of five citrus viroids in China. Eur. J. Plant Pathol. 124, 175–180. https://doi.org/10.1007/s10658-008-9386-y (2009).
Acknowledgements
The authors would like to acknowledge the Faculty of Graduate Studies (MSc program in Biology) at An-Najah National University.
Author information
Authors and Affiliations
Contributions
A.A.; O.A.; R.A.; S.N., and R.A. wrote the main manuscript text. A.A., O.A. and R.A. did the molecular detection work. O.A. and R.A. designed the experiment work. All authors shared data analysis and result interpretation including figures and tables.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Abualrob, A., Alabdallah, O., Kubaa, R.A. et al. Molecular detection of Citrus exocortis viroid (CEVd), Citrus viroid-III (CVd-III), and Citrus viroid-IV (CVd-IV) in Palestine. Sci Rep 14, 423 (2024). https://doi.org/10.1038/s41598-023-50271-5
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-023-50271-5
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.