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European Journal of Plant Pathology

, Volume 153, Issue 1, pp 251–264 | Cite as

Onion yellow dwarf virus ∆∆Ct-based relative quantification obtained by using real-time polymerase chain reaction in ‘Rossa di Tropea’ onion

  • Antonio TiberiniEmail author
  • Rossella Mangano
  • Giuseppe Micali
  • Giovanna Leo
  • Ariana Manglli
  • Laura Tomassoli
  • Giuliana Albanese
Article
  • 75 Downloads

Abstract

As part of a plant-pathogen interaction study between Onion yellow dwarf virus (OYDV) and onion cultivar Rossa di Tropea, a ΔΔCt-based relative quantification of OYDV was investigated to relate OYDV titer to accumulation of secondary metabolites in onion bulbs. An appropriate reference gene (RG) was required to achieve data normalization. Since no single internal control gene is universally used as an RG, multiple stably expressed reference genes were investigated. In particular, elongation factor (Elf), protein phosphatase 2A (PP2A), helicase (Hel-1), 5.8S rRNA, ubiquitin (UBQ) and ß-Actin (ß-Act) were compared one to another in both leaf and bulb tissues, at different growth and development stages, and with different infection status (healthy/OYDV-infected). Preliminary gene screening was carried out using an RT-qPCR assay (SYBR chemical), assessing both Ct values and melting curves. Expression stability of the reference genes in the sample sets was independently determined by three different software packages: geNorm, NormFinder and Bestkeeper. In contrast to Elf, PP2A, Hel-1 and ß-Act, 5.8S rRNA and UBQ proved to be the most stable RGs. An OYDV specific RT-qPCR TaqMan® assay was also developed and validated for relative quantification of OYDV titer. The assay was shown to be specific and sensitive, able to identify virus presence up to 10−6 dilution, representing a rapid and sensitive diagnostic tool for OYDV detection for application in field surveys. Finally, a ∆∆Ct method was developed, to be applied in future studies describing the molecular interaction between OYDV and onion cv. ‘Rossa di Tropea’. This approach was used to provide relative quantification of OYDV titer in samples obtained from different experimental trials.

Keywords

Plant virology Onion bulb Rossa di Tropea OYDV Reference gene ∆∆Ct 

Notes

Acknowledgments

The authors wish to thank Adrian Fox, Fera Science Ltd., York (UK) for English and scientific revision of the paper.

This study was carried out in the frame of the project: Study on Interaction between Onion yellow dwarf virus and nutraceutical compounds of ‘Rossa di Tropea’ Onion (SIR-MIUR grant – SIORTO-RBSI149LD5), funded by the Italian Ministry of Education, University and Research – MIUR. And, it is part of a wider initiative called Scientific Independence of your Researcher – SIR. We thank ‘Dolce Rossa’ farm for supporting this study.

Compliance with ethical standards

Animal and/or human studies

This research did not involve any animal and/or human participant.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10658_2018_1560_MOESM1_ESM.docx (93 kb)
ESM 1 (DOCX 93 kb)

References

  1. Abd El-Wahab, A. S. (2009). Aphid-transmission efficiency of two main viruses on garlic in Egypt, Onion Yellow Dwarf Virus (OYDV-G) and Leek Yellow Stripe Virus (LYSV-G). Academic Journal of Entomology, 2(1), 40–42.Google Scholar
  2. AbdEl-Wahab A. S., Elnagr. S., & El-Sheikh M. A. K. (2009). Incidence of aphid-borne Onion yellow dwarf virus (OYDV) in alliaceae crops and associated weeds in Egypt. In: 4th Conference on Recent Technologies in Agriculture, 21–33.Google Scholar
  3. Andersen, C. L., Jensen, J. L., & Ørntoft, T. F. (2004). Normalization of real-time quantitative reverse transcription-PCR data: A model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Research, 64, 5245–5250 PMID: 15289330.CrossRefGoogle Scholar
  4. Benmalek, Y., Yahia, O. A., Belkebir, A., & Fardeau, M. L. (2013). Anti-microbial and anti-oxidant activities of Illicium verum, Crataegus oxyacantha ssp monogyna and Allium cepa red and white varieties. Bioengineered, 4(4), 244–248.CrossRefGoogle Scholar
  5. Bos, L. (1976). Onion yellow dwarf virus. CMI/AAB Descriptions of Plant Viruses, 158(4) http://www.dpvweb.net/dpv/showdpv.php?dpvno=158.
  6. Brunner, A. M., Yakovlev, I. A., & Strauss, S. H. (2004). Validating internal controls for quantitative plant gene expression studies. BMC Plant Biology, 2229, 4–14.Google Scholar
  7. Bustin, S. A., Benes, V., Garson, J. A., Hellmans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M. W., Shipley, G. L., Vandesompele, J., & Wittwer, C. T. (2009). The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 55, 611–622.CrossRefGoogle Scholar
  8. Dai, J., Peng, H., Chen, W., Cheng, J., & Wu, Y. (2013). Development of multiplex real-time PCR for simultaneous detection of three Potyviruses in tobacco plants. Journal of Applied Microbiology, 114(2), 502–508.CrossRefGoogle Scholar
  9. Drake, C. J., Tate, H. D., & Harris, H. M. (1933). The relationship of aphids to the transmission of yellow dwarf of onion. Journal of Economic Entomology, 26, 841–846.CrossRefGoogle Scholar
  10. El Morsi, A., Abdelkhalek, A., AlShehaby, O., & Hafez, E. E. (2015). Pathogenesis-related genes as tools for discovering the response of onion defence system against Iris yellow spot virus infection. Botany, 93, 1–10.CrossRefGoogle Scholar
  11. Elnagar, S., El-Sheikh, M. A. K., & Abd El-Wahab, A. S. (2011). Effect of natural infection with Onion yellow dwarf virus (OYDV) on yield of onion and garlic crops in Egypt. Journal of Life Science, 5, 634–638.Google Scholar
  12. Grzelak, K., Milala, J., Krol, B., Adamicki, F., & Badelek, E. (2009). Content of quercetin glycosides and fructooligosaccharides in onion stored in a cold room. European Food Research and Technology, 228, 1001–1007.CrossRefGoogle Scholar
  13. Guo, J., Ling, H., Wu, Q., Xu, L., & Que, Y. (2014). The choice of reference genes for assessing gene expression in sugarcane under salinity and drought stresses. Scientific Reports, 4, 7042.CrossRefGoogle Scholar
  14. Harper, S. J., Delmiglio, C., Ward, L. I., & Clover, G. R. G. (2011). Detection of Tomato black ring virus by real-time one-step RT-PCR. Journal of Virological Methods, 171, 190–194.CrossRefGoogle Scholar
  15. Huggett, J., Dheda, K., Bustin, S., & Zumla, A. (2005). Real-time RT-PCR normalisation; strategies and considerations. Genes and Immunity, 6, 279–284.CrossRefGoogle Scholar
  16. Jarošová, J., & Kundu, J. (2010). Validation of reference genes as internal control for studying viral infections in cereals by quantitative real-time RT-PCR. BMC Plant Biology, 10, 146.CrossRefGoogle Scholar
  17. Katis, N. I., Maliogka, V. I., & Dovas, C. I. (2012). Viruses of the genus Allium in the Mediterranean region. In H. Lecoq & G. Loebenstein (Eds.), Viruses and virus diseases of vegetables in the Mediterranean Basin (pp. 163–208). San Diego: Academic Press.CrossRefGoogle Scholar
  18. Kumar, P., Dhawan, P., & Mehra, R. (2011). Characterization, transmission and host range of onion yellow dwarf virus. Plant Disease Research, 26(2), 176.Google Scholar
  19. Kumar, P., Dhawan, P., & Mehra, R. (2012). Symptoms and losses caused by Onion yellow dwarf virus and Iris yellow spot virus diseases of onion crop in Northen India. Journal of Mycology and Plant Pathology, 42(1), 153–160.Google Scholar
  20. Lanzotti, V. (2006). The analysis of onion and garlic. Journal of Chromatography A, 1112, 3–22.CrossRefGoogle Scholar
  21. Lilly, S. T., Drummond, R. S., Pearson, M. N., & Macdiarmid, R. M. (2011). Identification and validation of reference genes for normalization of transcripts from virus-infected Arabidopsis thaliana. Molecular Plant Microbe Interaction, 24, 294–304.CrossRefGoogle Scholar
  22. Liu, W., Zhao, X., Zhang, P., Mar, T. T., Liu, Y., Zhang, Z., Han, C., & Wan, X. (2013). A one step real-time RT-PCR assay for the quantitation of Wheat yellow mosaic virus (WYMV). Virology Journal, 10, 173.CrossRefGoogle Scholar
  23. Livak, K. J., & Schmittgen, T. (2001). Analysis of relative gene expression data using real-tme quantitative PCR and the 2-∆∆Ct method. Methods, 25, 402–408.CrossRefGoogle Scholar
  24. Mafra, V., Kubo, K. S., Alves-Ferreira, M., Ribeiro-Alves, M., Stuart, R. M., & Boava, L. P. (2012). Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions. PLoS One, 7(2), e31263.CrossRefGoogle Scholar
  25. Manglli, A., Mohamed, H. S., El Hussein, A. A., Agosteo, G. E., Albanese, G., & Tomassoli, L. (2014). Molecular analysis of the 3’terminal region of Onion yellow dwarf virus from onion in southern Italy. Phytopathologia Mediterranea, 53(3), 438–450.  https://doi.org/10.14601/Phytopathol_Mediterr-14027.Google Scholar
  26. Marani F., Bertaccini A. (1983) Virosi delle liliaceae ortive: cipolla, aglio, porro. In: Le virosi delle piante ortive. Reda Editore per l'Agricoltura (Ed), Roma Italy, 104–111.Google Scholar
  27. Mascia, T., Santovito, E., Gallitelli, D., & Cillo, F. (2010). Evaluation of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in infected tomato plants. Molecular Plant Pathology, 11(6), 805–816.CrossRefGoogle Scholar
  28. Melhus, I. E., Reddy, C., Shenderson, W. J., & Vestal, E. (1929). A new virus disease epidemic on onions. Phytopathology, 19, 73–77.Google Scholar
  29. Moreno, I., Gruissem, W., & Vanderschuren, H. (2011). Reference genes for reliable potyvirus quantitation in cassava and analysis of cassava brown streak virus load in host varieties. Journal of Virological Methods, 177, 49–54.CrossRefGoogle Scholar
  30. Obrero, A., Die, J. V., Roman, B., Gomez, P., Nadal, S., & González-Verdejo, C. I. (2011). Selection of reference genes for gene expression studies in zucchini (Cucurbita pepo) using qPCR. Journal of Agriculture and Food Chemestry, 59(10), 5402–5411.CrossRefGoogle Scholar
  31. Parrella, G., De Stradis, A., Volvas, C., & Agosteo, G. E. (2005). Outbreaks of Onion yellow dwarf virus (OYDV) on onion crops in Calabria (southern Italy). Journal of Plant Pathology, 87(4), 302.Google Scholar
  32. Pasquini, G., Barba, M., Hadidi, A., Faggioli, F., Negri, R., Sobol, I., Tiberini, A., Caglayan, K., Mazyad, H., Anfoka, G., Ghanim, M., Zeidan, M., & Czosnek, H. (2008). Oligonucleotide microarray-based detection and genotyping of Plum pox virus. Journal of Virological Methods, 147(1), 118–126.CrossRefGoogle Scholar
  33. Pfaffl, M. W., Tichopad, A., Prgomet, C., & Neuvians, T. P. (2004). Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–excel-based tool using pairwise correlations. Biotechnology Letters, 26, 509–515 PMID: 15127793.CrossRefGoogle Scholar
  34. Petriccione, M., Mastrobuoni, F., Zampella, L., & Scortichini, M. (2015). Reference gene selection for normalization of RT-qPCR gene expression data from Actinidia deliciosa leaves infected with Pseudomonas syringae pv. Actinidiae. Scientific Reports, 5, 16961.  https://doi.org/10.1038/srep16961.CrossRefGoogle Scholar
  35. Radonić, A., Thulke, S., Mackay, I. M., Landt, O., Siegert, W., & Nitsche, A. (2004). Guideline to reference gene selection for quantitative real-time PCR. Biochemical Biophysical Research Communication, 313, 856–862.CrossRefGoogle Scholar
  36. Robene, I., Perret, M., Jouen, E., Escalon, A., Maillot, M. V., Chabirand, A., Moreau, A., Laurent, A., Chiroleu, F., & Pruvost, O. (2015). Development and validation of a real-time quantitative PCR assay to detect Xanthomonas axonopodis pv. allii from onion seed. Journal of Microbiological Methods, 114, 78–86.CrossRefGoogle Scholar
  37. Robledo, D., Hernandex-Urcera, J., Cal, R. M., Pardo, B. G., Sanchez, L., Martinez, P., & Vinas, A. (2014). Analysis of qPCR reference gene stability determination methods and a practical approach for efficiency calculation on a turbot (Scophthalmus maximus) gonad dataset. BMC Genomics, 15, 648.CrossRefGoogle Scholar
  38. Rubio-Pina, J. A., & Zapata-Perez, O. (2011). Isolation of total RNA from tissues rich in polyphenols and polysaccharides of mangrove plants. Electronic Journal of Biotechnology, 14, 5.Google Scholar
  39. Schwartz, H. F., & Mohan, S. K. (2008). Compendium of onion and garlic diseases and pests. 2nd ed., APS Press. Google Scholar
  40. Shon, M. Y., Choi, S. D., Kahng, G. G., Nam, S. H., & Sung, N. J. (2004). Antimutagenic, antioxidant and free radical scavenging activity of ethyl acetate extracts from white, yellow and red onions. Food and Chemical Toxicology, 42, 659–666.CrossRefGoogle Scholar
  41. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., & Van Roy, N. (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology, 3, research0034 PMID: 12184808.CrossRefGoogle Scholar
  42. Van Dijk, P. (1993). Survey and characterization of potyviruses and their strains of Allium species. Netherlands. Journal of Plant Pathology, 99 (suppl.)(2), 1–48.Google Scholar
  43. Zhu, J., Zhang, L., Li, W., Han, S., Yang, W., & Qi, L. (2013). Reference gene selection for quantitative real-time PCR normalization in Caragana intermedia under different abiotic stress conditions. PLoS One, 8, e53196.CrossRefGoogle Scholar

Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2018

Authors and Affiliations

  • Antonio Tiberini
    • 1
    Email author
  • Rossella Mangano
    • 1
  • Giuseppe Micali
    • 1
  • Giovanna Leo
    • 1
  • Ariana Manglli
    • 2
  • Laura Tomassoli
    • 2
  • Giuliana Albanese
    • 1
  1. 1.Dipartimento di AGRARIAUniversità degli Studi Mediterranea di Reggio CalabriaReggio CalabriaItaly
  2. 2.Consiglio per la Ricerca in Agricoltura e l’Analisi dell’Economia Agraria, Centro di Ricerca Difesa e CertificazioneRomeItaly

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