Metabolomics

, 12:41 | Cite as

Citrus tristeza virus infection in sweet orange trees and a mandarin × tangor cross alters low molecular weight metabolites assessed using gas chromatography mass spectrometry (GC/MS)

  • Alberto Pasamontes
  • William H. K. Cheung
  • Jason Simmons
  • Alexander A. Aksenov
  • Daniel J. Peirano
  • Elizabeth E. Grafton-Cardwell
  • Therese Kapaun
  • Abhaya M. Dandekar
  • Oliver Fiehn
  • Cristina E. Davis
Original Article

Abstract

Citrus tristeza virus (CTV) (genus Closterovirus) is a plant pathogen which infects economically important citrus crops, resulting in devastating crop losses worldwide. In this study, we analyzed leaf metabolite extracts from six sweet orange varieties and a mandarin × tangor cross infected with CTV collected at the Lindcove Research and Extension Center (LREC; Exeter, CA). In order to analyze low volatility small molecules, the extracts of leaf metabolites were derivatized by N-methyl-N-trimethylsilyl-trifluoracetamide (MSTFA). Chemical analysis was performed with gas chromatography/mass spectrometry (GC/MS) to assess metabolite changes induced by CTV infection. Principal Component Analysis (PCA) and Hotelling’s T2 were used to identify outliers within the set of samples. Partial Least Square Discriminant Analysis (PLS-DA) was applied as a regression method. A cross-validation strategy was repeated 300 times to minimize possible bias in the model selection. Afterwards, a representative model was built with a sensitivity of 0.66 and a specificity of 0.71. The metabolites which had the strongest contribution to differentiate between healthy and CTV-infected were found to be mostly saccharides and their derivatives such as inositol, d-fructose, glucaric and quinic acid. These metabolites are known to be endogenously produced by plants, possess important biological functions and often found to be differentially regulated in disease states, maturation processes, and metabolic responses. Based on the information found in this study, a method may be available that can identify CTV infected plants for removal and halt the spread of the virus.

Keywords

Citrus tristeza virus (CTV) Feature selection Cross-validation Partial least square discriminant analysis (PLSDA) Mass spectrometry Gas chromatography Biomarker discovery 

Supplementary material

11306_2016_959_MOESM1_ESM.docx (125 kb)
Supplementary material 1 (DOC 126 kb)

References

  1. Aksenov, A., et al. (2014). Detection of Huanglongbing disease using differential mobility spectrometry. Analytical Chemistry, 86, 2481–2488. doi:10.1021/ac403469y.CrossRefPubMedGoogle Scholar
  2. Barbarossa, L., & Savino, V. (2006). Sensitive and specific digoxigenin-labelled rna probes for routine detection of citrus tristeza virus by dot-blot hybridization. Journal of Phytopathology, 154, 329–335.CrossRefGoogle Scholar
  3. Bar-Joseph, M., et al. (2002). The continuous challenge of citrus tristeza virus molecular research. In Paper presented at the Fifteenth IOCV Conference.Google Scholar
  4. Bar-Joseph, M., et al. (1979). The use of enzyme-linked immunosorbent assay for detection of citrus tristeza virus. Phytopathology, 69, 190–194.CrossRefGoogle Scholar
  5. Cambra, M., et al. (2000b). Routine detection of Citrus tristeza virus by direct immunoprinting-ELISA method using specific monoclonal and recombinant antibodies. In Proceedings 14th International Conference of the Organization of Citrus Virologists, Riverside, pp. 34–41.Google Scholar
  6. Cambra, M., Camarasa, E., Gorris, M. T., Garnsey, S. M., & Carbonell, E. (1991). Comparison of different immunosorbent assays for Citrus tristeza virus (CTV) using CTV-specific monoclonal and polyclonal antibodies. In R. H. Brlansky, R. F. Lee, & L. W. Timmer (Eds.), Proceedings XI International Organization of Citrus Virologist, IOCV, Riverside, CA, pp. 38–45.Google Scholar
  7. Cambra, M., et al. (2000a). Incidence and epidemiology of Citrus tristeza virus in the Valencian community of Spain. Virus Research, 71, 85–95.CrossRefPubMedGoogle Scholar
  8. Cevallos-Cevallos, J. M., Garcia-Torres, R., Etxeberria, E., & Reyes-De-Corcuera, J. I. (2011). GC-MS analysis of headspace and liquid extracts for metabolomic differentiation of citrus Huanglongbing and zinc deficiency in leaves of ‘Valencia’ sweet orange from commercial groves. Phytochemical Analysis, 22, 236–246. doi:10.1002/pca.1271.CrossRefPubMedGoogle Scholar
  9. Chaouch, S., & Noctor, G. (2010). Myo-inositol abolishes salicylic acid-dependent cell death and pathogen defence responses triggered by peroxisomal hydrogen peroxide. New Phytologist, 188, 711–718. doi:10.1111/j.1469-8137.2010.03453.x.CrossRefPubMedGoogle Scholar
  10. Cheung, W., et al. (2015). Volatile organic compound (VOC) profiling of Citrus tristeza virus (CTV) infection in Valencia citrus varietals using thermal desorption gas chromatography time of flight mass spectrometry (TD-GC/TOF-MS). Metabolomics, 11(6), 1514–1525.CrossRefGoogle Scholar
  11. Clements, R. S, Jr, & Darnell, B. (1980). Myo-inositol content of common foods: development of a high-myo-inositol diet. American Journal of Clinical Nutrition, 33, 1954–1967.PubMedGoogle Scholar
  12. Donike, M. (1969). N-Methyl-N-trimethylsilyl-trifluoracetamid, ein neues Silylierungsmittel aus der reihe der silylierten amide. Journal of Chromatography A, 42, 103–104.CrossRefGoogle Scholar
  13. Donike, M., & Zimmermann, J. (1980). Zur Darstellung von Trimethylsilyl-, Triethylsilyl- und tert. Butyl-dimethylsilyl-enoläthern von Ketosteroiden für gaschromatographische und massen-spektrometrische Untersuchungen. Journal of Chromatography A, 202, 483–486.CrossRefGoogle Scholar
  14. Fares, S., Gentner, D. R., Park, J. H., Ormeno, E., Karlik, J., & Goldstein, A. H. (2011). Biogenic emissions from Citrus species in California. Atmospheric Environment, 45, 4557–4568. doi:10.1016/j.atmosenv.2011.05.066.CrossRefGoogle Scholar
  15. Fiehn, O. (2006). Metabolite profiling in arabidopsis. In J. Salinas & J. Sanchez-Serrano (Eds.), Arabidopsis protocols. Methods in molecular biology™ (Vol. 323, pp. 439–447). New York: Humana Press.CrossRefGoogle Scholar
  16. Fiehn, O., Kopka, J., Doermann, P., Altmann, T., Trethewey, R. N., & Willmitzer, L. (2000a). Metabolite profiling for plant functional genomics. Nature Biotechnology, 18, 1157–1161. doi:10.1038/81137.CrossRefPubMedGoogle Scholar
  17. Fiehn, O., Kopka, J., Trethewey, R. N., & Willmitzer, L. (2000b). Identification of uncommon plant metabolites based on calculation of elemental compositions using gas chromatography and quadrupole mass spectrometry. Analytical Chemistry, 72, 3573–3580. doi:10.1021/ac991142i.CrossRefPubMedGoogle Scholar
  18. Folimonova, S. Y., et al. (2010). Infection with strains of Citrus tristeza virus does not exclude superinfection by other strains of the virus. Journal of Virology, 84, 1314–1325. doi:10.1128/JVI.02075-09.CrossRefPubMedPubMedCentralGoogle Scholar
  19. Futch, S. H., & Brlansky, R. H. (2004). Field Diagnosis of Citrus Tristeza Virus. Citrus Industry Magazine, 85, 22–23.Google Scholar
  20. Gandia, M., et al. (2007). Transcriptional response of Citrus aurantifolia to infection by Citrus tristeza virus. Virology, 367, 298–306. doi:10.1016/j.virol.2007.05.025.CrossRefPubMedGoogle Scholar
  21. Hijaz, F., & Killiny, N. (2014). Collection and chemical composition of phloem sap from Citrus sinensis L. Osbeck (sweet orange). PLoS One, 9, e101830. doi:10.1371/journal.pone.0101830.CrossRefPubMedPubMedCentralGoogle Scholar
  22. Hijaz, F. M., Manthey, J. A., Folimonova, S. Y., Davis, C. L., Jones, S. E., & Reyes-De-Corcuera, J. I. (2013). An HPLC-MS characterization of the changes in sweet orange leaf metabolite profile following infection by the bacterial pathogen Candidatus Liberibacter asiaticus. PLoS One, 8, e79485. doi:10.1371/journal.pone.0079485.CrossRefPubMedPubMedCentralGoogle Scholar
  23. Khakimov, B., Motawia, M. S., Bak, S., & Engelsen, S. B. (2013). The use of trimethylsilyl cyanide derivatization for robust and broad-spectrum high-throughput gas chromatography-mass spectrometry based metabolomics. Analytical and Bioanalytical Chemistry, 405, 9193–9205. doi:10.1007/s00216-013-7341-z.CrossRefPubMedGoogle Scholar
  24. Kind, T., et al. (2009). FiehnLib: Mass spectral and retention index libraries for metabolomics based on quadrupole and time-of-flight gas chromatography/mass spectrometry. Analytical Chemistry, 81, 10038–10048.CrossRefPubMedPubMedCentralGoogle Scholar
  25. Kitajima, E. W., Silva, D. M., Oliveira, A. R., Muller, G. W., Costa, A. S. (1964). Electron microscopical investigations of tristeza. In Proceedings of the 3rd conference of the international organization of citrus virologists, 1–9.Google Scholar
  26. Kushalappa, A. C., Lui, L. H., Chen, C. R., & Lee, B. (2002). Volatile fingerprinting (SPME-GC-FID) to detect and discriminate diseases of potato tubers. Plant Disease, 86, 131–137. doi:10.1094/Pdis.2002.86.2.131.CrossRefGoogle Scholar
  27. Leiss, K. A., Maltese, F., Choi, Y. H., Verpoorte, R., & Klinkhamer, P. G. (2009). Identification of chlorogenic acid as a resistance factor for thrips in chrysanthemum. Plant Physiology, 150, 1567–1575. doi:10.1104/pp.109.138131.CrossRefPubMedPubMedCentralGoogle Scholar
  28. Liu, G., Dong, X., Liu, L., Wu, L., Peng, S., & Jiang, C. (2015). Metabolic profiling reveals altered pattern of central metabolism in navel orange plants as a result of boron deficiency. Physiologia Plantarum, 153, 513–524. doi:10.1111/ppl.12279.CrossRefPubMedGoogle Scholar
  29. Mann, R. S., et al. (2012). Induced release of a plant-defense volatile ‘deceptively’ attracts insect vectors to plants infected with a bacterial pathogen. PLoS Pathogens, 8, e1002610. doi:10.1371/journal.ppat.1002610.CrossRefPubMedPubMedCentralGoogle Scholar
  30. Meijer, H. J., & Munnik, T. (2003). Phospholipid-based signaling in plants. Annual Review of Plant Biology, 54, 265–306. doi:10.1146/annurev.arplant.54.031902.134748.CrossRefPubMedGoogle Scholar
  31. Moreno, P., Ambros, S., Albiach-Marti, M. R., Guerri, J., & Pena, L. (2008). Citrus tristeza virus: a pathogen that changed the course of the citrus industry. Molecular Plant Pathology, 9, 251–268. doi:10.1111/j.1364-3703.2007.00455.x.CrossRefPubMedGoogle Scholar
  32. Narvaez, G., Skander, B. S., Ayllon, M. A., Rubio, L., Guerri, J., & Moreno, P. (2000). A new procedure to differentiate citrus tristeza virus isolates by hybridisation with digoxigenin-labelled cDNA probes. Journal of Virological Methods, 85, 83–92.CrossRefPubMedGoogle Scholar
  33. Nolasco, G., de Blas, C., Torres, V., & Ponz, F. (1993). A method combining immunocapture and PCR amplification in a microtiter plate for the detection of plant viruses and subviral pathogens. Journal of Virological Methods, 45, 201–218.CrossRefPubMedGoogle Scholar
  34. Olmos, A., Cambra, M., Esteban, O., Gorris, M. T., & Terrada, E. (1999). New device and method for capture, reverse transcription and nested PCR in a single closed-tube. Nucleic Acids Research, 27, 1564–1565.CrossRefPubMedPubMedCentralGoogle Scholar
  35. Perez, J. L., Jayaprakasha, G. K., Yoo, K. S., & Patil, B. S. (2008). Development of a method for the quantification of D-glucaric acid in different varieties of grapefruits by high-performance liquid chromatography and mass spectra. Journal of Chromatography A, 1190, 394–397. doi:10.1016/j.chroma.2008.03.026.CrossRefPubMedGoogle Scholar
  36. Roman, M. P., et al. (2004). Sudden death of citrus in Brazil: A graft-transmissible bud union disease. Plant Disease, 88, 453–467. doi:10.1094/Pdis.2004.88.5.453.CrossRefGoogle Scholar
  37. Rosner, A., & Bar-Joseph, M. (1984). Diversity of citrus tristeza virus strains indicated by hybridization with cloned cDNA sequences. Virology, 139, 189–193.CrossRefPubMedGoogle Scholar
  38. Sankaran, S., Mishra, A., Ehsani, R., & Davis, C. (2010). A review of advanced techniques for detecting plant diseases. Computers and Electronics in Agriculture, 72, 1–13. doi:10.1016/j.compag.2010.02.007.CrossRefGoogle Scholar
  39. Tsuchizaki, T., Sasaki, A., & Saito, Y. (1978). Purification of citrus tristeza virus from diseased citrus fruits and the detection of the virus in citrus tissues by fluorescent antibody techniques. Phytopathology, 68, 139–142.CrossRefGoogle Scholar
  40. Vela, C., et al. (1986). Production and characterization of monoclonal-antibodies specific for Citrus tristeza virus and their use for diagnosis. Journal of General Virology, 67, 91–96. doi:10.1099/0022-1317-67-1-91.CrossRefGoogle Scholar
  41. Westerhuis, J. A., et al. (2008). Assessment of PLSDA cross validation. Metabolomics, 4, 81–89. doi:10.1007/s11306-007-0099-6.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Alberto Pasamontes
    • 1
  • William H. K. Cheung
    • 1
  • Jason Simmons
    • 1
  • Alexander A. Aksenov
    • 1
  • Daniel J. Peirano
    • 1
  • Elizabeth E. Grafton-Cardwell
    • 2
  • Therese Kapaun
    • 2
  • Abhaya M. Dandekar
    • 3
  • Oliver Fiehn
    • 4
  • Cristina E. Davis
    • 1
  1. 1.Department of Mechanical and Aerospace EngineeringUniversity of California, DavisDavisUSA
  2. 2.University of California, Lindcove Research and Extension Center (LREC)ExeterUSA
  3. 3.Department of Plant SciencesUniversity of California, DavisDavisUSA
  4. 4.UC Davis Genome Center-MetabolomicsUniversity of California, DavisDavisUSA

Personalised recommendations