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)
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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.
KeywordsCitrus tristeza virus (CTV) Feature selection Cross-validation Partial least square discriminant analysis (PLSDA) Mass spectrometry Gas chromatography Biomarker discovery
This manuscript is based upon work supported by the California Citrus Research Board [CED, OF, AMD], the Industry-University Cooperative Research Program [CED, OF, AMD], and the Florida Citrus Production Advisory Council [CED]. Partial support was provided by National Institutes of Health (NIH) grant number #UL1 TR000002 [CED]. Student support was partially provided by the US Department of Veterans Affairs, Post-9/11 GI-Bill [DJP], and the National Science Foundation grant #1343479 Veteran’s Research Supplement [DJP]. The research was supported by UC ANR at Lindcove Research and Extension Center (Exeter, CA). Opinions expressed in this publication are those of the authors and do not necessarily reflect the view of the funding agencies.
Compliance with ethical standards
Conflict of interest
Authors AP, WHKC, DJP, AMD, OF and CED performed initial work on plant VOC biomarkers of citrus infection that pre-dated this manuscript, and they have a patent pending noting the identity of potential CTV-related VOC biomarkers (#WO2012129341 A2; priority filing date 21MAR2011).
All authors confirm that they have adhered to all required ethical standards for this research.
- 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
- 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
- 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
- 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
- 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
- Futch, S. H., & Brlansky, R. H. (2004). Field Diagnosis of Citrus Tristeza Virus. Citrus Industry Magazine, 85, 22–23.Google Scholar
- 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
- 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
- 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
- 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