Abstract
Huanglongbing (HLB) or citrus greening is a devastating disease of citrus trees that is caused by the gram-negative Candidatus Liberibacter spp. bacteria. The bacteria are phloem limited and transmitted by the Asian citrus psyllid, Diaphorina citri, and the African citrus psyllid, Trioza erytreae, which allows for a wider dissemination of HLB. Infected trees exhibit yellowing of leaves, premature leaf and fruit drop, and ultimately the death of the entire plant. Polymerase chain reaction (PCR) and antibody-based assays (ELISA and/or immunoblot) are commonly used methods for HLB diagnostics. However, they are costly, time-consuming, and destructive to the sample and often not sensitive enough to detect the pathogen very early in the infection stage. Raman spectroscopy (RS) is a noninvasive, nondestructive, analytical technique which provides insight into the chemical structures of a specimen. In this study, by using a handheld Raman system in combination with chemometric analyses, we can readily distinguish between healthy and HLB (early and late stage)-infected citrus trees, as well as plants suffering from nutrient deficits. The detection rate of Raman-based diagnostics of healthy vs HLB infected vs nutrient deficit is ~ 98% for grapefruit and ~ 87% for orange trees, whereas the accuracy of early- vs late-stage HLB infected is 100% for grapefruits and ~94% for oranges. This analysis is portable and sample agnostic, suggesting that it could be utilized for other crops and conducted autonomously.
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References
How to Feed the World 2050. In: Food and Agriculture Organization of the United Nations. 2009. http://www.fao.org/fileadmin/templates/wsfs/docs/expert_paper/How_to_Feed_the_World_in_2050.pdf. Accessed 11 Feb 2019.
Savary S, Ficke A, Aubertot J-N, Hollier C. Crop losses due to diseases and their implications for global food production losses and food security. Food Secur. 2012;4:519–37.
McClean APD, Oberholzer PCJ. Citrus psylla, a vector of the greening disease of sweet orange. S Afr J Agric Sci. 1965;8:297–8.
Capoor SP, Rao DG, Viswanath SM. Diaphorina citri Kuway., a vector of the greening disease of citrus in India. Ind J Agric Sci. 1967;37:572–6.
Bové JM. Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. J Plant Pathol. 2006;88:7–37.
Tsai JH, Liu YH. Biology of Diaphorina citri (Homoptera:Psyllidae) on four host plants. J Econ Entomol. 2000;93:1721–5.
Morgan JK, Zhou L, Li W, Shatters RG, Keremane M, Duan YP. Improved real-time PCR detection of ‘Candidatus Liberibacter asiaticus’ from citrus and psyllid hosts by targeting the intragenic tandem-repeats of its prophage genes. Mol Cell Probes. 2012;26:90–8.
Lee JA, Halbert SE, Dawson WO, Robertson CJ, Keesling JE, Singer BH. Asymptomatic spread of huanglongbing and implications for disease control. Proc Natl Acad Sci U S A. 2015;112:7605–10.
Chitarra LG, Bulk RW. The application of flow cytometry and fluorescent probe technology for detection and assessment of viability of plant pathogenic bacteria. Eur J Plant Pathol. 2003;109:407–17.
Wallner G, Amann R, Beisker W. Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry. 1993;14:136–43.
Hocquellet A, Toorawa P, Bove JM, Garnier M. Detection and identification of the two Candidatus Liberobacter species associated with citrus huanglongbing by PCR amplification of ribosomal protein genes of the beta operon. Mol Cell Probes. 1999;13:373–9.
Kim J, Wang N. Characterization of copy numbers of 16S rDNA and 16S rRNA of Candidatus Liberibacter asiaticus and the implication in detection in planta using quantitative PCR. BMC Res Notes. 2009;2:37.
Schaad NW, Frederick RD. Real-time PCR and its application for rapid plant disease diagnostics. Can J Plant Pathol. 2002;24:250–8.
Wang Z, Yin Y, Hu H, Yuan Q, Peng G, Xia Y. Development and application of molecular-based diagnosis for ‘Candidatus Liberibacter asiaticus’, the causal pathogen of citrus Huanglongbing. Plant Pathol. 2006;55:630–8.
Trivedi P, Sagaram US, Brlansky RH, Rogers M, Stelinski LL, Oswalt C, et al. Quantification of viable Candidatus Liberibacter asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide (EMA). Eur J Plant Pathol. 2009;124:553–63.
Almeida MR, Alves RS, Nascimbem LB, Stephani R, Poppi RJ, de Oliveira LF. Determination of amylose content in starch using Raman spectroscopy and multivariate calibration analysis. Anal Bioanal Chem. 2010;397:2693–701.
Zeng ZC, Hu S, Huang SC, Zhang YJ, Zhao WX, Li JF, et al. Novel electrochemical Raman spectroscopy enabled by water immersion objective. Anal Chem. 2016;88:9381–5.
Virkler K, Lednev IK. Blood species identification for forensic purposes using Raman spectroscopy combined with advanced analytical statistics. Anal Chem. 2009;81:7773–7.
López-López M, Delgado JJ, García-Ruiz C. Analysis of macroscopic gunshot residues by Raman spectroscopy to assess the weapon memory effect. Forensic Sci Int. 2013;231:1–5.
Cantarero A. Raman scattering applied to materials science. Procedia Mater Sci. 2015;9:113–22.
Kurouski D, Washington J, Ozbil M, Prabhakar R, Shekhtman A, Lednev IK. Disulfide bridges remain intact while native insulin converts into amyloid fibrils. PLoS One. 2012;7:e36989.
Bueno J, Lednev IK. Advanced statistical analysis and discrimination of gunshot residue implementing combined Raman and FT-IR data. Anal Methods. 2013;5:6292–6.
Farber C, Kurouski D. Detection and identification of plant pathogens on maize kernels with a hand-held Raman spectrometer. Anal Chem. 2018;90:3009–12.
Egging V, Nguyen J, Kurouski D. Detection and identification of fungal infections in intact wheat and sorghum grain using a hand-held Raman spectrometer. Anal Chem. 2018;90:8616–21.
Sanchez L, Farber C, Lei J, Zhu-Salzman K, Kurouski D. Noninvasive and nondestructive detection of cowpea bruchid within cowpea seeds with a hand-held Raman spectrometer. Anal Chem. 2019;91:1733–7.
Chiong Kelvin T, Mona B Damaj, Carmen S Padilla, Carlos A Avila, Shankar R Pant, Kranthi K Mandadi, Ninfa R Ramos, Denise V Carvalho, and T. Erik Mirkov (2017) Reproducible genomic DNA preparation from diverse crop species for molecular genetic applications. Plant methods, 13 (1), 106.
Rezadoost, M H, Kordrostami M, & Kumleh HH (2016). An efficient protocol for isolation of inhibitor-free nucleic acids even from recalcitrant plants. 3 Biotech, 6(1), 61.
Mafra V, Kubo K.S, Alves-Ferreira M, Ribeiro-Alves M, Stuart R.M, Boava L.P, Rodrigues CM, and Machado, M.A. (2012). Reference Genes for Accurate Transcript Normalization in Citrus Genotypes under Different Experimental Conditions. PLoS ONE 7, e31263.
Synytsya A, Čopíková J, Matějka P, Machovič V. Fourier transform Raman and infrared spectroscopy of pectins. Carb Polym. 2003;54:97–106.
Edwards HG, Farwell DW, Webster D. FT Raman microscopy of untreated natural plant fibres. Spectrochim Acta A Mol Biomol Spectrosc. 1997;53A:2383–92.
Tschirner N, Brose K, Schenderlein M, Zouni A, Schlodder E, Mroginski MA, et al. The anomaly of the ν1-resonance Raman band of bβ-carotene in solution and in photosystem I and II. Phys Stat Solid. 2009;246:2790–3.
Kurouski D, Van Duyne RP, Lednev IK. Exploring the structure and formation mechanism of amyloid fibrils by Raman spectroscopy: a review. Analyst. 2015;140:4967–80.
Agarwal UP. 1064 nm FT-Raman spectroscopy for investigations of plant cell walls and other biomass materials. Front Plant Sci. 2014;5:1–12.
Mary YS, Panicker CY, Varghese HT. Vibrational spectroscopic investigations of 4-nitropyrocatechol. Orient J Chem. 2012;28:937–41.
Yu MM, Schulze HG, Jetter R, Blades MW, Turner RF. Raman microspectroscopic analysis of triterpenoids found in plant cuticles. Appl Spectrosc. 2007;61:32–7.
Cao Y, Shen D, Lu Y, Huang JA. Raman-scattering study on the net orientation of biomacromolecules in the outer epidermal walls of mature wheat stems (Triticum aestivum). Ann Bot. 2006;97:1091–4.
Devitt G, Howard K, Mudher A, Mahajan S. Raman spectroscopy: an emerging tool in neurodegenerative disease research and diagnosis. ACS Chem Neurosci. 2018;9:404–20.
Adar F. Carotenoids - their resonance raman spectra and how they can be helpful in characterizing a number of biological systems. Spectroscopy. 2017;32:12–20.
Kang L, Wang K, Li X, Zou B. High pressure structural investigation of benzoic acid: Raman spectroscopy and x-ray diffraction. J Phys Chem C. 2016;120:14758–66.
Agarwal UP. Raman imaging to investigate ultrastructure and composition of plant cell walls: distribution of lignin and cellulose in black spruce wood (Picea mariana). Planta. 2006;224:1141–53.
Pompeu DR, Larondelle Y, Rogez H, Abbas O, Pierna JAF, Baeten V. Characterization and discrimination of phenolic compounds using Fourier transformation Raman spectroscopy and chemometric tools. Biotechnol Agron Soc Environ. 2017;22:1–16.
Liu Q, Luo L, Zheng L. Lignins: biosynthesis and biological functions in plants. Int J Mol Sci. 2018;19:335.
Bennett RN, Wallsgrove RM. Secondary metabolites in plant defence mechanisms. New Physiol. 1994;127:617–33.
Treutter D. Significance of flavonoids in plant resistance: a review. Environ Chem Lett. 2006;4:147–57.
Skadhauge B, Thomsen KK, Von Wettstein D. The role of the barley testa layer and its flavonoid content in resistance to Fusarium infections. Hereditas. 1997;126:147–60.
Sankaran S, Ehsani R, Etxeberria E. Mid-infrared spectroscopy for detection of Huanglongbing (greening) in citrus leaves. Talanta. 2010;83:574–581.
Shashilov VA, Lednev IK. Advanced statistical and numerical methods for spectroscopic characterization of protein structural evolution. Chem Rev. 2010;110:5692–713.
Eriksson L, Byrne T, Johansson E, Trygg J, Vikstrom C. Multi- and megavariate data analysis basic principles and applications, 3rd edn. Umetrics Academy; 2013.
Sankaran S, Mishra A, Maja JM, Ehsani R. Visible-near infrared spectroscopy for detection of Huanglongbing in citrus orchards. Comp Electron Agricult. 2011;77:127–34.
Hawkins SA, Park B, Poole GH, Gottwald T, Windham WR, Lawrence KC. Detection of citrus Huanglongbing by Fourier transform infrared-attenuated total reflection spectroscopy. Appl Spectrosc. 2010;64:100–3.
Vallejo-Pérez MR, Mendoza MG, Elias MG, Gonzalez FJ, Contreras HR, Servin CC. Raman spectroscopy an option for the early detection of citrus Huanglongbing. Appl Spectrosc. 2016;70:829–39.
Acknowledgements
The authors thank Texas A&M University-Kingsville, Citrus Center, and Riofarms, TX, for access to the citrus orchards.
Funding
This study was supported by funds from Texas A&M AgriLife Research, Texas A&M University Governor’s University Research Initiative (GURI) grant program (12-2016/M1700437) to DK, and USDA-NIFA-AFRI (2018-70016-28198) to KKM.
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Sanchez, L., Pant, S., Xing, Z. et al. Rapid and noninvasive diagnostics of Huanglongbing and nutrient deficits on citrus trees with a handheld Raman spectrometer. Anal Bioanal Chem 411, 3125–3133 (2019). https://doi.org/10.1007/s00216-019-01776-4
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DOI: https://doi.org/10.1007/s00216-019-01776-4