Abstract
Citrus huanglongbing (HLB) as a devastating disease seriously affects the advance in agriculture, so early detection or accurate diagnosis is the key to control its spread. This paper reported a method for early, rapid, and non-destructive detection of HLB using microscopic confocal Raman (MCR). The spectra of healthy (HE), HLB-asymptomatic (HA), and HLB-symptomatic (HS) leaves were very different at 730–810 cm−1, 866 cm−1, 942 cm−1, 1082 cm−1, 1250 cm−1, 1455 cm−1, and 1510–1630 cm−1, which could be clearly distinguished mutually. Meanwhile, the spectra of relative compounds inside leaves were connected to further analyze spectral differences. The contents of glucose, sucrose, carotene, and chlorophyll in HA leaves were distinctly decreased, but increased in starch and polyphenols compared with HE leaves. In addition, three types of leaves could be well classified by principal component analysis (PCA) whose cumulative percentage variance (CPV) accounted for about 91.01% (three principal components). Partial least square discriminant analysis (PLS-DA) also demonstrated the good clustering effect with an accuracy of 97.2%. Finally, BP-artificial neural network (BP-ANN) model was utilized to evaluate datasets (75% for training, 25% for testing). The low root mean square errors (RMSE 0.0616) and high squared correlation coefficients (R2 0.9598) values showed the high prediction accuracy and stability of the classification model. These results indicated that MCR had excellent practical values for horticulturists to constantly and early detect HLB, which was conducive to prevent and timely control of the spread of HLB.

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Baldwin E, Plotto A, Manthey J, McCollum G, Bai J, Irey M, Cameron R, Luzio G (2009) Effect of Liberibacter infection (huanglongbing disease) of citrus on orange fruit physiology and fruit/fruit juice quality: chemical and physical analyses. J Agric Food Chem 58:1247–1262
Baldwin E, Plotto A, Bai J, Manthey J, Zhao W, Raithore S, Irey M (2018) Effect of abscission zone formation on orange (Citrus sinensis) fruit/juice quality for trees affected by huanglongbing (HLB). J Agric Food Chem 66:2877–2890
Clark K, Franco JY, Schwizer S, Pang Z, Hawara E, Liebrand TW, Pagliaccia D, Zeng L, Gurung FB, Wang P (2018) An effector from the huanglongbing-associated pathogen targets citrus proteases. Nat Commun 9
De Gelder J, De Gussem K, Vandenabeele P, Moens L (2007) Reference database of Raman spectra of biological molecules. J Raman Spectrosc 38:1133–1147
Ding F, Allen V, Luo W, Zhang S, Duan Y (2018) Molecular mechanisms underlying heat or tetracycline treatments for citrus HLB control. Hortic Res 5:30
do Brasil Cardinali MC, Boas PRV, Milori DMBP, Ferreira EJ, e Silva MF, Machado MA, Bellete BS (2012) Infrared spectroscopy: a potential tool in huanglongbing and citrus variegated chlorosis diagnosis. Talanta 91:1–6
Fu X, He X, Xu H, Ying Y (2016) Nondestructive and rapid assessment of intact tomato freshness and lycopene content based on a miniaturized Raman spectroscopic system and colorimetry. Food Anal Methods 9:2501–2508
Gottwald TR (2010) Current epidemiological understanding of citrus huanglongbing. Annu Rev Phytopathol 48:119–139
Grafton-Cardwell EE, Stelinski LL, Stansly PA (2013) Biology and management of Asian citrus psyllid, vector of the huanglongbing pathogens. Annu Rev Entomol 58:413–432
Guzman JCV, Basu S, Rabara R, Huynh LK, Basu GC, Nguyen HB, Gupta G (2018) Liposome delivery system of antimicrobial peptides against huanglongbing (HLB) citrus disease. Biophys J 114:266a
He Y, Li X, Deng X (2007) Discrimination of varieties of tea using near infrared spectroscopy by principal component analysis and BP model. J Food Eng 79:1238–1242
Hu J, Jiang J, Wang N (2017) Control of citrus huanglongbing via trunk injection of plant defense activators and antibiotics. Phytopathology 108:186–195
Jagoueix S, Bové JM, Garnier M (1996) PCR detection of the two “Candidatus” liberobacter species associated with greening disease of citrus. Mol Cell Probes 10:43–50
Khan KM, Krishna H, Majumder SK, Gupta PK (2015) Detection of urea adulteration in milk using near-infrared Raman spectroscopy. Food Anal Methods 8:93–102
Koester S, Rim K, Chu J, Mooney P, Ott J, Hargrove M (2001) Effect of thermal processing on strain relaxation and interdiffusion in Si/SiGe heterostructures studied using Raman spectroscopy. Appl Phys Lett 79:2148–2150
Li W, Hartung JS, Levy L (2007) Evaluation of DNA amplification methods for improved detection of “Candidatus Liberibacter species” associated with citrus huanglongbing. Plant Dis 91:51–58
Li X, Lee WS, Li M, Ehsani R, Mishra AR, Yang C, Mangan RL (2012) Spectral difference analysis and airborne imaging classification for citrus greening infected trees. Comput Electron Agric 83:32–46
Martinelli F, Reagan RL, Dolan D, Fileccia V, Dandekar AM (2016) Proteomic analysis highlights the role of detoxification pathways in increased tolerance to Huanglongbing disease. BMC Plant Biol 16:167
Movasaghi Z, Rehman S, Rehman IU (2007) Raman spectroscopy of biological tissues. Appl Spectrosc Rev 42:493–541
Najbjerg H, Afseth NK, Young JF, Bertram HC, Pedersen ME, Grimmer S, Vogt G, Kohler A (2011) Monitoring cellular responses upon fatty acid exposure by Fourier transform infrared spectroscopy and Raman spectroscopy. Analyst 136:1649–1658
Peng Q, Zeng C, Zhou Y, Lian S, Nie G (2015) Rapid determination of turmeric roots quality based on the Raman spectrum of curcumin. Food Anal Methods 8:103–108
Pérez MRV, Mendoza MGG, Elias MGR (2016) Raman spectroscopy an option for the early detection of citrus huanglongbing. Appl Spectrosc 70:829–839
Pérez-Enciso M, Tenenhaus M (2003) Prediction of clinical outcome with microarray data: a partial least squares discriminant analysis (PLS-DA) approach. Hum Genet 112:581–592
Ranulfi AC, Cardinali MC, Kubota TM, Freitas-Astua J, Ferreira EJ, Bellete BS, da Silva MFG, Boas PRV, Magalhaes AB, Milori DM (2016) Laser-induced fluorescence spectroscopy applied to early diagnosis of citrus Huanglongbing. Biosyst Eng 144:133–144
Sankaran S, Ehsani R (2013) Detection of huanglongbing-infected citrus leaves using statistical models with a fluorescence sensor. Appl Spectrosc 67:463–469
Sankaran S, Ehsani R, Etxeberria E (2010) Mid-infrared spectroscopy for detection of huanglongbing (greening) in citrus leaves. Talanta 83:574–581
Sankaran S, Mishra A, Maja JM, Ehsani R (2011) Visible-near infrared spectroscopy for detection of huanglongbing in citrus orchards. Comput Electron Agric 77:127–134
Tenenbaum JB, De Silva V, Langford JC (2000) A global geometric framework for nonlinear dimensionality reduction. Science 290:2319–2323
Vasko P, Blackwell J, Koenig J (1972) Infrared and raman spectroscopy of carbohydrates: Part II: normal coordinate analysis of α-D-glucose. Carbohydr Res 23:407–416
Wang N, Trivedi P (2013) Citrus huanglongbing: a newly relevant disease presents unprecedented challenges. Phytopathology 103:652–665
Wang Y, Zhou L, Yu X, Stover E, Luo F, Duan Y (2016) Transcriptome profiling of huanglongbing (HLB) tolerant and susceptible citrus plants reveals the role of basal resistance in HLB tolerance. Front Plant Sci 7:933
Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703
Yang H, Zhao C, Li R, Shen C, Cai X, Sun L, Luo C, Yin Y (2018) Noninvasive and prospective diagnosis of coronary heart disease with urine using surface-enhanced Raman spectroscopy. Analyst 143:2235–2242
Acknowledgments
The authors would like to express their gratitude to the National Navel Orange Engineering Research Center (China) for supplying samples.
Funding
This work was supported by the National Science Foundation of China (61575065). The authors would like to thank the MOE Key Laboratory of Laser Life Science & Laboratory of Photonic Chinese Medicine for funding this research.
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Kangkang Wang declares that he has no conflict of interest. Yubo Liao declares that he has no conflict of interest. Yaoyong Meng declares that he has no conflict of interest. Xianzhi Jiao declares that she has no conflict of interest. Wei Huang declares that she has no conflict of interest. Timon Cheng-yi Liu declares that he has no conflict of interest.
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Highlights
• Verified a method for early, rapid, and non-destructive detection of HLB with MCR.
• Raman spectra of HE, HA, and HS leaves differ clearly from each other.
• The better classification (PCA) and clustering (PLS-DA) effects compared with other methods.
• BP-ANN model could accurately predict the kinds of samples.
• It might promote the protection and safety of the citrus industry.
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Wang, K., Liao, Y., Meng, Y. et al. The Early, Rapid, and Non-Destructive Detection of Citrus Huanglongbing (HLB) Based on Microscopic Confocal Raman. Food Anal. Methods 12, 2500–2508 (2019). https://doi.org/10.1007/s12161-019-01598-1
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DOI: https://doi.org/10.1007/s12161-019-01598-1


