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Detection of Clavibacter michiganensis subsp. michiganensis Assisted by Micro-Raman Spectroscopy under Laboratory Conditions
Plant Pathol. J. 2018;34:381-392
Published online October 1, 2018
© 2018 The Korean Society of Plant Pathology.

Moisés Roberto Vallejo Pérez1*, Hugo Ricardo Navarro Contreras2, Jesús A. Sosa Herrera3, José Pablo Lara Ávila4, Hugo Magdaleno Ramírez Tobías4, Fernando Díaz-Barriga Martínez2, Rogelio Flores Ramírez1, and Ángel Gabriel Rodríguez Vázquez2

1CONACyT- Universidad Autónoma de San Luis Potosí. Álvaro Obregón #64, Col. Centro, C.P. 78000, San Luis Potosí, S.L.P. México
2Universidad Autónoma de San Luis Potosí. Coordinación para la Innovación y la Aplicación de la Ciencia y la Tecnología (CIACyT). Av. Sierra Leona #550, Col. Lomas 2a. Sección, C.P. 78210, S.L.P., México
3CONACyT- Centro de Investigación en Ciencias de Información Geoespacial A.C. Circuito Tecnopolo Norte 117, Col. Fraccionamiento Tecnopolo Pocitos, CP. 20313, Aguascalientes, Ags. México
4Universidad Autónoma de San Luis Potosí. Facultad de Agronomía y Veterinaria. Km. 14.5 Carretera San Luis Potosí, Matehuala, Ejido Palma de la Cruz, Soledad de Graciano Sánchez, C.P. 78321. S.L.P. México
Correspondence to: Phone) +52 (444) 826 2300, FAX) +52 (444) 826 8410
E-mail) vallejo.pmr@gmail.com
Received February 7, 2018; Revised May 10, 2018; Accepted May 31, 2018.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Clavibacter michiganensis subsp. michiganesis (Cmm) is a quarantine-worthy pest in México. The implementation and validation of new technologies is necessary to reduce the time for bacterial detection in laboratory conditions and Raman spectroscopy is an ambitious technology that has all of the features needed to characterize and identify bacteria. Under controlled conditions a contagion process was induced with Cmm, the disease epidemiology was monitored. Micro-Raman spectroscopy (532 nm λ laser) technique was evaluated its performance at assisting on Cmm detection through its characteristic Raman spectrum fingerprint. Our experiment was conducted with tomato plants in a completely randomized block experimental design (13 plants × 4 rows). The Cmm infection was confirmed by 16S rDNA and plants showed symptoms from 48 to 72 h after inoculation, the evolution of the incidence and severity on plant population varied over time and it kept an aggregated spatial pattern. The contagion process reached 79% just 24 days after the epidemic was induced. Micro-Raman spectroscopy proved its speed, efficiency and usefulness as a non-destructive method for the preliminary detection of Cmm. Carotenoid specific bands with wavelengths at 1146 and 1510 cm-1 were the distinguishable markers. Chemometric analyses showed the best performance by the implementation of PCA-LDA supervised classification algorithms applied over Raman spectrum data with 100% of performance in metrics of classifiers (sensitivity, specificity, accuracy, negative and positive predictive value) that allowed us to differentiate Cmm from other endophytic bacteria (Bacillus and Pantoea). The unsupervised KMeans algorithm showed good performance (100, 96, 98, 91 y 100%, respectively).
Keywords : chemometrics, epidemiology, KMeans, LDA, PCA


October 2018, 34 (5)
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