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Detection of Clavibacter michiganensis subsp. michiganensis Assisted by Micro-Raman Spectroscopy under Laboratory Conditions.

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 ? 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).

SUBMITTER: Perez MRV

PROVIDER: S-EPMC6200046 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Detection of Clavibacter michiganensis subsp. michiganensis Assisted by Micro-Raman Spectroscopy under Laboratory Conditions.

Pérez Moisés Roberto Vallejo MRV Contreras Hugo Ricardo Navarro HRN Sosa Herrera Jesús A JA Ávila José Pablo Lara JPL Tobías Hugo Magdaleno Ramírez HMR Martínez Fernando Díaz-Barriga FD Ramírez Rogelio Flores RF Vázquez Ángel Gabriel Rodríguez ÁGR

The plant pathology journal 20181001 5

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 ...[more]

PMID: 30369848

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Project description:Tomato bacterial canker caused by Clavibacter michiganensis subsp. michiganensis is one of the most important seed-borne tomato diseases around the globe. The disease was initially reported in 1993 in Iran, and it became a rising threat for the multibillion dollar tomato industry of the country during the last decade. In this study, using phylogeographic analyses, we determined genetic diversity and geographic distribution of C. michiganensis subsp. michiganensis in Iran. Our field surveys showed that the pathogen is expanding into the southern and eastern areas of the country. Furthermore, multilocus sequence analysis and typing (MLSA/MLST) using the sequences of five housekeeping genes (atpD, gyrB, ppk, recA, and rpoB) revealed that 37 C. michiganensis subsp. michiganensis strains isolated in Iran had high genetic diversity and placed in 15 sequence types (STs), while all the available 184 worldwide C. michiganensis subsp. michiganensis sequences were placed in 43 STs. MLSA divided the worldwide C. michiganensis subsp. michiganensis strains into two phylogroups (I and II). Among the 37 strains isolated in Iran, 30 strains clustered in phylogroup I, while 7 strains clustered in phylogroup II. Phylogeographic data inferred from the allelic profile of the five housekeeping genes suggested multiple introductions of C. michiganensis subsp. michiganensis inoculum into Iran, while the geographic origin of the Iranian C. michiganensis subsp. michiganensis strains remains undetermined. Further analyses using higher numbers of strains are warranted to decipher the evolutionary history of C. michiganensis subsp. michiganensis in Iran. Additionally, stricter seed/transplant inspections are recommended to reduce the risk of pathogen expansion to areas with no history of the disease.IMPORTANCEClavibacter michiganensis subsp. michiganensis, the causal agent of tomato bacterial canker disease, is one of the economically important pathogens of solanaceous crops (e.g., eggplant, pepper, and tomato) around the world. The disease occurs in many countries, with a particular importance in regions characterized by high precipitation and humid environmental conditions. As a seed-borne pathogen, C. michiganensis subsp. michiganensis is included in the A2 (high risk) list of quarantine pathogens by the European and Mediterranean Plant Protection Organization (EPPO). Bacterial canker disease was reported for the first time in 1993 in Iran, while the geographic distribution, genetic diversity, and phylogenetic position of the causal agent remain undetermined. In this study, using the multilocus sequence analysis and typing (MLSA/MLST) approach, we provided a phylogeographic scheme for the C. michiganensis subsp. michiganensis strains isolated in Iran. Furthermore, global-scale phylogenetic analyses led to determination of phylogenetic position of Iranian C. michiganensis subsp. michiganensis strains among worldwide population of the pathogen. Based on diversity parameters and population structure, we suggest relatively higher genetic diversity of the bacterial canker pathogen in Iran than has so far been observed in the other areas of the world. Results obtained in this study provide a novel insight into the genetic diversity and population structure of the bacterial canker pathogen on a global scale.

Dataset Information

Detection of Clavibacter michiganensis subsp. michiganensis Assisted by Micro-Raman Spectroscopy under Laboratory Conditions.

Publications

Detection of <i>Clavibacter michiganensis</i> subsp. <i>michiganensis</i> Assisted by Micro-Raman Spectroscopy under Laboratory Conditions.

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