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Comparison of loop-mediated isothermal amplification, polymerase chain reaction, and selective isolation assays for detection of Xanthomonas albilineans from sugarcane

  • Research article, Bacteriology
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Abstract

A loop-mediated isothermal amplification (LAMP) assay was developed and compared to polymerase chain reaction (PCR), nested PCR, and selective isolation assays for detection of Xanthomonas albilineans, the causal agent of sugarcane leaf scald. The pathogen was isolated on selective medium from 44 out of 45 (98%) samples taken from symptomatic stalks, and from 44 out of 70 (63%) samples from asymptomatic stalks that were collected from plots with symptomatic stalks. Forty-two (93%), 41 (91%), and 42 (93%) symptomatic samples tested positive by LAMP, PCR and nested PCR, respectively. The pathogen was detected in 19 (27%), 8 (11%), and 25 (36%) of the 70 asymptomatic samples by LAMP, PCR and nested PCR, respectively. Symptomatic stalks were mainly, but not always, associated with high populations of the pathogen (107–109 CFU/ml), and asymptomatic stalks with low populations (<103 CFU/ml) or no bacteria. Although our LAMP and nested PCR methods detected 10 CFU/ml of X. albilineans in suspensions prepared with pure culture, they sometimes failed to detect the pathogen in samples with low pathogen populations. Isolation on selective medium along with another method should therefore be used for detection of the pathogen in asymptomatic stalks, especially in quarantine programs.

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Acknowledgements

Vanessa Duarte Dias was supported by a fellowship from the Brazil Scientific Mobility Program sponsored by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). We thank Dr. Alexandre S. G. Coelho for his help in the statistical analysis of data. This work would not have been possible without the financial support of the Florida Sugar Cane League. This work is supported by the USDA National Institute of Food and Agriculture (project Hatch/Rott FLA-BGL-005404).

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Authors and Affiliations

  1. IFAS, Everglades Research & Education Center, University of Florida, Belle Glade, FL, 33430, USA

    Vanessa Duarte Dias, Martha Hincapie & Philippe Rott

  2. Universidade Federal de Goiás, Goiânia, GO, 74690-900, Brazil

    Vanessa Duarte Dias & Marcos G. Cunha

  3. BGPI, CIRAD, Montpellier, France

    Emmanuel Fernandez, Isabelle Pieretti & Philippe Roumagnac

  4. BGPI, INRA, CIRAD, SupAgro, Univ Montpellier, Montpellier, France

    Emmanuel Fernandez, Isabelle Pieretti & Philippe Roumagnac

  5. USDA-ARS, Sugarcane Field Station, Canal Point, FL, 33438, USA

    Jack C. Comstock

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  1. Vanessa Duarte Dias
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  2. Emmanuel Fernandez
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  3. Marcos G. Cunha
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  4. Isabelle Pieretti
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  7. Jack C. Comstock
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  8. Philippe Rott
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Correspondence to Philippe Rott.

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Section Editor: Jorge T. De Souza

Electronic supplementary material

Supplementary Fig. 1

Detection of Xanthomonas albilineans by PCR (A) and nested PCR (B) in water suspensions from a pure culture of the pathogen (10-fold dilutions). Lane 1 = 100 bp ladder; lanes 2 to 8 = 106, 105, 104, 103, 102, 10, and 1 CFU/ml, respectively. The sizes of the PCR (A) and nested PCR (B) amplicons are 440 and 308 bp, respectively (PPTX 339 kb)

Supplementary Table 1

Characteristics of the 115 sugarcane samples used in this study (DOCX 19 kb)

Supplementary Table 2

Primers used to detect Xanthomonas albilineans by loop-mediated isothermal amplification (LAMP), polymerase chain reaction (PCR) and nested polymerase chain reaction (Nested PCR) (DOCX 12 kb)

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Duarte Dias, V., Fernandez, E., Cunha, M.G. et al. Comparison of loop-mediated isothermal amplification, polymerase chain reaction, and selective isolation assays for detection of Xanthomonas albilineans from sugarcane. Trop. plant pathol. 43, 351–359 (2018). https://doi.org/10.1007/s40858-018-0216-2

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  • DOI: https://doi.org/10.1007/s40858-018-0216-2

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