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Multilocus Sequence Typing reveals high variability among Clavibacter michiganensis subsp. michiganensis strains affecting tomato crops in Uruguay

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Abstract

Bacterial canker caused by Clavibacter michiganensis subspecies michiganensis (Cmm) has been a threat to tomato production in Uruguay for several years. In this study, 39 Cmm strains were collected from several outbreaks and production areas in Uruguay, and were identified by molecular assays and pathogenicity tests on a susceptible cultivar of tomato. In addition, a TaqMan assay targeting a putative two-component system sensor kinase gene demonstrated good specificity with all strains tested and gave no false negative results. The first epidemiological study of Cmm in this country was carried out in order to elucidate the origin of outbreaks and sources of infection and dissemination of the pathogen. Strains from Uruguay showed high genetic diversity based on a Multi Locus Sequence Typing analysis of five housekeeping genes. This approach revealed 36 sequence types (STs) within a worldwide collection of 108 Cmm strains. Ten STs correspond to strains solely isolated in Uruguay, including eight novel STs for the subspecies michiganensis. This high diversity reflects the introduction of new strains from different origins that most probably results from seed importation. This study provides relevant information about the distribution and origin of strains causing bacterial canker in Uruguay and will pave the way for the establishment of preventive measures to control the disease.

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Abbreviations

Cmm:

Clavibacter michiganensis subsp. michiganensis

ST:

Sequence Type

CC:

Clonal complex

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Acknowledgments

This work was supported by CSIC I + D Grupos N° 652 and MSc. scholarship provided to V. Croce by the National Research Council in Uruguay (ANII).

We thank Enrique Verdier from DGSA-MGAP and Elisa Silvera and María José Montelongo from Agronomy Faculty for providing Cmm strains isolated in Uruguay; Ana Arruabarrena for helping in the isolations of strains from Salto; Sophie Bonneau for her useful advices to build the trees; Eliana Richard for technical support in figure editing; Federico Boschi and Diego Maeso for useful discussions; and Maria Inés Lapaz and Analía Sanabria for critically reviewing the manuscript.

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Authors and Affiliations

  1. Laboratorio de Microbiología Molecular, Cátedra de Microbiología, Departamento de Biociencias, Facultad de Química, Universidad de la República, CP 11800, Montevideo, Uruguay

    Valentina Croce, María Julia Pianzzola & María Inés Siri

  2. INRA, UMR1345 IRHS Institut de Recherches en Horticulture et Semences, F-49071, Beacouzé, France

    Karine Durand & Marie-Agnès Jacques

  3. Instituto Nacional de Investigación Agropecuaria, INIA Salto Grande, Salto, Uruguay

    Matías González-Arcos

Authors
  1. Valentina Croce
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  2. María Julia Pianzzola
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  3. Karine Durand
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  4. Matías González-Arcos
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  5. Marie-Agnès Jacques
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  6. María Inés Siri
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Corresponding author

Correspondence to María Inés Siri.

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Figure S1

qPCR amplification with primers designed by Luo et al. (2008) showing the results of six strains as an example. A. Fluorescence vs. cycles graph showing the inefficiency of the CMM probe by atypical curves (strains MAI 1001, MAI 1003 and MAI 1027). Strains showing typical curves (MAI1031, MAI1029 and MAI1039) are displayed in this graph in order to compare both kinds of curves. B. The six products sequences of the strains showed in (a) aligned and mapped with the probe shows a mismatch in the sequences of strains showing atypical curves. (GIF 80 kb)

High resolution image (TIFF 32807 kb)

Figure S2

Maximum likelihood trees based on partial sequences of genes atpD, dnaK, gyrB, ppk and recA. Bootstrap values higher than 50 are displayed at each node in red. (PDF 423 kb)

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Croce, V., Pianzzola, M.J., Durand, K. et al. Multilocus Sequence Typing reveals high variability among Clavibacter michiganensis subsp. michiganensis strains affecting tomato crops in Uruguay. Eur J Plant Pathol 144, 1–13 (2016). https://doi.org/10.1007/s10658-015-0738-0

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