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Low recovery frequency of Gluconacetobacter diazotrophicus from plants and associated mealybugs in Cuban sugarcane fields

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Abstract

This study was aimed to isolate and identify the N2-fixing bacterium Gluconacetobacter diazotrophicus from 11 sugarcane varieties, grown under field conditions in four Cuban provinces, and from their associated mealybugs Saccharicoccus sacchari. Identification was based on morphological and biochemical tests and PCR-amplification of 16S rRNA genes using species-specific primers. From all sugarcane varieties and numerous mealybug colonies sampled, G. diazotrophicus isolates were recovered from inside sugarcane stems of only three varieties, and one from S. sacchari colony. These four isolates showed acetylene reduction activity in nitrogen-free media and contained nifH genes which were PCR-amplified using specific primers. ERIC-PCR fingerprinting was used to compare the Cuban G. diazotrophicus isolates with type and reference strains of N2-fixing Gluconacetobacteria. The very low frequency of G. diazotrophicus isolates recovered is probably related with the high doses of nitrogen fertilizers applied to the sugarcane in the Cuban fields for almost 30 years. Some genetic differences, using ERIC-PCR, were detected among G. diazotrophicus strains, which could be related with its source.

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Acknowledgements

This research was supported by the grant for the joint Project Cub00/02 between the Cuban Ministry for Science and Environment (CITMA) and German Bundenministerium fur Bildung und Forschung (BMBF). The authors also appreciate the support of the grant C/4226-1 from the International Foundation for Science (IFS). The technical assistance of Milagros Diez-Cabezas and Helga Castorph is also acknowledged. Authors appreciate very much the critical review of Dr. Juan Sanjuan at CSIC. Authors dedicate this paper to the Memory of Jesús Caballero-Mellado, excellent scientist and wonderful person.

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Authors and Affiliations

  1. Lab. Fisiología Vegetal, Dpto. Biología Vegetal, Facultad de Biología, Universidad de La Habana, La Habana, Cuba

    Patricia Ortega-Rodés, Eduardo Ortega, F. G. Loiret & Rosa Rodés

  2. University of Bayreuth, Bayreuth, Germany

    Diethelm Kleiner

  3. Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Ap. Postal 565-A, Cuernavaca, Morelos, Mexico

    Jesús Caballero-Mellado

Authors
  1. Patricia Ortega-Rodés
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  2. Eduardo Ortega
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  3. Diethelm Kleiner
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  4. F. G. Loiret
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  5. Rosa Rodés
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  6. Jesús Caballero-Mellado
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Corresponding author

Correspondence to Eduardo Ortega.

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Jesús Caballero-Mellado: In memoriam

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ESM. 1

Electrophoresis of nifH PCR amplification products. Primers used were NifH-F and NifH-R. Lane 1, 100 bp Ladder Plus; lane 2, FV-MBF-1; lane 3, FV-ML-1; lane 4, T2; lane 5, FV-B42-1; lane 6, FV-CP34-1; lane 7, PAl5T; lane 8, UAP-5560; lane 9, CFNE 501; lane 10, E. coli K12. Arrow points 500 bp band (JPEG 3 kb)

High resolution image (TIFF 49 kb)

ESM. 2

DNA electrophoresis of PCR amplification products obtained with the Gluconacetobacter diazotrophicus 23S rRNA gene specific primers, AD and 1440. Lane 1, 100 bp Ladder Plus; lane 2, FV-MBF-1; lane 3, FV-ML-1; lane 4, T2; lane 5, FV-B42-1; lane 6, FV-CP34-1; lane 7, G. diazotrophicus PAl5T; lane 8, G. azotocaptans CFN-Ca54T; lane 9, G. johannae CFN-Cf 55T; lane 10, CFNE 550; lane 11,UAP 5560; lane 12, E. coli K12; lane 13, 100 bp Ladder Plus. Arrow points 500 bp band. (JPEG 9 kb)

High resolution image (TIFF 410 kb)

ESM. 3

DNA electrophoresis of PCR amplification products obtained with the Gluconacetobacter diazotrophicus 16S specific primers (Franke-Whittle et al. 2005). Lane 1, 1 kb Ladder Plus; lane 2, G. diazotrophicus PAl5T; lane 3, FV-CP34-1; lane 4, FV-ML-1; lane 5, FV-MBF-1; lane 6, FV-B42-1; lane 7, T2; lane 8, G. azotocaptans CFN-Ca54T; lane 9, G. johannae CFN-Cf55T; lane 10, Control (water) (JPEG 12 kb)

High resolution image (TIFF 172 kb)

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Ortega-Rodés, P., Ortega, E., Kleiner, D. et al. Low recovery frequency of Gluconacetobacter diazotrophicus from plants and associated mealybugs in Cuban sugarcane fields. Symbiosis 54, 131–138 (2011). https://doi.org/10.1007/s13199-011-0133-3

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